The Bird's Nest Fungi 9781442632516

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The Bird's Nest Fungi

HAROLD j. BRODIE is Professor Emeritus of Botany, University of Alberta, Edmonton, and now lives in Victoria, BC. He has received many honours and awards as a result of his researches on Bird's Nest Fungi and many other groups of fungi. The intriguing Bird's Nest Fungi (Nidulariaceae) of forest, meadow-, and garden have been familiar to botanists since 1601, but only relatively recently has the significance of their peculiar form been realized. Dr Brodie traces the long controversy that arose when Bird's Nest Fungi were first classified as seed plants because of the numerous seed-like bodies contained in their small cup-shaped fruit bodies. The 'seeds' are now known to contain spores like those of other fungi such as puffballs, to which the Nidulariaceae are related. Present-day research has

shown that certain Bird's Nest Fungi produce chemicals having previously unrecognized molecular structure. Between these milestones Dr Brodie reveals the solution to the mystery of the dispersal of the 'eggs'from the'bird's nest': the fruit bodies are splash guns from which the reproductive spores are ejected by the force of falling raindrops. This explanation of the phenomenon is supported by copious observations and hitherto unpublished experiments. All known species of Nidulariaceae, including many only recently recognized, are described in this volume. All aspects of growth, structure, development, and life-cycle of these fungi, both in nature and in laboratory culture, are reported in a modern, comprehensive treatment of a subject which is of interest not only to mycologists but to amateur naturalists as well.

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The Bird's Nest Fungi

HAROLD J. BRODIE

U N I V E R S I T Y OF T O R O N T O PRESS TORONTO AND BUFFALO

University of Toronto Press 1975 Toronto and Buffalo Printed in Canada

Library of Congress Cataloging in Publication Data Brodie, Harold Johnston, 1907The bird's nest fungi. Bibliography: p. Includes index. i. Nidulariaceae. i. Title. QKÓ29.N5B7

589'.221

ISBN 0-8020-5307-6

75-19476

TO THE M E M O R Y OF A.H. REGINALD BULLER 1874 - 1944 whose boundless enthusiasm concerning the world of the fungi made everlasting debtors of all who knew him or were privileged to hear him.

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Contents

Foreword i ix Preface / xi Organization and Use of the Book i xiv 1 General Account of the Nidulariaceae / 3 A / Introduction and general structure / 3 B / Dispersal / 7 c / Nuclear behaviour /10 D / Other aspects of bird's nest fungi /10 2 Brief Historical Outline /14 A / Early studies /14 B / Unravelling of the dispersal problem /17 c / Recent studies / 20 D / Conclusion/ 22 3 Basidiospore Form, Development, and Germination / 23 A / Form and structure / 23 B / Development / 25 c / Germination / 26 D / Germ tubes and germlings / 29 4 Characteristics and Interactions of Homokaryotic My celia/ 32 A / Characteristics / 32 B / Nutritional requirements / 36 c / Mycelial interactions / 37 5 Heterokaryotic Mycelium and Fruit Body Formation / 44

A / Characteristics of heterokaryotic mycelium / 44 B / Formation of sporocarps / 46 c / Conditions known to affect fruiting / 47 D / Early signs of fruiting / 52 E / Opening of mature sporocarps / 52 F / Orientation of sporocarps / 57 G / Irregular fruiting / 58 H / Variation in form of sporocarps / 61 i / Details of sporocarp development / 63 6 Morphology and Behaviour of Nuclei / 71 7 The Fruit Body as a Spore Dispersal Mechanism / 80 A / The mechanism / 80 B / The funiculus / 80 c / The wall of the fruit body / 88 D / Shape of fruit body and angle of its sides / £ E / Plications and setae / 89 F / Position of fruit body / 90 G / Surface and arrangement of pendióles / 91 8 Experiments on Splash Dispersal of Pendióles / 93 A / The germ of the idea / 93 B / The experiments / 94 9 Occurrence, Distribution, and Ecology/101 A / General /101 B / Occurrence /101 c / Distribution and ecology /109

10 Miscellaneous Observations and Notes /119 A / Ingestion of bird's nest fungi by humans /119 B / Some metabolites of Cyathus /120 c / Rain-splash dispersal in other plants /122 D / Other cupulate fruit bodies and some thoughts on evolution /123 11 Review of Taxonomic Characters /126 A / Introduction /126 B / Macroscopic characteristics /127 c / Microscopic characteristics /129 12 The Genera Nidularia and Mycocalia /133 A / Distinction between the two genera /133 B / The genus Nidularia /133 c / The genus Mycocalia /137 D / Notes on doubtful species of Nidularia /140

viii / Contents

13 The Genera Nidula and Crucibulum 1142 A / Nidula 1142 B / The genus Crucibulum /147 14 The Genus Cyathus /150 A / The genus /150 B / The groups of Cyathus /150 c / Cyathus species and synonyms listed alphabetically/151 D / Species descriptions by groups /154 E / Doubtful species /180 Nidulariana /182 Selected Bibliography /188 Glossary /193 Index 1195

Foreword

This book, which represents the work of a distinguished investigator's professional lifetime, will be welcomed by all mycologists, teachers, and researchers alike, for it is as complete a treatise on a group of relatively little known but most interesting fungi as our present knowledge permits. The Bird's Nest Fungi is not a taxonomic revision but a monograph in every sense of the term. In fact, the author emphasizes the biology of these fungi and more especially the interesting mechanism they employ in releasing their propagules more than their classification, which, however, he does not neglect completely. Although throughout his discussion Dr Brodie is meticulously careful to credit others for their discoveries, his own long-term involvement with this group and his personal knowledge of all phases of the life of the Bird's Nest Fungi are evident in every chapter. The book is clearly written and the author's scholarship and vibrant personality shine throughout. His sense of humour is particularly evident in the closing section of the book, the Nidulariana, which only Brodie would

think of including in a monograph of this kind. And we are grateful that he did. It adds more sparkle in an already scintillating work. This is a much needed exposition of the Nidulariaceae which will take its place next to Tulasne's as the classic that it is, for Professor Brodie's expertise in the field, where he finds his pet fungi, in the herbarium, where he preserves them, and in the laboratory, where he studies their living processes, is evident throughout this book. Some of us should have liked to have Brodie's keys a little more detailed to enable us to identify Nidulariaceae a little more rapidly and a bit more certainly than is possible with the ones he provides, but the author has chosen to emphasize biology over taxonomy and this is his prerogative. One more word - I think if Dr Buller could read this work in its final form, he would say: 'You are sure, aren't you?'

CONSTANTINE J. ALEXOPOULOS

Professor of Botany University of Texas at Austin

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Preface

Anyone may become impatient if asked, too often, to explain that bird's nest fungi have never been found in bird's nests and have nothing at all to do with bird's nest soup. In the laboratory, surrounded by preserved specimens, living cultures, photographs, and drawings of the intriguing little bird's nests, not much time and energy are needed to tell the story briefly. A convert to Nidulariology may not be made on the spot, but at least it is possible to offer some reasons for one's eccentric taste: to explain that the bird's nests in question are fungi is not difficult, for everyone has some acquaintance with mushrooms, bread moulds, and cheese moulds, all of which are fungi ; to explain that the small fungus cups are the 'nests,' that the 'eggs' are masses of spores to serve for propagation, that the eggs are propelled from their nests by the force of falling raindrops, and that the entire fungus structure is a marvellous adaptation for accomplishing reproduction and dispersal - all this is not difficult. And, if time is a consideration, one tries to justify one's own interest in the bird's nest fungi by pointing out that when these organisms are grown in laboratory culture they produce, by their mysterious chemistry, a whole battery of chemical compounds that have not been known to chemists before and that may eventually be shown to have value as drugs to combat disease. But to tell more than this in sufficient detail

to imbue the reader or observer with pleasure and satisfaction requires time and patience. Over thirty years ago, my interest was aroused in a group of small goblet-shaped fungi which, except for the classic study of Tulasne in 1844, had remained in obscurity although botanists had been aware of their existence since bird's nest fungi were first described as long ago as 1601. The fungi assigned by mycologists to the family Nidulariaceae produce disseminules or propagating units large enough to have been mistaken, in the past, for seeds. Even when the controversy as to whether the Nidulariaceae are seed-plants or not ended, there still remained unsolved the problem of how the spores are dispersed and of how they escape from within the enclosing disseminules or pendióles. Tulasne demonstrated that each peridiole is firmly attached to the inner surface of the vase-like fruiting body by a very elegant and complex piece of mycelial apparatus which later became known as the funiculus. The very complexity of this umbilical attachment seemed to suggest some unique mechanism of spore dispersal, but the true operation of the funiculus long remained a mystery. Not until 1941 was it possible to fit together the numerous pieces of an intriguing jig-saw puzzle. It was finally apparent that the fruit body of the Nidulariaceae is an apparatus not only developed for the production of the prop-

agating spores but beautifully constructed and modified to serve as a means of utilizing the important natural force of falling raindrops to bring about the ejection of the seed-size disseminules to a distance of several feet. Recognition of the importance of rain in the dispersal of spores in the Nidulariaceae constitutes what is doubtless the most significant contribution that has been made by studies of these fungi to biological knowledge as a whole ; for, from it, came the realization that rainsplash is a very important aspect of the plant dispersal mechanism, it having been evolved in one form or another in the majority of plant phyla. But if the Nidulariaceae have taught us mainly about rain-splash dispersal in plants, it is nevertheless true that studies of these fungi have also been rich in interest in other directions: the development of their fruit bodies displays interesting and unique features; the interactions of their mycelia provide an example of an unexplained phenomenon of nuclear migration; study of their nuclei has provided some facts that may help to solve the problem of whether or not fungal nuclei are different in behaviour and structure from those of other plants ; study of their taxonomy has revealed previously unrecognized species ; and, most recently, mycelia of some species have been shown to produce a series of hitherto unrecognized chemical substances having antimicrobial properties and possible therapeutic value. Beyond this, there is much delight and satisfaction in contemplation of bird's nest fungi in their own right and it is in an effort to share such pleasure with others that this account of their forms and lives has been written. The intention has not been to deal exhaustively with all that has been written about the Nidulariaceae, or to establish with finality the true status of those species which are as yet imperfectly known. Rather, the aim has been to describe the chief aspects of the biology of the Nidulariaceae with which the author has had the greatest amount of first-hand experience, in the hope that others may be tempted to xii / Preface

probe whatever secrets may still be hidden in nature's fairy goblets. Another intention, which generally has been secondary to the first (in places it may even have been uppermost), has been to point out, wherever possible and as explicitly as possible, those dark corners of our knowledge about bird's nest fungi into which the torch of research should still be made to shine ; and if the impression is produced, here and there, that at present more is unknown than is actually known, no apology is offered. The world association of scientists (may it never be given a name !) confers upon each of its members complete and unstinting cooperation and mutual assistance and expects no acknowledgment. I have been no less fortunate than my fellows in this regard ; and thus this book is possible because of a multitude of helping hands, including those of small smiling boys in the jungles of Guadeloupe who were eager to look for bird's nest fungi, as well as those of directors of museums and herbaria who never refused all possible assistance. All helpers should recognize themselves somewhere in these pages. Where copyright and special circumstances required it, specific acknowledgment is made. The major acknowledgment I wish to make is of my debt to the late Professor A.H.R. Buller, who first aroused my interest in the Nidulariaceae late in 1941, and bequeathed to me about a hundred pages of notes recording the early stages of the first modern investigation of the bird's nest fungi. Buller did not live to see into press more than one small note concerning his observations and ideas. I have been privileged to carry the torch but it was lighted by another. Special thanks are due to Dr D. B. O. Savile of the Plant Research Institute (Canada Department of Agriculture) who generously devoted much time and energy to a meticulous reading of the manuscript and made numerous suggestions for its improvement. Dr R.W.G. Dennis (Herbarium, Kew Gardens) has frequently given unstinting assistance in such matters as

rechecking details regarding type material and literature references not readily available to me. Dr Dennis's help has been of the utmost value to me and is gratefully acknowledged. Mr J.T. Palmer of Liverpool has generously given the benefit of his advice and criticism concerning what has been recorded herein about species of the genera Nidularia and My co calía. Illustrations have come from many sources. Most photographers who encountered bird's nest fungi for the first time begged to be allowed to take a shot and many of their shots are included. True' scientists who deplore the inclusion in this book of a bit of leaven labelled 'Nid-

xiii / Preface

ulariana' can easily eliminate it by means of scissors. These notes are included to record, in part, the pleasures encountered as the scientist gropes his way towards new discoveries. In dedicating this work to my teacher, critic and long-time friend, the late Dr A.H.R. Buller, I am deeply conscious of what he would have said if I had asked him to examine the manuscript - 'Are you sure?' Publication of this book has been made possible by grants from the National Research Council of Canada and the publications fund of the University of Toronto Press. Victoria, BC

HAROLD J. BRODIE

Organization and Use of theBook

Glossary A partial glossary is provided at the end of the book for amateur botanists and persons interested in natural history who generally welcome the definition of certain terms used herein. Cross-references These are given as follows: an arabic numeral indicates a Chapter ; this may be followed by a capital letter indicating the major section of the chapter ; subdivisions of the latter then follow in numerals and lower case letters, e.g., 7, A, i. A complete listing of the chapter contents is provided at the beginning of the book. Index For species names, where several references are given, the principal taxonomic reference is given in bold face numerals, e.g. 274. An asterisk * indicates reference to an illustration of the indexed item. Names of species or genera in the Index regarded as synonyms are enclosed in brackets, e.g. (albosaccum).

Synonymy A complete synonymy in a work of this size is impractical. In the chapters dealing with taxonomy, the principal synonyms are given, together with references to fuller or complete synonymy. Palmer (1968) provides a useful guide to the literature dealing with the Gasteromycetes.

Herbaria and Location of Specimens The largest and most important collection of the present century is the Lloyd Herbarium, included in the National Fungus Collections, Plant Industry Station, Belts ville, Maryland, USA. The Nidulariaceae known to Tulasne and Montagne are to be found in the herbarium of the Musée de l'Histoire Naturelle in Paris. The Royal Herbarium in Kew contains a large number of collections of early date as well as isotypes of much European material. Those species from South America described by Spegazzini are located in the Instituto de Botánica Spegazzini, Universidad Nacional de La Plata, Argentina. A large proportion of the species described in this book are also in the author's own herbarium (including all types of species named by the author). Photographs of the species included Not all the species described in the book are illustrated, partly for reasons of economy but to a large extent because fresh undamaged specimens of undoubted authenticity were not available for some species. Even when photographs or drawings of certain species could have been provided, these are not included because such illustrations were considered to be of little value for identification purposes. Keys For the largest genus Cyathus, a key is provided only to the sections. In the author's ex-

peri ence, long and complex key s co ve ring large numbers of species are difficult to use and are too frequently used as a substitute for a careful

xv / Organization and use of the book

comparison of the specimen with the full description.

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The Bird's Nest Fungi

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I/ General Account of the Nidulariaceae

A / I N T R O D U C T I O N AND GENERAL STRUCTURE The first encounter with a member of the gasteromycete fungi known as the Nidulariaceae or bird's nest fungi usually engenders in the mind of the observer, whether a professional botanist or an amateur nature lover, a feeling of delight and wonder: delight certainly, because of the symmetry and artistic form of the small vase-shaped or bell-shaped fruit bodies; wonder probably, if the observer is at all curious as to why the little cups should be filled with small lentil-shaped bodies resembling seeds. The whole fruit body bears a general resemblance to a miniature bird's nest containing eggs, whence the common English name bird's nest fungi (figure i). The Latin name for the family to which these fungi belong is Nidulariaceae, which is derived from nidula, meaning a little nest. These small treasures of the fungus world are often found inadvertently and sometimes in strange places. To set out deliberately to find them is, however, another matter: most bird's nest fungi are small, often less than 1/4 inch or about 7 mm in height or breadth ; most of them are light brown or the colour of old wood, although a few species are white, grey, yellow, or rust-brown. They may therefore easily be overlooked. All species are saprophytic and are usually 3 / General account of the Nidulariaceae

found in moist, often shaded locations, growing upon decaying wood, on old sacking or other similar fibrous material, on the excrement of horses and cows, or directly upon soil, especially if the latter is rich in humus. In temperate regions, bird's nest fungi are most abundant in the early autumn; in the tropics, the rainy season is the most productive period. However, because the fruit bodies are tough and leathery and seldom decay, old dry specimens may be found at almost any season of the year. The order Nidulariales belongs to the Gasteromycetes, those basidiomycetes that release their spores in a closed sporocarp, rather than discharging them forcibly in the open air as do most of the class. The Nidulariales are widely distributed, some species having been found in most countries, although a few are at present known to exist in only one or two localities. The order comprises two families: the Nidulariaceae includes the five genera Mycocalia, Nidularia, Nidula, Crucibulum, and Cyathus ; the apparently related genus Sphaerobolus was at one time included in the Nidulariaceae, but was placed by E. Fischer (1933) in a separate family, the Sphaerobolaceae. This classification has been followed by most present-day mycologists (Alexopoulos, 1962). In this book, we are concerned only with the Nidulariaceae. In structure, the fruit bodies of the five gen-

Newly opened fruit bodies of Cyathus striatus showing plication or fluting of cups and moist FIGURE i glistening pendióles. On the right are three unopened cups still covered by the white epiphragm. Xz.5

era are all basically alike. A typical fructification consists of a so-called peridium (figure 2), a term which in a strict sense applies to the outer bounding wall but which has also frequently been used to designate the fructification as a whole. Within the peridium are numerous lenticular bodies (figure 2) or 'pendióles' (also termed pendióla, or sporangioles). Within each peridiole there is a hymenial or fertile layer (figure 2) consisting of basidia, which bear basidiospores, intermingled with paraphyses. The mature fruit body in all genera except Nidularia and Mycocalia most commonly is in 4 / The bird's nest fungi

the form of a cup, a vase, or an inverted bell. Nidularia and. Mycocalia are subcylindrical to globose. In Cyathus the internal surface of the cup may be longitudinally ridged (usually referred to as 'plicate' or 'striate/ figures i, 2) ; in such species, the external surface is also ridged and, in some species, both surfaces are ridged, although the external ridges may be hidden because of the woolly exterior of the fruit body. In Nidula and Crucibulum the cups appear, to the unaided eye, smooth externally and internally. Often, especially in Cyathus, the fruit body is expanded at the base into a broad or rounded

striae

setae

peridium

funicular - cord peridiole purse sheath

peridiole

hapteron tunica

purse

middle piece stipe sheath

funicular ^ cord hapteron

emplacement middle piece

FIGURE 2 Section through fruit body of Cyathus striatus (semi-diagrammatic) showing names of structures. Basidiospores are borne inside the lenticular pendióles, each of which is attached to the inner wall of the fruit body by a funiculus consisting of a sheath, middle piece, and purse. When a raindrop lands in a peridiole, the purse is torn open at its lower end, freeing the long funicular cord. Central figure X10, approximate. Section at right X25, approximate. Figure courtesy Can. J. Bot., 1951, 29: 224 5 / General account of the Nidulariaceae

mycelial pad called the 'emplacement' (figure 2). The rim or lip of the cup is smooth or unadorned in some species, whereas in others it is beset with hyphal bristles or setae, which are minute in some species and conspicuous even to the unaided eye in others (figure 2). The wall of the peridium of Crucibulum consists of a single thick layer of closely interwoven hyphae. In Cyathus, the wall is composed of three distinct layers. The apex of a young fruiting body is at first covered by a special membrane of hyphae called the 'epiphragm' (figures i, 24) which, however, is absent in Nidularia and Mycocalia. As the result of many changes during development, the peridial wall gradually expands laterally and the epiphragm ruptures, thus opening the cup and exposing its contents. In a freshly opened cup of Cyathus, the pendióles may be seen at first to be lying in a clear gelatinous material (figure 23c) which soon dries. The internal structure of each peridiole is complex and will be dealt with in detail in Chapter 5, i, 2. At this point it is sufficient to understand that, when the peridiole is mature, the central portion of it contains a mass of basidiospores, most of which have become separated from their basidia and lie free from one another or, to a greater or less degree, lie intermingled with hyphal fragments. In Cyathus and Crucibulum, each peridiole is firmly attached to the inner wall of the peridium by a funiculus (figures 2,36) consisting of a complex cord of interwoven hyphae. This will be discussed in more detail later (Chapter 7, B). Using a stereoscopic microscope, the funiculus may be seen to be differentiated into three regions: (i) a somewhat conical tube, whose distal end merges with the inner wall of the peridium (figure 365) and which is called the basal piece ; (2) a median constricted portion called the middle piece (figure 36m) ; (3) an upper sheath (the purse, figure 36p), which is connected at its base to the constricted portion and at its apex to the lower surface of the peridiole. Within the purse there is a thread composed 6 / The bird's nest fungi

of long hyphae wound around one another like the strands of a cable. This structure, the funicular cord, is coiled up within the purse ; it is attached at one end to the peridiole, whereas at the other it becomes the so-called hapteron and lies just inside the lower end of the purse (figure 36h). In Crucibulum a funiculus is also present, but it is simpler in structure than that of Cyathus. In Nidula, Nidularia, and Mycocalia there is no funiculus and, especially in the last two genera, the peridioles are covered with an adhesive substance. Using only the simple features described above, we can characterize the genera1 of the Nidulariaceae, about which detailed information will be given in the following pages. The structural relationships between the genera can be shown in a key. KEY TO THE G E N E R A i Fruit body globose at maturity, dehiscing irregularly, epiphragm absent Nidularia (figure 50) and Mycocalia (see Chapter 12) i

Fruit body cup-shaped or vase-shaped at maturity, when young covered by an epiphragm 2 2 Peridioles free within the peridium, not attached by funiculi. Nidula (figures 51, 52) 2 Peridioles attached by funiculi to inner wall of peridium 3 3 Fruit body wall of one layer, funiculus a simple cord attached to a button-like mass Crucibulum (figure 38c) 3 Fruit body wall composed of three distinct layers, funiculus a complex cord made up of several distinct parts Cyathus (figures i, 2)

The small globose fruit bodies of Nidularia are usually brown or buff coloured. The broad rather shallow fruit bodies of Crucibulum vary from a rich ochre-yellow in fresh specimens to grey or dirty-white in old weathered specimens i The genus Mycocalia is omitted from this chapter henceforth because of the difficulty of separating it on a simple non-microscopic basis from Nidularia. Mycocalia will be dealt with in Chapter 12.

and the pendióles have a conspicuous white outer covering. In Ni dula, fruit bodies of three species vary from snow-white to grey, and in one species they are tawny. Fruit bodies of the many species of Cyathus vary in colour from almost white, through light buff and many shades of brown, to very deep brown or almost black. The genus Cyathus has by far the largest number of species: more than 70 have been named. Only four species oiNidula are known at present. Crucibulum has long been considered monotypic, but two more species have been named recently. B / DISPERSAL With the foregoing simplified account of the appearance and structure of the Nidulariaceae in mind, attention may now be given to one of the most interesting aspects of these fungi, namely the mechanism for the dispersal of pendióles in nature through the agency of falling raindrops. Much of the above information concerning the structure of bird's nest fungi was known to Tulasne as early as 1844. Tulasne (1844) had turned the searchlight of his wonderful powers of observation to the fruit body of Cyathus, revealing every detail with all the accuracy which his integrity, his microscope, and his brother's talents as an illustrator made possible. However, although Tulasne observed and recorded so much, he did not understand everything. He did not comprehend the relationship of the beautiful and complex structure of the fruiting body of Cyathus to the problem of how the pendióles that enclose the basidiospores escape from the fruit body. Being honest and humble, he admitted his failure in this respect and refrained from making any of the ludicrous guesses which had been made frequently by his predecessors. Lloyd (1906), in his monograph of the Nidulariaceae, dedicated his own work, with every justification, to Tulasne with the following line: 'L.R. Tulasne, who first made a thorough study of the Nidulariaceae and wrote 7 / General account of the Nidulariaceae

the first monograph on the subject and whose careful accurate work will always remain as the highest authority/ Although the necessary morphological evidence had been provided by Tulasne and although many bits and pieces of observation were recorded subsequently, still the clue necessary for full understanding of the mystery of dispersal continued to remain hidden for almost another century. The funnel-shaped or vase-shaped sporocarps of most members of the Nidulariaceae are splash-cups, from which the pendióles (containing basidiospores) are ejected to a distance of several feet by the force of falling raindrops which land in the open mouths of the cups. If the common species Cyathus striatus be taken as an example (figures i, 2), the following points may be regarded as important in the operation of this fungus splash-cup in nature (Brodie, i95ia, 1956, 1957). The cup is about 6-8 mm in diameter at the mouth. Large raindrops have a diameter of about 3-4 mm and such drops are effective in discharging the pendióles from a cup of comparable dimensions. The walls of the fruit body are elastic and consequently able to resist being deformed by the battering of raindrops. Also, the fruit body is provided with a relatively large solid mass of hyphae around its base which acts as an 'emplacement7 and prevents the splash-cup from being knocked over by rain. When large raindrops, having a terminal velocity of about 4-8 metres per second, fall into the fruit body, the displacement of water in the cup creates a strong thrust upward and outward along the inclined sides, and the pendióles are forcibly ejected (figure 4). The fungus cup has sides which form an angle of 60 to 70° with the horizontal. This angle is probably related to the splash action, for it will be shown that the maximum ejection of water drops from model cups occurs when such cups are constructed with the sides inclined at an angle of the same order. When a peridiole is being splashed from

fruit body

peridiole

young binucleate

heterokaryotic mycelium (2n)

basidium

HETEROKARYQTIC PHASE

diploid (2n) fusion nucleus meiosis

HOMOKARYOTIC homokaryotic mycelia (n)

Nt

mature basidiospores (binucleate)

PHASE

haploid (n) basidiospores

young basidiospores (uninucleate)

FIGURE 3 Diagram representing nuclear events in the life cycle of most of the Nidulariaceae: homokaryotic basidiospores (haploid) give rise to homokaryotic mycelia which must unite in appropriate pairs to produce the heterokaryotic (diploid) mycelium ; the fruit body which develops from the latter produces peridioles within which basidiospores are borne ; young basidia contain a pair of haploid sexually compatible nuclei which fuse; the diploid fusion nucleus undergoes meiosis resulting in haploid basidiospores. 8 / The bird's nest fungi

the fruit body (figure 4) the force jerks the funiculus, and the purse is torn open at its lower end where it is attached to the middle piece. Rupture of the purse causes the almost instantaneous expansion of a special mass of hyphae which had been coiled under pressure within the lower part of the purse. The hyphae of this mass are highly adhesive and together form an attachment organ, the 'hapteron' (figure 2). If a peridiole in flight from a fruit body strikes an object such as a leaf, petiole, or stem, the hapteron (then consisting of a mass of loosely interwoven and adhesive hyphae) becomes entangled in epidermal hairs or other projections (figures 4, 38b, 39). The peridiole may continue in flight and, as it does so, the long funicular cord is pulled out from the upper part of the purse (figure 4). This cord is a miniature cable, composed of many hyphae spirally twisted about one another. By virtue of its structure, the cord has considerable strength and elasticity and can be extended unbroken as much as three inches or more. When the hapteron end of the cord becomes caught, the peridiole is ultimately checked in flight by the extended funicular cord and is jerked backward (figure 4). As a result, the peridiole tends to swing around its attachment point and to become fastened securely to whatever object it may strike (figure 4). Even when a peridiole glides along an object such as a leaf, it easily becomes attached because of the adhesive nature of the hapteron (figure 4). Subsequently it remains there because, when the adhesive hapteron dries, it cannot easily be washed away by rain. Conjecture as to the fate of a peridiole thus fixed to vegetation rests at present largely upon circumstantial evidence which will be reviewed in detail in Chapter 9, c, i. Some of the principal observations pointing to the belief that individual, widely separated basidiospores from within pendióles do germinate in nature may be noted briefly. Horses and cows and other herbivorous animals doubtless frequently eat vegetation to which pendióles are attached. 9 / General account of the Nidulariaceae

Add to this the fact that Cyathus stercoreus is an almost strictly coprophilous species and that a number of other species such as Crucibulum la eve are commonly found growing upon dung, and there emerges a strong possibility that herbivorous animals do help to spread the Nidulariaceae subsequent to the initial dispersal by rain. It cannot be said with certainty, however, that the basidiospores of all bird's nest fungi must pass through the alimentary tract of some herbivorous animal for germination and large-scale dispersal to occur, although there is evidence to suggest that this is so for some species. Peridioles variously attached to vegetation by their funicular cords and haptera can be found easily by anyone who troubles to examine foliage and stems within two or three feet of fruit bodies after a heavy rainstorm (figures 38b, 39). Finally it may be noted that there is extreme variation in the form and colour of the fruit bodies of such species as Cyathus stercoreus (Brodie, 1948^. This would not be expected unless basidiospores were eventually freed from one another so that haploid (homokaryotic) mycelia having different genetic constitutions could be widely distributed and could recombine to produce the large amount of morphological variation which exists and which, at least in C. stercoreus, is known to be the result of genetic recombination. If one examines the different species of Cyathus or compares this genus with Crucibulum, Nidula, and Nidularia, it will immediately be recognized that there is great variation in the extent to which the fruit bodies may be interpreted as highly developed structures 'ideally' adapted for rain-splash dispersal. In Crucibulum the peridiole is attached to the fruit body by a funiculus, but a funiculus of much simpler structure than that of Cyathus. In Nidula, although the fruit body is cupshaped, there is no funiculus, and pendióles are covered with an adhesive coating which enables them to stick to vegetation. In Nidularia the globose fruit bodies open to form only a crude approximation to a cup; pendióles in

this genus are also devoid of funiculi but, as in Nidula, are provided with an adhesive coating. It is tempting to regard such variation as representing a phylogenetic series, at least as far as the evolution of the splash dispersal mechanism in these fungi is concerned. C / NUCLEAR BEHAVIOUR Except for the possible spread of the vegetative mycelium by insects or other agencies, the basidiospores are the chief means of reproduction in the Nidulariaceae. Fragmentation of mycelium into oidia has been reported to occur in four species but, as will be shown, this is probably an artifact of culture in vitro for at least two of the species. Four unicellular hyaline basidiospores are commonly developed on each basidium, although there is some variation as to the number of spores. As in other gasteromycetous fungi, basidiospores of the Nidulariaceae are not forcibly separated from their basidia and, as noted above, at maturity lie free from one another to form the central mass of each peridiole. Each basidiospore receives a single haploid nucleus from its basidium (figure 3). Although basidiospores are usually binucleate when mature, the two nuclei are the products of a mitotic division within the maturing spore and each spore is genetically haploid (i.e., homokaryotic) in almost all species. With two exceptions, all members of the family when grown in pure culture display a 'tetrapolar' or four-mating-type form of sexuality or self-sterility in the completion of their life cycle (figure 30). Only one species has ever been reported to be self-fertile, i.e., to have the capacity to complete its life-cycle starting from the germination of a single basidiospore; and one species has the bipolar mating system. Moreover, in the self-sterile species, the production of fruit bodies by a single isolated homokaryotic mycelium has never been reported in this group. In all except one species, homokaryotic mycelia of two appropriate or compatible mating types must unite, appar10 / The bird's nest fungi

ently solely by fortuitous growth and hyphal anastomosis, before the heterokaryotic phase of these fungi is established (see Chapter 4, c,

i).

For most species, the mycelium derived from a single basidiospore (homokaryotic, see figure 3) is slow-growing, is provided with simple septa, has numerous nuclei which are irregularly distributed, and is self-sterile. Heterokaryotic mycelium which results from the union of compatible homokaryotic mycelia bears clamp connections (figure 3) and has its nuclei disposed in pairs or dikaryons. In nature, or under certain conditions in artificial culture, the heterokaryotic mycelium grows rapidly and usually (not invariably) tends to produce conspicuous mycelial cords (figures i4g, 2ia, b), which are the forerunners or primordia of fruit bodies. Small lumps or knots appear on the mycelial cords and, under favourable conditions, these knots become differentiated into fruit bodies. The whole process of fruiting in pure culture requires about forty days from the time of transfer of a diploid mycelium to fresh nutrient medium. No doubt, in nature, the time required for the development of fruit bodies may vary widely from that required in culture. The fusion of sexually compatible nuclei takes place in young basidia (figure 3) and, as in most basidiomycete life-cycles, nuclear fusion is followed directly by meisosis, which results in the production of four haploid nuclei. D / O T H E R A S P E C T S OF BIRD'S NEST F U N G I In the preceding pages, the Nidulariaceae have been examined in summary fashion, mainly from the viewpoint of what can be learned from a study of whole or dissected fruit bodies with the unaided eye or under the microscope. Some attention will now be given, again in survey form, to a few of the characteristics of these fungi when they are grown in pure culture in the laboratory. Attempts to obtain cultures have had the following principal objectives : (i)

FIGURE 4 Diagram of splashing of peridiole from fruit body of Cyathus striatus : a and b, raindrop lands in cup; c, splash ejects peridiole which flies out with adhesive hapteron exposed; d, hapteron sticks to stem as peridiole is carried forward by momentum and funicular cord is extended by pull ; e, peridiole jerked back by limited extension of cord ; f, j erk causes peridiole to swing around a point of attachment and cord is wrapped around plant stem. Figure courtesy Can. J. Bot., 1951,29: 228 11 / General account of the Nidulariaceae

to learn the life cycle, sexuality pattern and the form and behaviour of nuclei during the vegetative phase ; (2) to learn whether or not mycelial characteristics of the different species are correlated with the morphology of the sporocarps so that mycelium colour, growth rate, nutritional requirements, etc. might possibly support the definition of species based solely on fruit bodies ; (3) to learn something of the nutritional and other physiological requirements of these fungi as an aid to understanding their ecology ; (4) to bring about fruiting in culture as a necessary part of genetic studies, and as a source of abundant fruit bodies and cultures of known mating type for teaching purposes. To those whose interest is primarily in the living organism, the Nidulariaceae prove enticing. Many of these fungi, though not all, have been cultured. Grown on synthetic media, their mycelia are fast-growing, sleek, and colourful. Their nutritional requirements are relatively simple and some species can, with some regularity, be induced to produce an abundance of normal sporocarps (Brodie, iQÓib, i9Ó8a). Some difficulties have stood in the way of attempts to achieve the objectives outlined above. The first difficulty is the matter of the germination of basidiospores. It has been found that if peridioles are subjected to surface sterilization and then macerated and the spore suspension so obtained is held at 40° c for two days, some spores of some species germinate although the percentage is usually low (10-20 %). However, some spore samples have been found to resist germination regardless of the various treatments applied to them. The second difficulty concerns fruit body formation. Cyathusstercoreus, C. pallidus, C. bullen , and some others will fruit on liquid or solid medium in about forty days, but others have resisted all efforts to induce fruiting. Despite the fact that fruiting has not yet been brought fully under control, there is a great deal of information available regarding the conditions necessary for fruiting and about the 12 / The bird's nest fungi

development of fruit bodies. It is known, for example, that light is absolutely necessary for fruiting and that the normal development of sporocarps is affected by photoperiod and by the wave length of light (Lu, 1965). It is also known that the element calcium plays some essential role in normal fruiting (Lu, S.-h., 1973). In addition, observation and experiment have shown that fruit bodies of some species are, at some early stages of their formation, phototropic and probably geotropic. Still another problem can be dismissed briefly since, at present, no way of solving it has been suggested. The basidiospores at maturity lie free from their basidia and well mixed within the peridiole. Thus there seems to be no simple means of isolating the four spores that together would represent a tetrad. Without the possibility of tetrad analysis, some kinds of genetic work on the Nidulariaceae will obviously remain difficult. Finally, under the list of obstacles, is cytology. Chromosome morphology and nuclear behaviour have been studied (Lu and Brodie, 1964) but, to say the least, the nuclei of the Nidulariaceae have not, so far, proven to be the most obliging objects for cytological study, although their chromosomes are larger than those of most fungi. One of the most important characteristics of the Nidulariaceae, which has resulted in an unsatisfactory state of their taxonomy, is the extreme variation displayed by many species as they are found in nature. One of the best examples of such variability is to be seen in the common coprophilous fungus Cyathus stercoreus. Often in a small garden plot one can find fruit bodies of this species ranging from 3 to 15 mm in height and from the palest grey to deep chocolate-brown in colour. This variation has been shown to be caused by genetic recombination (Brodie, 1948^. A similar variation of genetic origin in C. olla has also been noted (Brodie, i952a) and presumably variation in other species is due, at least in part, to the existence of different genotypes. Even if part of the variation is caused by differences in envi-

ronment (and this has indeed been observed), the practical problem of defining species is a very real one. For example, many of the apparently very distinct forms of C. stercoreus, which may be found in nature and can be produced by genetic recombination in the laboratory, have in fact been described as species, although it is doubtful that such variants should even be designated as forms. Enough has been stated to indicate that every effort should be made to recognize the range and limits of variation of each valid species and especially to exercise caution in the naming of new species. Finally, one other aspect of the Nidulariaceae in culture, which apparently has not been observed in nature, is the existence of certain strains which produce remarkably aberrant fruit bodies. The latter bear little resemblance to normal fruit bodies, being spherical, completely closed and each containing but a single peridiole devoid of a funiculus (Brodie, i955a). If, as seems probable, such aberrations are the

13 / General account of the Nidulariaceae

result of mutation, survival in nature might well be in danger. If, however, the aberrations are able to survive, it is tempting to suggest that such globose aberrant fruit bodies may actually be, or may be related to, certain supposedly simple gasteromycetes such as Protogaster. As yet, there is little evidence to support such a view. This overall view of the bird's nest fungi may serve as a basis for a more thorough examination of many interesting aspects of these fascinating organisms. A hundred years ago, Tulasne was unable to disclose as much as has been outlined above: nevertheless our debt to him is great and we can still be stimulated by a consideration of the motivation which he expressed thus in the Carpología Fungorum (Tulasne, 1861-5): 'For today we are eager for new things no less than were our fathers ... but we ... are richer in tools and means of observation by the aid of which all of us can investigate the many secrets and wonders of nature ...'

2/ Brief Historical Outline

A / EARLY STUDIES The Nidulariaceae have received more attention from botanists from the time of Clusius (1601) (see end of this chapter) to the present than is commensurate with their size and their seemingly modest role in nature. The reason for this interest is plainly that they, unlike most fungi, possess reproductive bodies which not only are large enough to be seen by the naked eye but which are sufficiently like seeds in size, shape, and colour to have been mistaken for them by those early botanists who were anxious to classify fungi as seed producers. The story of the growth of knowledge concerning the bird's nest fungi up to his own time was told in detail by Tulasne (1844) in his monographic work, and a briefer account in English of essentially the same facts was given by White (1902) in her paper on the North American species. Since these summaries are available to all, the following account is intended not to present a complete history of the subject, but rather to refer mainly to those publications bearing most directly upon the problem of the understanding of the operation of the spore dispersal mechanism in the Nidulariaceae. A few other works, chiefly those relating to taxonomic and to pure culture studies, are also reviewed briefly. The earliest undoubted reference to a fungus 14 / The bird's nest fungi

belonging to the Nidulariaceae seems to have been made by Clusius (1601) (see White, 1902). After having made its debut, the little fungus nest with its eggs then became the object of the long controversy found in the writings of Camerarius, Marsigli, Tournefort, Antoine de Jussieu, Bulliard, Micheli, and many others. The matter was supposedly settled when Hoffman (1790) showed that the 'eggs' or peridioles could not be seeds since they themselves contained the true spores. However, the spores had not been observed to germinate, and this led Tulasne, as late as 1844, to refrain from being dogmatic, and to write 'car, si les corpuscules contenus dans les sporanges des Cyathus, ne peuvent être, suivant nous, eut égard au mode de leur génération, à la régularité de leur forme, à leur nombre, regardés que comme de véritables spores, on ne comprendrait pas que la nature les eut multipliés avec tant de prodigalité dans chaque conceptacle pour n'en faire sortir qu'un individu solitaire.' The solution of the problem as to whether a peridiole was to be regarded as a seed or not hinged partly upon two others, namely, the means whereby the peridioles are disseminated in nature, and their fate subsequent to dispersal. Concerning the latter, we find Haller in 1768 adhering to the idea that a peridiole is really an embryonic plant, a belief also shared by Schrank. Bulliard, in 1791, thought that if a

peridiole were placed on soil, roots would grow from one surface while the funiculus would develop into the rudiment of the epiphragm. Camerarius much earlier (1688) had planted some pendióles during wet weather, but failed to obtain recognizable fruit bodies of bird's nest fungi from his plantings. The problem of dispersal of the pendióles was even more vexatious. Several of the early botanists, e.g. Paulet, subscribed to the erroneous view that the peridioles are shot from the cup by some kind of elastic or spring mechanism present either in the cup or in the peridioles themselves. Some survival of this idea may also be traced in speculations of a much later date; thus Kickx, in 1841, tried to draw comparisons between Cyathus and Sphaerobolus in the manner of the ejection of the peridioles. It is interesting to note that Tulasne, who did more than any of the early investigators to reveal the extraordinarily complex structure of the fungus cup and especially of the funiculus, did not allow himself to speculate beyond the bounds set by what he had actually observed. Accurate in almost all details concerning the structure of the funiculus, his studies did not lead him to an understanding of the operation of the mechanism that he had revealed, and one cannot but be impressed by the intellectual integrity that forced him to write what may be freely translated thus (Tulasne, 1844): 'Certainly it would be extremely difficult for us to accept the opinion of Paulet, because we cannot find in Cyathus any curious structure such as that which is responsible for the ejection of the glebal mass of Sphaerobolus ... the truth still remains to be discovered/ Still another possibility was considered by Brefeld (1877) who, in the third volume of his Botanische Untersuchungen, suggested that animals (presumably small animals such as insects) carry the peridioles out of the cups by seeking the peridioles for food and getting their legs entangled in the hyphae of the funicular cord. Although Brefeld was wrong in this deduction, for which he had made no direct ob15 / Brief historical outline

servation, yet he based it upon two points, both of which had merit: (i) the spores usually do not germinate well unless they have been heated, suggesting that they might have to pass through the alimentary tract of an animal ; and (2) the spores are surrounded by the thick indurated walls of the peridioles and the peridioles do not dehisce, suggesting that in nature the wall of the peridiole might need to be dissolved by the digestive processes of an animal. Closer to the truth, though by no means the whole truth, was the suggestion of rain as an agency of dispersal. The first to offer this explanation was Ray (1686) who believed that rain, having filled the cup, would overflow and carry the peridioles with it. It is possible that Sowerby held a similar view, for he referred to Cyathus olla (his Nidularia campanulata) and described seeing the peridioles hanging out of the cups attached only by their funiculi and seeing peridioles scattered on the ground. Berkeley illustrated C. olla in his Outlines of British Fungology (1860) showing peridioles hanging out of the cups; he did not, however, explain how the peridioles got there. Persoon, who was at first inclined to accept Paulet's notion of a spring mechanism, in the Synopsis Fungorum, expressed more credence in the possibility of rain as the dissemination agent. Schmitz (1842), who studied the structure of Cyathus striatus just before Tulasne, also supported this hypothesis. Sachs (1855) dealing with Crucibulum laeve, made an interesting observation. For three successive years he had found fruit bodies of this fungus growing on the wood of a small bridge in the Canal Garden in Prague. During spring floods, this part of the bridge was frequently completely under water. Sachs noticed, however, that the fungus did not spread to another bridge a hundred yards away downstream. He had previously observed that the peridioles could float in water and that the spores germinate after being inundated. He therefore concluded that the peridioles are not as easily washed out of the fruit bodies as had been supposed.

The first detailed report on the structure of one of the Nidulariaceae was the study of J. Schmitz (1842). Schmitz's work, however, left much unsolved until the problem was taken up by Tulasne in 1844. In 1855, Julius Sachs gave an account of the development and morphology of the fruit body of Crucibulum laeve, one of the chief aims of which was to clear up certain details of the formation of the pendióles which Tulasne had not studied. Sachs did not understand fully the relationship of the cord of the funiculus to the covering or 'purse' within which it lies. Except for this detail, he added considerably to knowledge of the fungus. Although both Hoffman (1859) and Hesse (1876) had made reference to the germination of the basidiospores of Cyathus striatus, Eidam (1877) was the first to observe the development of mycelium. Eidam contrived to get some spores of both C. striatus and Crucibulum laeve to germinate at about 25° c in horse-dung decoction. He also described what he interpreted as the formation of fruit body rudiments on the clamp-bearing mycelium of both species. Brefeld (1877) made an examination of the structure of the fruit body of Crucibulum laeve. His ideas about the dissemination of the peridioles have already been mentioned. Concerning the morphology, Brefeld agreed, in the main, with Sachs. On one point, however, he differed substantially ; Brefeld was emphatic in his assertion that the funicular cord which can be pulled out of the little button that lies below the pendióle in Crucibulum is not attached to the wall of the peridium. Although he saw the button, he did not interpret correctly the cord that it contains. Brefeld thought that, upon placing the peridiole in water, the button developed into a long cord. He insisted, however, that under natural conditions in the cup, the button did not assume this form. Doubtless Brefeld's insistence upon this point was connected with his belief that animals disseminate the peridioles. If the peridioles were attached to the cups, the funiculus would hinder the dissemination of the peridioles. The only other work of what may be called 16 / The bird's nest fungi

the early period, to which reference should be made, is that of R.E. Fries on Nidularia, published in 1910. Fries's investigations showed that Nidularia is simpler in structure than either Cyathus or Crucibulum. The development of the fruit body of Nidularia, however, corresponds closely to that of Crucibulum in its general course. Although a considerable amount had been written concerning the Nidulariaceae by early botanists, it was Tulasne who first brought order out of confusion by his monograph of 1844. As we have seen, Tulasne made a detailed and accurate study of the three common species of Europe. By means of this study he became much more able than his predecessors to undertake a critical investigation of the entire group of the bird's nest fungi. He had only seventeen collections from foreign countries in addition to the three European species, yet with this material he described the genera and thirteen species. The first monographic work on the North American species was done by Miss V.S. White (1902). She placed the North American material in nineteen species and established a new genus, Nidula. C.G. Lloyd (1906) produced a world monograph of the Nidulariaceae which has long served as a standard work. Lloyd's studies were based on extensive collections of his own and on studies of the more important herbarium material of Europe. Numerous illustrations accompanied the descriptions. Lloyd placed the known Nidulariaceae in thirty-two species. The conservatism of his species concept seems to have been justified, at least if we can judge by his treatment of the extremely variable Cyathus stercoreus. This is a fungus which exhibits great diversity in different collections in the size, shape, and colour of the cups. Lloyd regarded the whole assemblage of different types of this Cyathus as representing but one variable species, and he reduced to synonymy approximately a half dozen similar fungi that had been described as distinct species. Cunningham (1924) gave an account of the

Nidulariaceae of New Zealand in which he recognized eight species within that country. The 'Gasteromycetes of the Eastern United States and Canada' by Coker and Couch (1928) dealt with twelve species of the Nidulariaceae in some detail. In 1920, Leva Walker published an account of the development and structure of Cyathus olla, C. striatus, and Crudbulum laeve. Miss Walker's investigation provided a confirmation, based on modern methods, of what had been described by Tulasne and others, and it serves today as the basis for exact knowledge of the developmental morphology of the group. Although Miss Walker succeeded in culturing Cyathus olla, she did not attempt to obtain single spore mycelia. Further, the important unsolved problem of the dissemination of the peridioles did not receive her attention. B / U N R A V E L L I N G OF THE DISPERSAL PROBLEM G.W. Martin (1927) published an important paper whose main purpose was to draw attention to the manner in which the basidiospores are developed in the Nidulariaceae. It was shown that the spores of the five species studied are all developed on basidia and liberated into the interior gelatinous matrix of the pendióle. An interesting and important new fact reported was that, in Cyathus stercoreus, the basidiospores are surrounded by 'nurse hyphae' which, by their gradual disintegration, make it possible for the basidiospores to increase greatly in size after they have been liberated from their basidia. A very important contribution made by Dr Martin was his observation regarding the dissemination of the peridioles of Crudbulum. After correctly noting that no account based upon experiment had ever been given of this phenomenon in nature, Martin recorded the following observation which, because of its importance, is quoted in full: Half buried in the ground were the bases of the old 17 / Brief historical outline

cornstalks, nearly every one of which supported a number of basidiocarps, of Crudbulum laeve. Above and around every cluster of basidiocarps, on twigs, dead leaves, and similar fragments, were numerous peridioles, obviously forced out of the cups by rain drops, and glued firmly to whatever they had hit. Debris of this sort, readily transported by wind and water, and often deposited in heaps of similar material, would afford highly efficient dissemination of the fungus. Some of the fruiting bodies were brought into the laboratory and soaked for a while. When removed from the water, the peridioles could easily be spattered out of the cups by dropping water into them from a pipette a couple of feet above them. In Cyathus stercoreus, as in Crudbulum, the funiculus tends to disappear as the peridioles age, and in this species, as in Nidularia, it is possible that rain may play the same role. In Cyathus striatus, on the other hand, where the funiculus is persistent and the peridioles are well down in the narrow part of the cup, this method of dissemination might be unavailable.

Martin was apparently the first to deduce that the peridioles of the bird's nest fungi are spattered out of the cups by rain and that they adhere to vegetation, and he was the first to test the hypothesis by actual experiment in the laboratory. Martin did not connect his knowledge of the structure of the funiculus with his observation and, as will be shown later (Chapter 8), was incorrect in assuming that dissemination by rain does not occur in Cyathus striatus. Curiously enough Martin's observation seems to have passed unnoticed for many years, or at least it did not stir the imagination of mycologists interested in dispersal. Beginning in 1936, Nils Fries of Sweden (Fries, 1936) reported studies of single-spore cultures and sexuality in Crudbulum laeve and Cyathus striatus. Although Fries initiated investigations into this field of the biology of the Nidulariaceae which have since been carried on by myself, he did not attack the problem of the spore dispersal mechanism. The next link in the chain of evidence in the

latter connection is a paper by Dr William W. Diehl (1941). In December 1931, Dr Diehl examined some specimens of a fungus on leaves of Camellia plants, sent to him from Mississippi. The fungus was considered to be identical with Leptostroma Camelliae described by Zenker (1834) on leaves of Camellia japónica in Germany. Small, black, button-like pads about 2 mm in diameter were seen firmly attached to the surface of the Camellia leaves. In microtome sections, no hyphae within the leaf tissue could be found. Diehl was interested in the reappearance of Zenker's fungus - this time in America - after the lapse of nearly a century. Cultures on oatmeal agar were easily obtained, and the mycelium was observed to bear clamp connections, indicating its basidiomycetous affiliation. In January 1933, Diehl examined plantings of Camellia at a commercial nursery at Savannah, Georgia, and there found similar fungus 'buttons' on the under surfaces of the lower leaves of many bushes. The buttons were smaller than those of the Mississippi material, but very similar. The leaves were noted to be exceptionally free from any insect that might have served as host for the fungus. Reasoning that since the buttons occurred chiefly on the lower sides of the leaves and mostly on leaves not more than two feet above the ground, they might have been projected there from below, Dr Diehl found his answer on fragments of boxes that had contained the Camellia cuttings and lay rotting on the ground. The boxes were covered with fruit bodies of Cyathus pallidus,1 and it appeared possible that the pendióles from these had been projected on to the Camellia leaves above. Diehl concluded that i Diehl identified his fungus correctly as Cyathus pallidus. As far as the writer is able to ascertain, this constitutes the first record of the finding of this species in the United States. Miss White reported it from Cuba and Puerto Rico, and Lloyd from Jamaica and Antigua. Dr Diehl kindly allowed me to examine the specimens collected at Savannah, Georgia, 6 January 1933. They seem to fit the descriptions perfectly and cannot be placed in any other species.

18 / The bird's nest fungi

Zenker's Leptostroma and the curious fungus buttons from Mississippi were pendióles of a species of Cyathus, and he ended his paper with this statement: 'No attempt is made at this time to explain how peridioles could be projected through the air to leaves above the sporocarps of this species, but G.W. Martin's account of the dissemination by spattering raindrops of peridioles in the related Crucibulum vulgare (laeve) is very suggestive/ Thus Diehl's observation not only settled the question of the identity of Zenker's Leptostroma, but it added a valuable contribution to Martin's evidence that the peridioles of bird's nest fungi are actually splashed from the cups, an idea, as we have seen, first suggested by Ray as early as 1686. To deal in chronological order with the events that follow Diehl's paper, will be to introduce here (to some extent) materials which must be dealt with more fully later. However, it is of some importance to follow a chronological order at this point in view of the fact that Professor A.H.R. Duller, who studied the problem at that time, did not live2 to publish his own extensive work upon the subject except in the form of a single abstract. We shall therefore digress momentarily to indicate precisely how Duller began his investigations of the bird's nest fungi at the point where Diehl's paper left the whole problem. Professor Duller had been invited to give a summer course at Louisiana State University during the summer of 1941 and there, in June, he began his study. The very first of his unpublished notes concerns this beginning and is quoted verbatim: Louisiana State University, ]une 13, 1941 -first finding of fungus and of its peridioles on leaves Went on excursion with Professor Forbes, Mrs Luke and Dr Dufrenoy on a trip northwest in Louisiana, an inspection trip for sugar cane at northern limit 2 Buller began his study in June 1941 and died 3 July 1944. I have examined Buller's unpublished notes, some of which are incorporated in the following pages.

where it is grown. Went through Bunkie to the Meeker Sugar Plantation, about eighty miles northwest of Baton Rouge, near Alexandria. At Bunkie, at the corner of a plot in which there were rows of corn and soya-beans, I found cups of a Cyathus.3 Knowing of Dr Diehl's work, and that heavy showers are frequent in Louisiana, I looked for the pendióles that might have been splashed out of the cups on to adjacent soya-beans (used in warm weather as green manure and cover crop). I showed cups on ground to Dr Dufrenoy and, realizing that after the late thunder shower, some of them might have been splashed upwards, I cut off near its base the nearest plant (more than one foot high) and, on looking it over, observed two pendióles (black, oval, flattened) hanging each by its funiculus, one to the stem about six inches above ground and the other to a leaflet about one foot above ground. Next day, Dr Edgerton kindly photographed these peridioles and their cords attached to the soya-bean, 51/2 times the natural size. Collected some cups from ground.

From June 1941 until near the end of August, Professor Duller worked intensively in following up the lead indicated by his observation. Before he left Louisiana State University, he had made a careful examination (as his notes show) of the cups and peridioles of Cyathus stercoreus, had studied sections of the peridium and of the peridioles and had verified much of what Tulasne had written concerning C. striatus. He had also carried out experiments in the laboratory which showed that the peridioles can be splashed from the cups and he had solved much of the mystery of the role of the funicular cord in the dispersal mechanism. Duller had further made a careful study of all the background literature pertaining to the subject during this time and, except for some minor details, had acquired a clear idea of how all the known facts fitted into his concept of the operation of the dissemination mechanism. On his way back to the University of Manitoba, Duller stopped at the University of Min3 Later identified by Buller as C. stercoreus

19 / Brief historical outline

nesota, where he discussed his investigations before Dr B.C. Stakman's Phytopathological Seminar (August 1941). Duller returned to Manitoba late in August, and at once took up the study with great energy. On 3 September I dined with him and was regaled with as much of the story as could be told at that time; and Duller requested help in collecting material and in carrying out certain proposed experiments. Dy October of that year enough had been done to establish the main ideas and, on 13 October 1941, Duller presented a fifteenminute paper by invitation before the Madison meetings of the National Academy of Sciences. Ordinarily an abstract of the paper would have been published, but that year it was not. The abstract was filed, however, with the Academy Secretary. The title of Duller's paper was published in the Annual Report of the National Academy of Sciences for the fiscal year 1941-2. Following Diehl's paper, the next publication dealing with the Nidulariaceae was one by Dr D.O. Dodge (1941) in the NovemberDecember issue of Mycologia entitled 'Discharge of the sporangioles of bird's nest fungi. ' In this paper, Dodge reported correspondence with a Mr John J. Shea who 'Several years ago called my attention to the fact that the sporangioles of species of Cyathus often hang suspended to leaves by the funicular threads which may vary in length up to two or three inches/ Dodge goes on to state that Mr Shea reported that the fungus shoots its sporangioles into the air and that they may be found attached to whatever plants are growing near the fungus cup. Shea also reported he had found peridioles 'attached to leaves of a scrub oak which were 13 feet above the ground/ Shea indicated that the peridioles are 'shot out at night and not when the peridia are wet, but some time afterwards/ Shea, as did Martin in 1927, observed that the peridioles, after being projected from the fungus cup may adhere to vegetation in the vicinity. We do not know precisely when Shea's observations were made: Dr Dodge re-

ferred to correspondence of 'several years ago' and to the fact that 'for twelve years or more' Shea had made a study of species of Cyathus. Unlike Martin, who deduced that rain was responsible for the forceful ejection of pendióles and who proved it by experiment in the laboratory, Shea does not appear to have realized that the force of falling raindrops causes the ejection of pendióles (as will be proven in the ensuing pages). Indeed, if one interprets Dr Dodge's report correctly, Shea seems to have had the idea that some other mechanism is involved when he states: (i) that the pendióles are not 'shot' out when the fruit bodies are wet; (2) that the pendióles are ejected at night; and (3) that pendióles were shot on to window panes and awnings from fruit bodies grown in the house in old cheese boxes (where, presumably, they could not be reached by rain). In any case, the chief point to record is Shea's report that, when the pendióles of the bird's nest fungi are ejected from the cups, those of Cyathus frequently become attached to vegetation by their funicular cords while those of Crucibulum may be found attached directly to the leaves. Dr Dodge included in his paper two photographs of Cyathus striatus and a text figure showing pendióles of this species and the funicular cord to illustrate his account of the funiculus. On 29 May 1942, Professor Buller reported his research before the meeting of Section v of the Royal Society of Canada under the title 'The Splash-cups of the bird's nest fungi, Liverworts and Mosses.' The one-hundredword abstract of this paper which was published in the Proceedings of the Royal Society of Canada (Buller, 1942) is the only printed account of his work that appeared in a scientific publication during his lifetime. A popular article based on Buller's Royal Society paper was written by Dora Smith Conover in the paper Saturday Night published in Toronto, Canada, 14 November 1942. Still another semi-popular article appeared in the Journal of the New York Botanical Garden under the title 'Egg-throwers of the Mush20 / The bird's nest fungi

room World.' Carol H. Woodward (1943) was the author of this article, with acknowledgment to Dr B.O. Dodge as co-operator. In Miss Woodward's article the story of how raindrops splash the peridioles from the cups of Cyathus striatus is revealed in some detail. Photographs and drawings illustrate the story. C / R E C E N T STUDIES In 1943 there appeared the third of a series of papers by Dr Nils Fries of Sweden (N. Fries, 1943) dealing with the sexuality of Crucibulum laeve and Cyathus striatus. This work is dealt with later (Chapter 4, c, i) and mention need be made here only of the fact that Fries did not concern himself at all with problems of morphology of the fruit bodies or the problem of the dissemination of peridioles. In May 1948, Brodie (i948a) published the results of investigations into the sexuality of Cyathus stercoreus, which had never previously been obtained in single spore culture. These investigations, like those of Fries, are reviewed elsewhere (Chapter 4, c, i). They did not include any studies of the morphology of the fruit bodies ; but it may be mentioned that the development of a technique for obtaining large quantities of normal fruit bodies of this splendid member of the bird's nest fungi was of great value in enabling me to keep on hand a supply of fresh, living material, from early 1948 until the present time, for experiments and for making numerous morphological studies. A second paper (Brodie, 1948^ dealing with Cyathus stercoreus has some bearing on attitudes towards problems of taxonomy in the Nidulariaceae. This paper reported upon the fruiting of different strains of diploid mycelium of C. stercoreus. Many different morphological types of fruit bodies were obtained which bore so little superficial resemblance to the wild type C. stercoreus that, had they been found in nature, they might well have been described as distinct species. From this work we know that the great variability of

C. stercoreus as it is observed in nature has, in part at least, a genetic basis. Further similar studies might help to clear up some of the taxonomic problems in other bird's nest fungi. The next paper in chronological order is one by Dr Nils Fries (1948), reporting that Nidularia farcta, like most other members of the Nidulariaceae thus far studied, is heterothallic and tetrapolar. A very low percentage of spore germination rendered culture studies of N. farcta difficult, and Dr Fries did not pursue investigations further. Since 1948, over fifty papers have been published dealing with the Nidulariaceae. The results of most of these are incorporated elsewhere in this book and will be referred to only briefly here. The information is dealt with by subject rather than chronologically. i / Recent contributions to taxonomy and distribution About twenty species of the Nidulariaceae have been described as new since 1948 and these are discussed in the chapters dealing with taxonomy. Cyathus pygmaeus, a species described by Lloyd (1906), but not recognized again until sixty years later was found to be fairly common in the Northwestern United States and elsewhere (Brodie, i966b). Cyathus olla forma anglicus, previously known only from England, Colorado and Oregon was found in abundance in Western Canada (Brodie, 196/d). Two species of Crucibulum, which was previously generally considered to be a monotypic genus, were described as new (Brodie, i9/oc; 19/ic). In 1961, J.T. Palmer set up a new genus of the Nidulariaceae which he called Mycocalia (Palmer, 19613). Palmer also undertook a taxonomic survey of Nidularia and published a number of critical studies of species of that genus (Palmer, 1957, 19583, 1958^ 19603, i96ob). The known distribution range of a number of species has been extended by recent collecting, and lists of the Nidulariaceae of various 21 / Brief historical outline

countries have been published from China (Tai and Hung, 1948), Japan (Kobayasi, 1937), the West Indies (Brodie and Dennis, 1954; Brodie 19671), Argentina (Wright, 1949), subarctic regions (Bowerman and Groves, 1962; Christiansen, 1941; Larsen, 1932; Lange, 1948), Canada (Brodie, i968b), Mexico (Guzman, 1969), and from other areas. 2 / Recent contributions to sexuality, morphology, and physiology Beginning about 1948, I published a series of papers reporting studies of the interactions of single spore mycelia of a number of species. These reports showed that tetrapolar or fourmating-type sexuality is almost invariably the established situation. The only known exception was reported by Burnett and Boulter (1963) who found both homothallism and bipolarity in Mycocalia. The so-called unilateral type of diploidization was found to occur in Cyathus stercoreus (Brodie, 1948; Fulton, 1950) and has since been recorded as occurring in Cyathus africanus (Brodie, 19676). The formation of interesting abnormal fruit bodies, each enclosing but a single peridiole, was reported (Brodie, 19553) to occur in Cyathus poeppigii and C. limbatus. Regener3tion of old fruiting bodies of Nidula, Crucibulum, and Cyathus and the abnormal twinning of fruiting bodies was described (Brodie, 1958). Studies of the problem of germination of basidiospores were undertaken by Brams (1950) 3nd Miles (1953) but these 3nd subsequent studies hsve Í3iled to provide a really S3tisÍ3ctory solution to the problem. The influence of light 3s the most important Í3Ctor sífecting the development of fruiting bodies W3S demonstrated through the experiments of Garnett (1958) 3nd Lu (1965). The first photographic demonstration of chromosomes in a member of the Nidularisceae was published by Lu and Brodie (1962), who reported that the chromosome number for Cyathus stercoreus is n — 12. A

chromosome count (not unequivocal) of n =12 was also recorded for C. olla (Lu and Brodie, 1964). The nuclear cycles in C. stercoreus were described and illustrated by B.C. Lu (I^^SL, b). He stated that 'meiosis and mitosis are essentially similar to those processes in higher organisms/ An important recent addition to knowledge about the Nidulariaceae was the announcement that mycelium of Cyathus helenae (a species described by me in 1966) produces a series of previously unrecognized chemical compounds and that these substances have some antimicrobial activity (Allbutt et al., 1971). The substances collectively were named cyathin. The same or very similar substances are produced by C. striatus, which is closely related taxonomically to C. helenae and, curiously enough, by C. africanus, which is a species far removed taxonomically from either of the other two species. D/ CONCLUSION In these pages have been traced the passages of the little bird's nest fungi across the stage of scientific investigation and even, at times, through its spotlight. To record these passages briefly is to lose much of the delight afforded by reading especially the old publications in their original form and entirety. To encourage

22 / The bird's nest fungi

the reader to delve more deeply, a single example follows from the writing of Clusius in 1601. Moreover this fungus, which I will call anonymous, is very different from the preceding ones, and I consider it to be the smallest of all, for it is barely half an inch high. In the fall a great many grow, without petiole, on wooden boards away from dust and sand. They have the colour of cinders or are of a lifeless colour ... Sometimes they grow alone or when in numbers two, three, or four adhering together, and when ripe they throw off the top part and appear full of a viscous juice, and of seeds which are about the size of the seeds of cyclamen, but have the outline of small fungi and are apparently cinder-coloured ... I remember that a friend of mine sent me, once upon a time ... some of these seeds dried and with a certain strange name, asking me if I could find out what they were. For there are certain characters who endeavour to catch and buy praise from the ignorance of others. [Quoted from White (1902)] It is a far cry from Clusius's quaint notes to some of the recent publications in Nidulariology that compare species by means of chromatographic analyses (Johri and Brodie, 19/ia) ; and it is a fact, often forgotten or too seldom stated, that not many of the steps in a path such as this were supported by, or even flanked by, the costly accoutrements of modern science.

3 / Basidiospore Form, Development, and Germination

A / F O R M AND STRUCTURE The basidiospores of the Nidulariaceae are unicellular and colourless with the exception of those of Mycocalia sphagneti which are reported to be yellowish-brown. In shape they vary from spherical, through ovoid to ellipsoid (figures 5, 8). Only in one species, Crucibulum cyathiforme (Brodie, 19/ic), they have been reported to be slightly curved. The greatest variation among spores of the different species is seen in their size, which ranges from 4 ^ in Cyathus microsporus to a remarkable 50 /¿ in diameter occasionally recorded for C. poeppigii. Moreover, in many species the spores, even in a single peridiole, may vary greatly in size. The reason for this variation has to do with the stage of their maturity; as will be explained, spores of some species are known to undergo enlargement, after they have first been formed. In some species this enlargement is slight, but in others it may be very great. This variation, a result of the spores' capacity to continue growth after detachment from the basidia, must be taken into consideration by anyone who is using the measurement of spores as one of the criteria for identifying species. Ignorance of this fact may have led to confusion in spore measurements in the past; the practice seems to have been to assume sometimes that the size characteristic 23 / Basidiospore form, development, and germination

of a species is that of the largest spores, which are probably the most mature; the matter is less important in descriptions which, as is preferable, indicate the range in spore size for a given species. Basidiospores of the Nidulariaceae seldom have conspicuous structures, discernible with a light microscope, that can be used taxonomically. Palmer (i96ib) has pointed out that spores of Cyathus striatus and C. triplex bear what he describes as a 'notch/ and Brodie (19676) has illustrated what was called an apiculus characteristic of the spores of Cyathus africanus (figure 8b). For most species, however, the basidiospores have a uniform appearance over their entire wall surface. If the ultrastructure of the apparently smooth surface of basidiospores of the Nidulariaceae were known (cf. Eckblad, 19/ib), differences between species might be discerned on the basis of the architecture of the spore surfaces, but so far the scanning electron microscope has not been applied to these fungi. The spore wall (figures 6, 7, 8) may be thin relative to the diameter of the spore, as it is in Cyathus olla, or it may be very thick, as in C. crassimurus and C. stercoreus. Nothing is known about the chemical nature of this wall and one can but suppose that it consists chiefly of chitin with a certain admixture of hemicellulose. Under the light microscope, spore walls of some thick-walled species give the impres-

FIGURE 5 Spores, sporelings, and mycelia of Cyathus stercoreus: a to i and k, 1, m, X5oo; j, Xi5o. Basidiospore before enlargement shown at g ; h and i spore enlarging by absorbing material from surrounding nurse hyphae. Germination of spore, a to f ; e, shows squid-like germling. Hyphal fusions between compatible monokaryon hyphae shown at m and k. Figure courtesy Am. J. Bot., 1948, 35: 313 24 / The bird's nest fungi

sion of being lamellate or made up of several layers. Individual species of the Nidulariaceae differ greatly in the number of spores developed (or matured) within each peridiole. Most species that have small basidiospores, e.g., Cyathus olla, produce them in such quantities that when a softened peridiole is squashed in water a turbid spore suspension is obtained. On the other hand, in some large-spored species, e.g., Cyathus stercoreus, so few spores mature that it may be necessary to hunt carefully for them in a squash preparation. Also, it has often been reported (Lloyd, 1906) that pendióles may contain no spores at all. No explanation can be given at present for this failure of spore production, although Martin (1927) observed that some basidia in Crudbulum laeve shrivel without producing basidiospores. B/DEVELOPMENT Martin (1927), who studied the development of the basidiospores of some members of the Nidulariaceae, stated that they are produced on basidia which line the cavity of the peridiole. Basidia vary in shape from narrowly clávate, as in Cyathus olla, to broadly clávate or nearly globose as in C. stercoreus (figure 6f-j). Each basidium usually bears a clamp connection at its base. Commonly four spores are borne on each basidium as in Cyathus striatus ; but 3-spored, 2-spored, and even i-spored basidia may be found in other species, and in C. stercoreus the basidia may produce as many as eight spores. Attachment of basidiospores to the basidia is by means of sterigmata in Nidularia and Crudbulum, but in all three species of Cyathus examined by Martin the spores were found to be sessile on the basidia. When spores have been freed from their basidia they are also freed from the sterigmata, except in Nidularia in which the sterigma may remain attached to the spore. In Mycocalia certain basidia appear to undergo morphological changes which have led to 25 / Basidiospore form, development, and germination

FIGURE 6 Basidiospores and basidia of the Nidulariaceae: a to e, Nidularia pulvinata showing gelatinous material around disintegrating basidia and variable number of basidiospores, X5oo, approximate; f to j, Cyathus stercoreus showing globoid basidia X5oo, approx.; k, basidiospores of C. striatus; 1, spores of C. olla ; m, spores of C. stercoreus ; n, spores of Crudbulum laeve ; o, Cyathus pallidus, 1 to o x 500 approx. their being designated as 'metamorphosed basidia/ They become swollen, remain sterile, and come to resemble large globose basidiospores, although they are more irregular in shape than true basidiospores and generally have thinner walls. Metamorphosed basidia were first described by Olive (1946) in M. duriaeana, and have been found by Palmer (1963) in M. denudata, M. minutissima, M. reticulata, and M. sphagneti. Palmer has added the measurements and shape of metamorphosed basidia to the characters used in describing the species of Mycocalia. There appears to be no published report that metamorphosed basidia are capable of germinating as do true basidiospores.

sh

si

Changes frequently take place in basidiospores after the disintegration of basidia. These changes may involve only a slight increase in size, or as in Cyathus striatus,the thickening of the spore wall; or it may involve the extraordinary development of special 'nurse hyphae/ which surround the spores (figure 5) and, by their gradual disintegration, allow the spores to increase tremendously in size, as in Cyathus stercoreus. Events in the basidium leading to passage of a single haploid nucleus into each young basidiospore will be described in detail in Chapter 6. It is necessary at this point to realize only that a mature spore, for nearly all species, is genetically haploid. The statement is often made in text books that basidiospores of the Nidulariaceae are regularly binucleate at maturity as the result of a mitotic division. This is true for some species, but others may have one nucleus in each spore and still others more than two nuclei, depending upon mitotic nuclear divisions occurring as spores mature; but the spores, and their resulting mycelia are always homokaryotic (i.e., all the nuclei are genetically identical). C/GERMINATION

FIGURE 7 Section through pendióle of Cyathus pallidus semidiagrammatic: h, hymenial layer with basidia and spores; gm, gelatinous matrix produced by disintegrating hyphae; sh, subhymenial layer; si, inner sclerotic layer of cortex, c; t, tunica. X5oo, approximate

Basidiospores of the Nidulariaceae, like those of other members of the Gasteromycetes, are not forcibly discharged from their basidia. As maturation proceeds, the basidia collapse and gelatinize to the point of being unrecognizable. Tissues lining the walls of the peridiole also gelatinize and the whole gelatinization process forces spores toward the center of the peridiole where they lie immersed in a gelatinous matrix. 26 / The bird's nest fungi

It has been common experience that basidiospores of the Gasteromycetes germinate, under laboratory conditions, in very low percentage or not at all ; and the Nidulariaceae are not exceptional in this regard. In 1962, reviewing twenty years of experience with the bird's nest fungi, I reported (Brodie, 1962^): We have found that if peridioles are macerated and the spore suspension so obtained is held at 40° c for two days, some spores usually germinate. But some spore samples, as fresh as others, have proved resistant to every trick in the books - enzymes, detergents and all the rest. It would be highly desirable to know the secret of the recalcitrance of these spores, but meanwhile we have cultured and studied those that do germinate.

FIGURE 8 Basidiospores of Nidulariaceae: a, Cyathus gracilis, Xijoo; b, Cyathus africanus showing apiculus, X24OO; c, Crucibulum cyathiforme showing curved spores, Xzooo; d, Cyathus pictus, globose spores, X5oo

In species whose pendióles are eaten by herbivorous animals, it would appear that spore germination in the animals' digestive tracts would be undesirable. A built-in dormancy mechanism may therefore exist, although this has not yet been established experimentally. Many pages could be devoted to an account of numerous efforts to induce basidiospores to germinate, but the reading would be tedious and the effort of doubtful value. It may serve some purpose, however, to record experiences leading to successful attempts with certain species, and a few instances of efforts that have produced negative results. The first major problem concerns the difference in the germinability of spore samples derived from different fruit bodies of any one species. A manipulation which has yielded a germination of 20 per cent of the spores of one sample (germination percentage is usually lower than that), when repeated exactly, using 27 / Basidiospore form, development, and germination

a different spore sample of the same species, often fails to evoke any germination in the second sample. Moreover, because the number of spores in any one fruiting body is limited, it is difficult to obtain a sufficient number of tests to permit evaluation of the results of germination studies. All germination tests carried out in my laboratory have involved a basic procedure as the first step. This procedure is as follows: 1 / Whole pendióles, removed from the freshest and most undamaged fruiting bodies available, are soaked in sterile distilled water for 10 minutes at room temperature. 2 / Pendióles, after soaking, are removed with sterile forceps to an aqueous solution of mercuric chloride of concentration i : 1000, ensuring that the peridioles are completely immersed; if peridioles were suspected of being badly contaminated on the surface, a mercuric chloride solution of 1:500 was occasionally

employed without any apparent deleterious effect upon the spores. Pendióles are allowed to stand in the solution for two minutes. Sterilization of the pendióle wall is effected by the treatment. 1 3 / Pendióles are removed aseptically from the mercuric chloride and passed through two separate rinses of sterile distilled water. 4 / Pendióles are removed again to fresh distilled water in a sterile dish and macerated with sterile scalpel and forceps to obtain a suspension of basidiospores. What has been done to the resultant sterile spore suspension (which, of course, includes fragments of pendióle wall and of some hyphae from the inside of the peridium), in efforts to obtain germination, can be summarized as follows : (a) Spores which germinate readily. In some species, notably Cyathus olla, C. earlei, C. pygmaeus, C. africanus, and C. setosus, spores germinate to some degree, depending upon species and spore sample, when the sterile aqueous spore suspension is merely allowed to stand at room temperature. At times, spores have germinated only after a lapse of time - as much as a week or ten days - but most often germination begins within 48 hours at room temperature. (b) Spores which require heat. Spores of the widely distributed coprophilous fungus Cyathus stercoreus and of the tropical species C. poeppigii and C. limbatus may germinate in fair proportion if the sterile spore suspension (prepared as indicated above) is subsequently placed in an incubator and held at 40° c for 48 hours. Holding spores at this temperature for longer periods has not been found to be more effective. (c) Spores which do not respond to (a) or (b) 1 It is conceivable that residual traces of mercury occa-

sionally inhibit the germination. However, there is no clear evidence of this inasmuch as different tests of identical spore samples may behave differently even when the washing technique is apparently a uniform practice.

28 / The bird's nest fungi

treatment. For a large number of species, neither (a) nor (b) treatment results in germination. Spores of such species, subsequent to the initial basic procedure, have been subjected to a great variety of conditions involving acidity, temperature, presence of enzymes and of such substances as detergents, mineral salts, vitamins, etc. Such tests have, for the most part, produced negative results. When germination did occur, frequently the particular operation was unsuccessful when repeated, at least partly because of the variability of spore samples, as already noted. A few examples only of the many efforts that have been made to cause the spores to germinate may be commented upon. i / Enzymes. Because of the strong possibility that basidiospores may pass through the alimentary tracts of some herbivorous animal, the possible effect of enzymes has been tested. A second reason for testing, which could be connected with the first or could be an unrelated problem, is the possibility that enzymes in the natural environment may be necessary for the breakdown of the hard peridiole wall within which basidiospores are enclosed. Brams (1950) studied the effect of the enzymes pepsin, trypsin, and lipase upon the germination of spores of Cyathus stercoreus and reported that the percentage of germination was increased and the time for germination decreased when the spores were immersed in solutions of these enzymes adjusted to the pH optimum for each enzyme. Pepsin was reported to have the greatest 'beneficiar effect. Miles (1953) attempted to verify Brams's results, using the same fungus and the same enzymes as well as others. Regardless of pH, temperature, or enzyme concentration, Miles failed to observe any enhancement of germination. Many efforts over many years, and involving other enzymes, have met with the same fate as Miles's experiments and one must conclude that, if enzymes do affect germination, the necessary conditions have not been met or the specific enzyme necessary has not been tested.

2 / Wetting agents. Various wetting agents have been used to try to induce germination but without significant or repeatable positive result. 3 / Chemical salts. Repeated attempts were made to ascertain whether or not the ordinary salts present in the nutrient media used to achieve vegetative mycelial growth were necessary for spore germination or had any marked effect upon germination. Again no promising leads have been obtained. 4 / Other tests. Because borax has been reported to promote germination of some kinds of fungus spores, various concentrations were added to spore suspensions, and at the same time other conditions such as temperature were varied. No stimulus to germination was observed in any test (Miles, 1953). That an osmotic problem might be involved was considered by Miles, who used solutions of sucrose and glycerol either alone or added to nutrient media. Although in a few tests it seemed that percentage germination was increased, Miles reported that the extremely small amount of germination obtained and the small numbers of spores involved made his experiments inconclusive. Whatever the environmental factor may be that initiates the germination process of basidiospores, it is obvious that it has not been found. Age of the spores does not appear to be important, for basidiospores of some species have been found to germinate well merely in distilled water, even when the spores had been taken from young sporocarps in which the epiphragm had barely ruptured and, as noted elsewhere (Chapter 9, c, 3c), basidiospores as much as four years old have been observed to germinate. D / G E R M T U B E S AND G E R M L I N G S The difficulty of bringing about germination adds interest to the appearance of germlings when they are finally obtained. The first indication of germination (after about 8-18 hours) is often, but not always, the development of a 29 / Basidiospore form, development, and germination

clear turgid appearance in certain spores. These contrast with the more optically dense ungerminated spores. This stage is followed rapidly by a marked swelling of germinating spores in certain species, notably Cyathus olla ; in other species no marked swelling is detected. Two moderately distinct principal types of morphology of young germlings have been observed, although intermediate conditions also exist. These types are, as far as present information goes, roughly correlated with the presumed taxonomic grouping of species, although it cannot be said that the correlation is high. 1 / Sporelings from small thin-walled spores. These are best exemplified by sporelings of Cyathus olla. In such species the thin-walled spore swells to an extent that can be readily observed and measured and emits a single rather thin germ tube (4-6 ¡JL in diameter) from one end. The germ tube most frequently reaches a length of five or more times the length of the spore before beginning to branch. In some species that have this type of germination, more than one germ tube may be produced by a single spore (e.g., Nidula spp. (figure 9)). There is, however, considerable variation, and the artificiality of association of species in this connection is realized fully. This type of germination has been seen in the following species: Cyathus olla, C. olla forma anglicus, C. earlei, C. microsporus, C. pallidus, C. striatus, Nidula spp., and Crucibulum laeve. 2 / Sporelings from large thick-walled spores. These are best represented by sporelings of Cyathus stercoreus (figure 5). In this type fully mature spores swell only slightly, or sometimes not at all. A single germ tube may emerge but, far more frequently, from four to six stout germ tubes emerge almost simultaneously and these are commonly 8 to 10 ¡m in diameter. Germ tubes may emerge chiefly from one end, giving the young sporeling a kind of squid-like appearance (figure 5), or they may emerge at several points over the spore surface. This kind of germination has been noted in the following: Cyathus sterco-

FIGURE 9 Spores and mycelial structures of two species of Nidula. From a to h (incl.), N. candida: a, b, spores germinating showing equatorial position of germ tubes, Xi3oo; c, young germling, hyphae still aseptate, x6oo; d, mature monokaryon hyphae and f, dikaryon hyphae, Xioo; e, oidia on monokaryon hyphae, X9oo; g, oidia in drops of exúdate, X5o; h, outline of pendióle, Xz. From i to q (incl.), N. niveo-tomentosa, structures and magnification corresponding to those in upper half of drawing ; in p and q, note position and arrangement of clamp connections. A difference between the two species is shown in the type of germlings developed - compare b and k. Figure courtesy of Mycologia, 1951, 43: 332 30 / The bird's nest fungi

reus, C.poeppigii, C. limbatus, andC. canna. Many other species whose germination has been studied do not conform completely to either of the above descriptions. The germination patterns of two species of Nidula, N. niveo-tomentosa and N. candida, are worthy of note (Brodie, 1951^. In both species, germlings are essentially of the first type described. Spores swell slightly, and from one to three slender germ tubes are produced. However, a marked difference in the morphology of germlings of the two species develops when they are two or three days old: germlings of N. candida usually have two (occasionally three) germ tubes that arise from the equatorial region of the spores; whereas those of N. niveo-tomentosa produce from one end of the spore a single germ tube which soon branches, giving the germling as a whole something of the squid-like appearance (figure 9) of the germlings of C. stercoreus. Here we have a clear difference between the germlings of two closely related species. As the two Nidula species are easily separated on the ordinary criteria applied to the fruit bodies, sporeling morphology is unnecessary as a taxonomic aid. However, the existence of such specific differences suggests that such studies, applied to species that are difficult to separate by sporocarp morphology, might yield criteria that could be of taxonomic value. But the investigator who wishes to obtain a series of vigorous mature mycelia, each derived from a single basidiospore, is not yet out of difficulty merely because sporelings as described above have been obtained. It has been noticed repeatedly that sporelings may die at a very early stage. Young mycelia may become highly vacuolate and cease to grow or, in some instances, they appear to undergo lysis. For any particular species, this difficulty has occasionally been overcome when another spore sample was used to obtain a fresh supply of germlings. For other species, death of sporelings has always taken place soon after spore germination. In the latter situation, transferring spore31 / Basidiospore form, development, and germination

lings to the surface of various common nutrient media (potato-dextrose agar, malt agar, etc.) has not sufficed to ensure continued growth of sporelings. Presumably some nutrient deficiency is responsible for such premature demise of the sporelings, but the matter has not been investigated further. If the spores are allowed to germinate on nutrient agar, or if a germinating spore suspension has been plated out on nutrient agar, the morphology of the very young developing mycelia may be studied conveniently. Sporelings of different species differ slightly in appearance, but all have some common characteristics. They vary considerably in growth rate, degree of branching and septation, and in diameter of hyphae. Germlings are almost invariably devoid of septa up to about the third or fourth day from the visible onset of germination (figure 5). One exception to this was observed in Cyathus striatus in which one or two septa were found in germ tubes scarcely more than twice the length of the spores from which they grew. When septation of sporelings does begin it is rapid and soon involves all hyphae. Sporelings of all species rapidly lose their juvenile characteristics (e.g., the squid form of C. stercoreus) and, because of the branching of hyphae, all assume a radially symmetrical form. Very young mycelia, up to about ten days from the sowing of spores for germination, are colourless, although older haploid mycelia of many species do become pigmented, most commonly in some shade of brown. No observation seems to have been made as to the number of nuclei present in very young sporelings of the Nidulariaceae prior to septation. For a variety of other basidiomycetes it is known that many nuclei may accumulate in young germlings in the early coenocytic stage as the result of repeated mitotic divisions, and it is probable that this is also true in the Nidulariaceae. However, Walker (1920) illustrated a young, but already septate, germling of Cyathus olla in which two nuclei are shown regularly disposed in each cell.

4 / Characteristics and Interactions of Homokaryotic Mycelia

A / CHARACTERISTICS If individual sporelings of the Nidulariaceae, each derived from a single basidiospore, are transferred to a suitable nutrient agar medium, most of them develop rapidly into homokaryotic (haploid) mycelia having many characteristics in common with those of other members of the Basidiomycetes grown in laboratory culture. Only a few bird's nest fungi have special features in culture. Most homokaryotic mycelia appear, to the unaided eye, fluffy, radially symmetrical, and of fine texture (figure 11). Most are snowwhite at first, but many, though not all, later develop brown pigments in varying degree, with the result that ten-day-old cultures are commonly light buff, golden brown, or dark brown. Brown pigments are developed (figure 11) by mycelia of large-spored species such as Cyathus stercoreus, C. poeppigii, and others, while lack of pigment is characteristic of small-spored species, including C. olla, C. pallidus, and others. The first deviation from the common mycelial type concerns the extent to which mycelium is produced aerially rather than on the surface of the nutrient medium or below it. In contrast to the fluffy mycelium (much aerial growth) of the majority of the Nidulariaceae, homokaryotic mycelia of Crudbulum laeve and of Nidula spp. have a flat, moist appearance (little aerial 32 / The bird's nest fungi

growth). Deviation of this sort has also been seen occasionally in individual homokaryotic mycelia of species in which other homokaryons are fluffy. Another deviation from the common mycelial type is in the degree of symmetry and in the texture of mycelia. In several species, of which Cyathus helenae is a good example, certain homokaryotic mycelia have a very irregular or 'blotchy' appearance (figure 10). Olchowecki and Brodie (1968) reported that some of the irregular mycelia maintain this characteristic in repeated transfers while others revert to the even or regular appearance of the majority of homokaryons. Whether these irregularities in morphology (which are probably accompanied by physiological deviations) are the result of the operation of some genetic mechanism or have some other cause is unknown. The brownish pigment so prevalent in mycelia of Cyathus has been assumed to be of the nature of a melanin, although no chemical examination has apparently been made. Aside from this brown pigment, the only other pigment reported is of a peculiar dull purplish brown (vinaceous) colour, which diffuses into the nutrient substrate from homokaryotic mycelia of Nidula niveo-tomentosa (Brodie, i95ic). Again, nothing is known of the chemical nature of the Nidula pigment, but it is worthy of note that it is not produced by the closely related species N. candida.

FIGURE 10 Homokaryotic mycelia of Cyathus helenae showing variation in morphology in différent strains: a, irregular growth pattern and a fast-growing sector, Xi.2; b, regular growth pattern and a slowgrowing sector, Xo-5 ; c, regular homokaryotic mycelium, Xioo; d, irregular and contorted hyphae found in some strains, Xioo. Photo reproduced courtesy of Can. J. Bot., 1968,46: 1427 33 / Characteristics and interactions of homokaryotic mycelia

FIGURE il Differences in colour and growth rates of homokaryotic and dikaryotic mycelia of Cyathus stercoreus : white homokaryon (a) and dikaryon (b) ; brown homokaryon (c) and dikaryon (d) ; all cultures of same age and all Xo.y 34 / The bird's nest fungi

Microscopically also, homokaryotic mycelia of the Nidulariaceae have few characteristics not shared with many other saprophytic basidiomycetous fungi. Hyphae, as seen through the light microscope, are of the order of 4 to 8 fji in diameter, are provided with simple perforate septa at fairly frequent intervals, and send out branches at an angle of 45-70° to the main axis. The appearance of the mycelium of Cyathus stercoreus is shown in figure 5 (see also Brodie, 19483, for description) and that of Ni dula candida in figure 9. Some deviation from the majority of homokaryons of a given species is often encountered in the regularity of form of the hyphae. In some homokaryotic cultures, hyphae may be irregularly sinuate and bear numerous knobs (figure 10). Development of the latter kind of mycelium does not seem to indicate unfavourable growth conditions, for the irregularity of form is maintained when the mycelium is grown on a wide variety of nutrient media. Because each monospore mycelium is homokaryotic, being derived from a single genetically haploid basidiospore, it should not be assumed that each cell of the mature mycelium contains but a single nucleus. Miles (1953) showed that each basidiospore of Cyathus stercoreus is uninucleate, but Fulton (1950) reported that in homokaryotic mycelia of the same species, although many cells do contain only a single nucleus, some cells contain more than one, and the terminal cells of most hyphae contain several nuclei. For C. striatus Walker (1920) illustrated many cells of homokaryotic mycelium containing two nuclei and the terminal cells of hyphae containing many nuclei. As stated above, most bird's nest fungi do not produce an abundance of hyphal segments of the type known as oidia. Straight simple oidia may occasionally be produced by homokaryotic mycelia of Crucibulum laeve and Cyathus striatus growing in hanging-drop cultures; but oidia do not appear in vigorous mycelia grown on agar plates. Only two species

of the Nidulariaceae are known to produce oidia regularly and in abundance under a wide variety of growth conditions. Homokaryons of Nidula niveo-tomentosa develop long straight oidia on aerial branches (figure 9) and those of N. candida form oidia in clusters on the ends of upright knobby branches (figure 9) accompanied by the formation of a shiny exúdate (Brodie, i95ic). It is not known whether or not any of the oidia noted here are produced on mycelia growing under natural conditions, or if they are, whether or not they serve for the asexual reproduction of these fungi. The oidia may also, as in many other higher fungi, facilitate dikaryotization of spatially separated mycelia through insect agency. In at least one member of the Nidulariaceae, hyphae of homokaryotic and, especially, heterokaryotic mycelia produce some kind of metabolite often seen under the microscope as irregular lumps on the surface. This is illustrated for Cyathus stercoreus (figure 5) in which it occurs regularly. In some species, the lumps appear more regular and may be crystalline. The chemical nature of these materials is not known. Mycelia of different species of bird's nest fungi, and even different monokaryons of the same species, differ greatly in their rate of growth in laboratory culture, depending upon temperature, pH, and the nature of the nutrient substrate. Most of the experimental work along these lines conducted in our laboratory has been utilitarian in plan: it was usually conducted merely to determine the optimum growth condition for mycelia intended to be used for sexuality pattern determinations, for efforts to induce fruiting, or for morphological study. There seems little use therefore in reporting the results of such work in detail: instead a few general comments are given. Some growth of homokaryotic mycelia of these fungi can take place at temperatures ranging from 5° to 30° c. For most species, growth is slow and sparse as either temperature extreme is approached. A temperature of 22° c appears to be the optimum for growth for the

35 / Characteristics and interactions of homokaryotic mycelia

majority of species examined. The only striking exception to this rule is that mycelia of Nidula candida andN. niveo-tomentosa have been shown to make more normal-looking growth at 8-15 °c than at room temperature (e.g., 22°c), although growth was slower at the lower temperature (Brodie, 1951^. One might expect tropical species to require higher temperatures for optimal mycelial growth than temperate species, but this is not borne out by the tests made, though future tests with other species may change the picture. The growth rate at optimal temperature also varies with species, strain, pH, nutrition, etc., as might be expected. Different homokaryotic mycelia of Cyathus stercoreus may grow at rates varying from 1.5 mm to 3 mm per day at 22° c (Brodie, i948a,b). It is worthy of note that, although exposure to a temperature of 40° c has been observed to stimulate spore germination in C. stercoreus and many other species, growth of the mycelium usually ceases and mycelia soon die at temperatures well below this figure. B/NUTRITIONAL REQUIREMENTS The nutritional requirements of the Nidulariaceae in culture do not appear to be unique. Again, experiments have been directed towards the solving of practical problems rather than towards specific studies of nutrition in relation to fundamentals of physiology. As the result of many trials and errors a medium was developed, with a composition only partially defined chemically, which allowed the growth of a variety of species. This medium (subsequently locally named 'Brodie Medium') has the following composition: Brodie Medium - Bacto agar, 20 g; maltose, 5 g; dextrose, 2 g; peptone, 0.2 g; asparagine, 0.2 g ; yeast extract, 2 g; magnesium sulphate, 0.5 g ; calcium nitrate, 0.5 g ; dihydrogen potassium phosphate, 0.5 g; ferrous sulphate, trace; distilled water to make -L litre (Brodie, 1949). After autoclaving, the medium has a pH of about 6-6.5.

36 / The bird's nest fungi

Mycelia of many species grow equally well upon this medium if the agar component is omitted, but omission of other ingredients has appeared to retard the growth of some species and to make it much less useful as a general medium for the growth of a large number of species. When a specific nutritional problem must be studied, it is necessary to employ a medium for which the chemical composition of the ingredients is fully known. One such medium was developed (Johri and Brodie, 1971^ which has the following composition: John s Defined Medium - Dextrose, 30 g; asparagine, 1.5 g; dihydrogen potassium phosphate, i.o g ; calcium nitrate [Ca(NO3)24H2O)], 0.5 g ; magnesium sulphate [MgSO4.2H2O], 0.5 g ; zinc sulphate [ZnSO4.2H2O], 250 mg; thiamine hydrochloride, 150 mg; distilled water to make i litre. As might be expected, alteration in the proportions of the substances supplied in the substrate can affect the growth of a particular species or a particular strain. However, it is difficult to draw any firm conclusions from the observations that have been reported and only a few examples will be given. Dextrose appears to be the carbohydrate best utilized by bird's nest fungi as a primary source of carbon (Fulton, i95ob; Garnett, 1958) although a wide variety of carbon compounds may support growth. In Brodie Medium, better growth is usually obtained when maltose is present in addition to glucose, but the reason for this is not clear as far as the Nidulariaceae are concerned. Of the several sugars that are utilizable, no single one, when provided as the only sugar, seems to have a profound effect upon mycelial growth, although Fulton (i95ob) showed that different homokaryotic strains of Cyathus stercoreus may differ in the particular sugar utilized to achieve optimum growth. Studying homokaryotic mycelia of Cyathus helenaef Johri and Brodie (i97ib) demonstrated that, among sugars utilized by the fungus, no particular sugar used in Johri's Defined Medium mat-

erially affected total growth as measured by dry weight. However, the particular sugar used did markedly affect the synthesis by the fungi of the various components of the antibiotic metabolite complex now called 'cyathin. ' In Brodie Medium and in Johri's Medium, nitrogen is supplied in organic as well as in inorganic form. Garnett (1958) reported that mycelial growth of some strains of Cyathus stercoreus was not as good when nitrogen was supplied to the fungus in such forms as potassium nitrate or ammonium nitrate; moreover, other strains failed to grow under these conditions. She concluded that organic nitrogen is required by this fungus. Johri (1969) reported that ammonium nitrate and ammonium sulphate are poor sources of nitrogen for homokaryotic mycelia of C. helenae, but that calcium nitrate is a good source. Total growth, as measured by dry weight, was not strikingly affected by the nitrogen source, but the production of cyathin was affected appreciably. It has not been established conclusively that vitamins have a noticeable effect upon mycelial growth; and Johri (1969) reported that seven common vitamins tested appeared not to affect the yield of cyathin materially. A relation between the carbon and nitrogen sources available to the fungus is suggested by Johri's conclusion that 'the appropriate combination of carbon and nitrogen sources makes possible a high yield of any particular component of the cyathin/ So-called 'minor elements/ that is, elements present in minute amounts, must certainly be present in Brodie Medium. Johri (1969) added copper, iron, manganese, and zinc separately in low concentration (e.g., o.i3-o.5Oppm) to a basal medium and observed that for Cyathus helenae, copper and iron were inhibitory, while zinc seemed to play an important nutritional role, especially in the production of cyathin. Regarding the effect of hydrogen ion concentration on growth, it appears that the optimum depends upon the particular fungus and

the particular nutrient involved. In any case, the fungi are able to grow at pH ranging from 4.8 to 7; for most species the optimum value has been found to be pH 6.0. There is no evidence to show that light has any effect upon the vegetative growth (as measured by dry weight) of homokaryotic or heterokaryotic mycelia of the Nidulariaceae. However, light has a profound effect upon fruiting as will be explained in Chapter 5. The production by Cyathus helenae, C. africanus, and C. striatus of a series of previously unrecognized chemical compounds which have antimicrobial properties (Johri and Brodie, i97ib; Allbutt et al., 1971) will be reported later (Chapter 10, B, i). Pertinent to the present discussion is the fact that the metabolite complex, now known as cyathin, is produced only by homokaryotic mycelia (Johri and Brodie, 1971^. This observation is an unexpected one, for it will be shown (Chapter 5, A) that, when two sexually compatible mycelia unite, the genotype of the individual mated homokaryons usually determines the phenotype of the resulting heterokaryons. Finally, under characteristics of homokaryotic mycelia of the Nidulariaceae, is the matter of the inability of homokaryons to form sporophores. I have seen no report indicating the occurrence of homokaryotic fruiting bodies and my own experience with many species leads me to the conclusion that homokaryotic mycelia never fruit. Indeed, they do not even produce the mycelial cords and mycelial knots that, for the species studied, have been shown to be the morphological precursors of sporophore development. C / MYCELIAL INTERACTIONS i / Regular behaviour With the single exception of the two species Mycocalia denudata and M. duriaeana (Burnett and Boulter, 1963) all members of the bird's nest fungi so far studied have been found

37 / Characteristics and interactions of homokaryotic mycelia

to display the 'tetrapolar' type of sex reaction when monospore cultures of any one species are paired in all possible combinations. A brief generalized account of the mechanics of sexuality determination and a description of the 'normal' or usual course of events will be useful as a background for consideration of some of the unusual phenomena that have been observed. For this purpose, reference is made briefly to the coprophilous fungus Cyathus stercoreus, which has had more laboratory study than most other species. It has been my practice to select 20 to 30 vigorously growing cultures from a series of single-spore mycelia, obtained as described in Chapter 3, leaving slow-growing or otherwise deviant cultures for later consideration when separate mating-type stocks had been obtained. To select 'healthy plants' in this way for experiment may be a mistake if one bears in mind the advice that used to be given to young scientists - 'treasure your exceptions/ Deviant cultures may conceivably be those which bear some genetically determined biochemical deficiency, whose study might prove interesting. Also the practice may have been at least one factor contributing to the unexplained observation that the four mating-types have seldom appeared in the equal proportions to be expected on a genetic basis. Single-spore mycelia are allowed to grow on nutrient agar slants of Brodie Medium until it is judged that there is a sufficient quantity of each to allow all mycelia to be transferred as fresh plantings paired in all possible combinations . Mycelial pairs are set at the centre of the surface of slants so that the mycelia being confronted are about one centimetre apart. When this procedure is used, physical union between mycelia requires more time than if the mycelia had been planted immediately beside one another, but any unusual reaction between mycelia can more readily be detected. In about two weeks, the paired mycelia in tubes are examined macroscopically for any interaction that may be observable with the unaided eye and, microscopically, for an as387 The bird's nest fungi

sessment of the results of anastomosis between the individuals of the mycelial pair. For the most part, the latter becomes a matter of determining whether or not clamp connections, indicating the presence of heterokaryotic (dikaryon) mycelium, can be seen. Those pairings from which clamp-bearing mycelium has been produced are recorded as (+) and those in which no clamp connections can be found in teased-out water mounts are recorded as (—). By recording the results in a checkerboard table, and grouping together those numbered cultures which show the same reaction pattern across the table, it is possible to sort out mating types. Usually it will be seen that cultures fall into four groups, which may be designated tentatively as i, n, m, and rv. It will be apparent that mycelia of Group i united only with mycelia of Group n to give rise to heterokaryotic mycelium, and that Group m united only with Group rv. If Group i mycelia be now designated by a formula such as AB, indicating the presence of two alíeles of the two pairs of genes determining sexual compatibility, then Group n mycelia (fertile when paired with those of Group i) are ab. Group m and Group rv, the other two sexually compatible heterozygous mycelia, are then A b and aB (figure 30). The basis for these assumptions may be found in any review or text book dealing with the genetics of fungi. Formulae such as ab, a'b', etc., and others have been commonly employed to avoid the supposed implication, inherent in the use of upper and lower case letters, that A is dominant and a recessive. I prefer the latter scheme merely because it is easier to follow visually than a, a', a", etc. Without at the moment considering details of the morphology and physiological characteristics of the heterokaryotic mycelium, it may be noted here that some mycelial pairings give immediate visible indications of some sort of interaction between the mycelia. Vigorous coarse hyphae may arise, indicating the presence of heterokaryotic mycelium to the unaided eye ; pigmentation may arise between the

TABLE 1

Mating systems in the Nidulariaceae Name

Origin of cultures

Investigator and reference

Nidularia far eta Mycocalia denudata* Mycocalia duriaeana^ Ni dula candida Nidula niveo-tomentosa Crucibulum laeve Crucibulum parvulum Cyathus africanus Cyathus bullen Cyathus canna Cyathus earlei Cyathus helenae Cyathus julietae Cyathus microsporus Cyathus olla Cyathus olla forma anglicus Cyathus pallidus Cyathus poeppigii Cyathus pygmaeus Cyathus stercoreus Cyathus striatus

Europe Europe Europe North America North America Europe North America Africa West Indies Costa Rica Mexico North America West Indies Costa Rica North America

N. Fries, 1948 Burnett & Boulter, 1963 Burnett & Boulter, 1963 Brodie, i95ic Brodie, i95ic N. Fries, 1936, 1943 Brodie, i97oc Brodie, i97ob Brodie, i9/ob Brodie, 19683 Brodie, 19623 Olchowecki & Brodie, 1968 Brodie, 19683 Brodie, 19683 Brodie, 1949

North America North America North America North America North America Europe

Brodie, 19523 Brodie, 19683 Brodie, 19683 Brodie, 1970!? Brodie, 19483 N. Fries, 1936

* Bipolar mating system, facultatively homothallic t Homothallic. All other species have tetrapokr systems paired homokaryons ; and various other effects may be noted. In many pairings, the two mycelia merely intergrow until no break between them can be discerned. Following essentially the procedure described above, the mating systems of 20 species and one clearly marked form of Nidulariaceae have been elucidated up to the present time. A list of these species is given in Table i. There are few groups of closely related fungi for which information regarding sexuality patterns is available for such a large proportion of the species, and it is interesting to note that, of the 21 entities listed in the table, 19 display tetrapolar heterothallism. Only the two species of Mycocalia have been recorded as having other mating systems: M. denudata is both bipolar-heterothallic and facultatively homothallic, while M. duriaeana is homothallic (Burnett and Boulter, 1963).

Heterothallism involving four mating types is thus almost exclusively the sexuality pattern that has been established in the Nidulariaceae, and only this pattern has been reported in the genus Cyathus. The genetic and cytological implications of these findings have been considered to some extent by Burnett and Boulter

(1963)One interesting aspect of the sexuality pat-

tern of the Nidulariaceae that emerges from a study of the publications referred to in Table i is the existence of alíeles of the genes (possibly groups of genes) that determine mating type. Different alíeles may exist within the population of a relatively limited geographical area, or differences may appear (on the basis of limited sampling) to be restricted geographically; in the latter case the mycelia bearing sex allelomorphs have been referred to as 'geographical races/ Burnett and Boulter (1963) reported

39 / Characteristics 3nd interactions of homokaryotic mycelia

from seven to thirteen mating-type 'factors' among a number of isolates of Mycocalia denudata. Fries, earlier, had reported four and five mating-type factors at the A and B loci respectively in nine fruit bodies of Cyathus striatus (N. Fries, 1940) and three and eleven respectively in fifteen fruit bodies of Crucibulum laeve (N. Fries, 1943). Fries also reported that some allelomorphs were either confined to or more prevalent in certain localities. Brodie (195 za) studied two forms of Cyathus olla sufficiently distinct in morphology for one to be recognized as C. olla forma anglicus, and demonstrated that both forms possess the A and a pair of sexuality genes (i.e., possess these in common) and that the two forms differ in the other pair of sexuality genes, B and b. A somewhat similar situation was studied by Olchowecki and Brodie (1968) who examined the possible interfertility between mycelia of Cyathus striatus and the closely related C. helenae. It was reported that 'Dikaryotic mycelia were developed in certain matings of monosporous mycelia of C. helenae with monosporous mycelia of C. striatus/ Although there was evidence of the common existence of some mating-type genes between C. helenae homokaryons and C. striatus homokaryons, the authors concluded that 'close genetic relationship is not taken as indicative of conspecificity/ From time to time the author and students have endeavoured to demonstrate linkage between mating-type and morphological and physiological features of monosporous mycelia. There are certainly great differences between individual homokaryons (e.g., in C. stercoreus) in colour, texture, growth rate, and nutritional requirements ; however, no one has yet shown any close association of any of these characteristics with any particular matingtype. Among C. stercoreus homokaryons there are white mycelia and brown mycelia and there are slow growers and fast growers; but brown and white, fast and slow appear randomly distributed throughout all four matingtypes. After a careful study of this situation 40 / The bird's nest fungi

in C. stercoreus, Fulton (i95ob) concluded that differences in nutritional requirements exist among monosporous cultures, but that there is as yet no evidence of an association of nutritional requirements with any particular mating-type. 2 / Some irregularities One unexpected and seemingly irregular feature of studies of sexuality in many species is the unequal representation of the four mating-types among series of monospore mycelia selected for analysis of the sexuality pattern. The diploid or zygote nucleus in young basidia may be represented (as to mating-type genes) as AaBb. Following normal meiosis, among numbers of basidia equal numbers of spores of all four haploid genotypes AB, ab, Ab, aB should be formed. Not only has regular distribution of monospore mycelia representing all mating-types seldom been observed, but it has been common experience to find skewed or irregular distributions. A few examples only are given here. Because mating-type is determined empirically for any one species, comparison between species has doubtful significance. It is noteworthy, however, that for a given species neither a large nor a small representation of mycelia is characteristic of any one mating-type. If small numbers were regularly associated with two mating-types for a given species (e.g., AB and Ab), then one might suspect linkage with some gene determining nutritional deficiency. At present, however, no explanation can be offered for the irregular distributions observed ; but it has been suggested (Brodie, 1953) that a sublethal factor may be involved. Still another irregularity that has been observed is the occasional occurrence of the complete lack of fertility among monospore cultures derived from a single fruit body of one species. One example of this was reported (Brodie, i968a) in Cyathus poeppigii. Spores from one particular fruit body yielded haploid mycelia which behaved according to the tetra-

TABLE 2

Mating-type (%) of cultures analysed Species

AB

ab

Ab

Cyathus poeppigii Cyathuspallidus Cyathus julietae Cyathus canna Cyathus pygmaeus Cyathus helenae

33 40 26 I 3 40 1.6

36 8 40 26 10

2l

10

10

32 7 13 20 24.7

67

27 48

3° 6.7

polar scheme when mated, but 29 monospore mycelia isolated from a different fruit body (from the same collection) were completely sterile when paired among themselves or with testers obtained from the first isolation. The fruit body that yielded the sterile homokaryotic isolates was found to be made up of binucleate heterokaryotic hyphae and the conclusion was given (Brodie, 19683, p. 205) that: 'It is apparent that some unusual phenomenon is involved here which requires further investigation/ 3 / Unilateral dikaryotization Although the phenomenon which came to be known as unilateral diploidization, but is more properly termed dikaryotization, is now known to occur in a number of basidiomycetes, one of the first examples to be investigated was that of a member of the Nidulariaceae, Cyathus stercoreus (Brodie, 19483; Fulton, i95ob ; Brodie, 1953). In most pairings of compatible homokaryotic mycelia of this fungus, dikaryotization is of the regular type. Beginning at the point of contact between two monosporous mycelia growing on an agar plate, dikaryotization (i.e., the conversion of haplophase into dikaryophase mycelium) spreads around the periphery of each member of the compatible pair until each homokaryotic mycelium lies surrounded by a ring of heterokaryotic dikaryon hyphae. In certain pairings, conversion of homokaryotic into heterokaryotic mycelium

aB

Reference

Brodie, 19683 Brodie, 19683 Brodie, 19683 Brodie, 19683 Brodie, i97ob Olchowecki & Brodie, 1968

takes place only on one side, or in one direction, when two compatible homokaryons are confronted with one another (figure 12). Nuclear migration is unilateral and one homokaryon remains unchanged, morphologically at least. Fulton (19503) (Mrs C. Ahlgren) showed that unilateral dikaryotization occurs most frequently in AB x ab combinations or in Ab x AB. Even in these, however, in some pairings, dikaryotization is of the 'regular' type. In unilateral pairings of AB x ab,theAB mycelium was always found to be the acceptor of nuclei (i.e., it became heterokaryotic), whereas the ab mycelium was always the donor of nuclei and remained homokaryotic. When monosporous mycelia that had displayed unilateral dikaryotization and had acted as acceptors in pairing were stained, the older cells of such mycelia were found to contain two nuclei each, although each monospore mycelium arose from a single uninucleate basidiospore. No explanation of the phenomenon of unilateral dikaryotization has yet been offered for Cyathus stercoreus. 4 / Interaction between species and varieties Over many years, many efforts have been made to ascertain whether or not any degree of fertility may exist between monosporous mycelia belonging to different species. Whenever sets of homokaryotic mycelia of known mating-type were obtained and there were at the same time available sets of homo-

41 / Characteristics and interactions of homokaryotic mycelia

FIGURE 12 Unilateral dikaryotization in Cyathus africanus. Of a sexually compatible pair of monokaryon mycelia, only the lower one has produced coarse conspicuous dikaryotic hyphae; Xi.8. Photo reproduced courtesy Svensk Bot. Tidskr., 1970, 64: Pi. ii, p. 52

FIGURE 13 Fruit bodies produced by matings of homokaryon mycelia of Cyathus olla with homokaryon mycelia of C. olla forma anglicus. The fruit bodies were unlike those of either parent; Xi.2. Photo reproduced courtesy Mycologia, 1952, 44 • 4*6

karyons of a different species, pairings were made. In no instance were fruiting bodies obtained that could be shown to be hybrids and in only two instances was any fertility between homokaryotic mycelia observed. At the outset, of course, there is the old problem of the definition of species in any particular group of organisms and the two observations reported here are considered in the light of that problem. (a) Cyathus olla x Cyathus olla forma anglicus. In 1952 it was reported (Brodie, 195 za) that when certain homokaryotic mycelia of the typical form of Cyathus olla were paired with homokaryons of a large and distinctive form of the same species known as

C. olla forma anglicus, heterokaryotic mycelia resulted. Analysis of the results of such pairings showed that the mycelia of the two forms possess the A and a pair of sexuality genes in common and that they differ in the other pair of sexuality genes. A few mycelia of the 'species X form' pairings produced fruit bodies unlike those of either parental strain (figure 13). The hybrid fruit bodies failed to open normally and none produced spores. In the assessment of these results, Brodie took the position that 'the two fungi are forms of one species' and not two separate species as had been assumed by some specialists. However, among flowering plants such a limited and irregular Fi, with little or no possible introgres-

42 / The bird's nest fungi

sion, would be taken as indicative of specific distinctness (effective genetic isolation), for without effective backcrossing the gene pools remain effectively separate. On this basis, it may be that anglicus should be recognized as a species. (b) Cyathus helenae x Cyathus striatus. Perhaps inconsistently, a different position was taken in interpreting the results of matings between haploid mycelia of Cyathus helenae andC. striatus (OlchoweckiandBrodie, 1968). Here, diploid mycelia were developed in certain matings between mycelia of the two species, but no fruiting of any sort was observed. In the absence of any fruit bodies resulting from pairings of C. helenae and C. striatus, the presence or absence of any internal isolating mechanism between the species remains mere speculation. However, the two species are very distinct as to habitat, and C.

helenae, although doubtless closely related to C. striatus, is a well-defined fungus - so much so that it has been collected and correctly identified by amateur naturalists. Other species pairs could be cited for which the morphological (and presumably taxonomic) relationship is just as close as in the examples just considered: Cyathus poeppigii seems to be the tropical counterpart of C. striatus of temperate regions; Cyathus pygmaeus is a closely related small version of C. olla; Crucibulum parvulum is certainly closely related to C. laeve. Yet for all these pairs and many more, attempts at hybridization have yielded only negative results. Especially in the tropics, many closely related species may grow in close proximity and one must conclude that strong genetic and other barriers maintain species isolation and identity.

43 / Characteristics and interactions of homokaryotic mycelia

5 / Heterokaryotic Mycelium and Fruit Body Formation

A / C H A R A C T E R I S T I C S OF HETEROKARYOTIC MYCELIUM Anastomosis between two monosporous homokaryotic mycelia of sexually compatible strains leads to the formation of the heterokaryotic (dikaryotic) phase of the Nidulariaceae. To the unaided eye, heterokaryotic mycelium of many species is readily distinguishable from homokaryotic in being fluffier, coarser, and more regular (figures 11, 14). In some species (e.g., Cyathus stercoreus) the heterokaryon may be more heavily pigmented than either parental homokaryon. One of the most striking examples of gross morphological difference is seen in the mycelia of C. olla: homokaryotic mycelium of this species is snow-white and composed of fine hyphae, and the edge of the colony appears regular; heterokaryotic mycelium is not so white, is composed of coarse hyphae which tend to form fascicles or visible cords, and the edge of the colony is uneven, some clusters of hyphae projecting beyond the main mass. With few exceptions, the growth rate of heterokaryotic mycelium is almost double that of the homokaryons, and this character alone often makes identification of the former a simple matter. In some species, such as C. stercoreus, there is great variation in colour among homokaryotic mycelia (all grown on the same nutrient 44 / The bird's nest fungi

medium), which may be white, light buff, or some shade of very deep brown. This variation results in differences among the heterokaryotic mycelia arising from differently coloured homokaryons. Ignoring the mating types involved, the following has been observed: HOMOKARYONS

HETEROKARYONS

PAIRED

PRODUCED

White x White White x Deep Brown Deep Brown x Deep Brown

Light to Medium Buff Medium Buff Intense Brown of some shade

From examination of many such pairings it seemed possible that the gene or genes for brown are dominant and able to affect the pigmentation of the resultant heterokaryotic mycelium (figure 11). Some years ago, the author attempted to investigate this display of colour and to determine the mechanism of its inheritance. The project was finally abandoned, first because (at the time) regular production of fruit bodies in culture had not been achieved and, second, because no combination of two pure white haploids was ever observed to yield a diploid as white as either parent ; in fact, most white X white crosses yielded buffcoloured diploids. Probably the brown pigmentation in C. stercoreus is under the control of several genes acting together. More will be recorded later regarding colour in this fungus

FIGURE 14 Structural characteristics of homokaryotic and dikaryotic mycelium of Cyathus stercoreus : a, homokaryotic hyphae, b, dikaryotic hyphae, a, b. X100 ; c, homokaryotic hypha and d, dikaryotic hypha, c, d X5oo; e, dikaryotic hypha with granules on surface, x/oo; f, irregular form of certain monokaryon hyphae, x 200 ; g, dikaryon hyphae forming mycelial cords, X4 ; h, dikaryon hyphae with rudiments of fruit bodies, X4« Figure courtesy Am. J. Bot., 1948,35: 314 45 / Heterokaryotic mycelium and fruit body formation

FIGURE 15 Effect of calcium on production of fruit bodies of Cyathus stercoreus, Xo.5: central flask, no calcium; left-hand flask, calcium chloride; right-hand flask, calcium nitrate. Photograph courtesy Mr S-h. Lu

when sporophores produced by the heterokaryotic my celia are described. As seen under the light microscope, heterokaryotic mycelium consists of coarser hyphae than the homokaryotic (6-10 ju, according to species). HeterokaTyotic hyphae are generally straight, are provided at regular intervals with clamp connections, and branch rather more sparsely than do homokaryotic mycelia with the branch hypha diverging at a smaller angle from the main hypha (figure 14). Nuclei of heterokaryotic mycelia are regularly disposed in pairs or dikaryons, one dikaryon lying in each cell (figure 33). No heterokaryotic mycelium of any bird's nest fungus has ever been reported to produce oidia under any conditions of growth. Little information can be given regarding the physiology of heterokaryotic as opposed to homokaryotic mycelia which has not already

46 / The bird's nest fungi

been stated in the previous chapter. The heterokaryotic mycelium must have some marked differences biochemically from the homokaryotic since it is faster growing and possesses the potentiality for sporophore production, which the homokaryon lacks completely. A detailed comparison of homokaryons and heterokaryons, therefore, should prove interesting, but this is but one of many studies that still remain unaccomplished. B / F O R M A T I O N OF S P O R O C A R P S There would be little to gain by reviewing the various reports in older literature of the more or less fortuitous fruiting of Nidulariaceae in the laboratory. Some of the more important of these reports are included in the bibliography of this book, but no attempt is made to assess their value because the nutrients and substrate

on which fruiting occasionally occurred nearly always involved materials which were chemically undefined and could not be duplicated precisely. They included, for example: carrot slices, prune juice agar, dung agar, malt agar, green pea agar, corn stalks, and other materials, only a few of which seem even to approach the chemical composition or physical texture of the substrates on which bird's nest fungi are usually found fruiting in nature. In passing, it may be noted that I have attempted (often in desperation) to fruit bird's nest fungi using the old recipes, and for the most part such efforts have met with outstanding lack of success. Before considering what is known at present of the conditions that do affect fruiting, it should be said that those few species that have been induced to fruit in the laboratory do so readily and on a variety of nutrient media; those that show no tendency to fruit have not been induced to do so under a variety of conditions which would take many pages to report in detail. Neither Garnett (1958) nor Miles (1953) obtained any conclusive evidence that any particular chemical substance present in the substratum induced fruiting of Cyathus stercoreus. Garnett made a study of the possible effects of the accumulation of 'staling products' in the nutrient medium and concluded that: Vegetative growth and fruit body formation can occur on medium which has supported vegetative growth up to the formation of mycelial knots (25 days). No evidence was found of the presence or absence of substances in such media which either stimulate or inhibit fruiting/ Shih-hsiung Lu (1973), working in my laboratory, observed that the element calcium appears to play some role in the normal development of fruit bodies. Lack of calcium does not appear to prevent fruiting, but when calcium is absent fruit bodies do not develop normally (figure 15). This study was a preliminary one and, until it has been extended, no categorical statement can be made about the possible role of calcium in fruit body development.

C / C O N D I T I O N S K N O W N TO AFFECT FRUITING The most important factor thus far clearly demonstrated to affect fruit body formation in the Nidulariaceae is light. Following the leads suggested by many casual observations made over a period of several years, Garnett (1958) was the first to demonstrate experimentally that it is necessary to expose heterokaryotic mycelium of Cyathus stercoreus to light for fruiting to occur. Not even the hyphal knots, which are the fruit body initials (see below), are formed in total darkness. Garnett reported that the light most effective in inducing fruiting lay in a wavelength band below 530 m/¿ and that wave lengths greater than 600 m/¿ were not effective. She observed also that continuous light is not necessary for fruit body development. Provided the mycelium has reached a certain degree of maturity in the dark, a short exposure to light stimulates fructification, which will then continue even if cultures are subsequently maintained in the dark. The role of light in fruiting in C. stercoreus was placed on a more quantitative basis by B. C. Lu (1965). Lu's conclusions may be stated briefly as follows: (i) light is a strict necessity for fruiting; (2) the amount of light energy required for fruiting to take place is a constant, approximately 17,200 foot-candle hours at 25° c; (3) Lu Abstract: 'It is hypothesized that photoinduction becomes operative when a hypothetical "photoreceptive precursor" develops in the mycelium. The development of such a precursor is believed to occur when conditions unfavourable for good vegetative growth (e.g. shortage of food supply) develop in the culture. Internal metabolic pathways then shift to favour the development of the photoreceptive precursor/ Lu observed also that morphological variations in fruit bodies are associated with different light treatments. The higher the light intensity, the smaller were the sporophores that developed; and it

47 / Heterokaryotic mycelium and fruit body formation

FIGURE 16 Fruiting of Cyathus olla on soil in greenhouse pots, xi.j

was also noticed that pendióles were nearly fifty per cent smaller in the small sporocarps than in the larger ones. Light is not only a necessity for fruiting and a factor which affects the manner of development of sporocarps as noted above ; it also affects the position which mature sporocarps ultimately assume, i.e., sporocarps are positively phototropic. This matter will be discussed more fully later (Chapter 5, F). Other factors affecting fruiting have been less fully studied. The time required depends upon temperature, upon the species involved, and even upon the particular strain of a species, and the only generality that can be stated is that most species that do fruit in laboratory culture do so best at about 25° c, in from 18 to 40 days. Species and strains of species have been found to differ markedly in the tendency they have exhibited to fruit in laboratory culture. A list of the species which have fruited in my laboratory is given herewith. Some others, notably Crucibulum laeve, Cyathus striatus, Nidularia spp. have been reported in publications to fruit but, as noted elsewhere, without regularity and under conditions and on substrates which are difficult to duplicate precisely. 48 / The bird's nest fungi

Cyathus stercoreus. Some strains fruited readily on a variety of solid or liquid media (Brodie, 1948^ Garnett, 1958; Lu, 1965). Cyathus bulleri. Fruited readily on solid Brodie Medium (Brodie, 1967^. Cyathus olla. Fruited on solid Brodie Medium, but better when mycelium transferred to soil in pots (Brodie, unpubl.). Cyathus pallidus. Fruited on solid Brodie Medium, but not regularly or abundantly; better when transferred to soil in pots (Brodie, 19683). Cyathus berkeleyanus. Abortive sporocarps on agar. Normal fruiting when transferred to soil. Cyathus poeppigii and Cyathus limbatus. Abortive sporocarps on agar. Normal fruiting on soil.

Concerning the remarks in the list above, it has been found that some species (e.g., C. olla and C. pallidus) fruit weakly and often abortively on agar media and certain cultures of these species fail to fruit. However, if cultures that have just overgrown culture plates are transferred to sterilized loamy soil, they may fruit abundantly and 'normally' (figures 16,17,18, 19). Four- or six-inch pots of soil are sterilized in steam and, when the soil is cool, cultures consisting of mature mycelium and the nutrient agar upon which the mycelium was grown are chopped into small pieces under

FIGURE 17 Fruiting of Cyathus pallidus on soil in greenhouse pots, X 2 . 5

FIGURE 18 Formation of fruit bodies of Cyathus olla attached to wooden pot label, X-L.J

aseptic conditions, mixed thoroughly with the top inch of soil in pots, and allowed to stand on the greenhouse bench. If the soil is kept moist with sterile distilled water, an abundant development of normal fruit bodies has frequently been observed to take place in as short a time as one week. The rapidity with which such mycelia produce sporocarps from agar cultures in which fruiting had not previously taken place, or only abortively, suggests that the response is not likely a matter of nutrition but is rather the result of the proper physical environment being provided. Whatever the explanation, the 'trick' of planting in soil has resulted in successful sporophore production when other efforts failed. One further observation in connection with fruiting is worthy of note; indeed it may suggest many lines of important investigation for 'Nidulariologists' new to the game! From the available evidence one is easily persuaded to

49 / Heterokaryotic mycelium and fruit body formation

FIGURE 19 Formation of fruit bodies of Cyatlnus bullen : a, on nutrient agar, X i ; b, on mycelium transferred to sterile soil in pots, Xo.25 50 / The bird's nest fungi

FIGURE 20 Formation of fruit bodies of Cyathus stercoreus in contact with roughened glass rods standing in liquid nutrient medium, Xi

believe that the physical structure of the substrate and the physical nature of the environment are stronger determiners of fruiting of the Nidulariaceae than is the chemical nature of the environment. Two pieces of evidence can be offered in this regard. First, it has been observed repeatedly that when the mycelium of Cyathus olla is growing in garden soil rich in organic matter (as this species regularly does) there is a strong tendency for sporocarps to be developed on the surface of various substrates away from the soil, on old dead stems of plants, for example, or on old boards. Similar be-

haviour has been noted when mycelium of this species is chopped up and mixed with soil: the sporocarps, very commonly, develop not on the surface of the soil but on the wooden label used to identify the experiment or on the soil immediately adjacent to the label (figure 18). Possibly more significant is the observation, involving some forty flasks, that when Cyathus stercoreus fruited in flasks of liquid Brodie Medium, there was a significantly greater number of sporocarps formed on or close to sintered glass rods standing in the liquid (figure 20) than elsewhere in the flasks. Because, over the brief duration of this experiment (S.h. Lu, 1972, unpublished), it is unlikely that nutriment could have been derived by the fungus from a clean sterile glass rod, one is forced to consider seriously the possibility that some physical attribute of the rough glass rods in contact with the mycelium stimulated the formation of sporocarps. It is difficult to imagine what role the nature of a surface, as such, may have as a stimulus for sporocarp production, but an investigation would appear to be inviting and challenging. For reasons that are completely unknown at present, it appears that the formation of fruit bodies under laboratory conditions occurs rather more readily in the morphologically simpler genera Nidularia and Mycocalia than in the more structurally complex genera Crucibulum and Cyathus. Many mycologists have reported that apparently normal fruit bodies of Nidularia pulvinata can be obtained if the heterokaryotic mycelium of this fungus is allowed to grow for from 4 to 6 weeks, in the light, upon pieces of sterilized corn stem (Zea mays] ; and I have fruited this species several times in this manner although I have never induced it to fruit on synthetic media. Species of Mycocalia are apparently more 'obliging/ for Cejp and Palmer (1963) have reported the development in culture of normal sporocarps of several species. Pendióles that had been subjected to surface sterilization were allowed to grown on 2 per cent maltose agar at

51 / Heterokaryotic mycelium and fruit body formation

25° c. Cejp and Palmer reported that 'typical' fruit bodies were formed for the species M. denudata andM. duriaeana. M. minutissima also fruited, but the fruit bodies were 'not perfectly formed/ The uniperidiolar species M. sphagneti also fruited when mycelium was grown on 2 per cent maltose agar. The species of Mycocalia fruit, in nature, in very wet locations and commonly upon dead stems of Juncus and grasses. What may be different chemically or physically in the natural environment of these fungi from that of species of Cyathus is unknown. D / E A R L Y S I G N S OF F R U I T I N G The first indication that the assiduous bird's nest fungus gardener may expect a harvest of fruit bodies to reward his patience and labour is a rather abrupt change in the appearance of the heterokaryotic mycelium. This is likely to occur from three to four weeks after the transfer of the mycelium to culture tubes or plates. If the young mycelium is already pigmented, as it is in the majority of Cyathus stercoreus heterokaryons, an abrupt intensification of the pigment may be observed unevenly over the mycelium. Even in species such as C. olla, in which the mycelium is whitish or only lightly pigmented, brownish blotches may appear. Almost at the same time as the pigment deepens, or even earlier, conspicuous mycelial cords appear among the aerial hyphae. These are best seen in Cyathus stercoreus (figures 14, 21) in which they are often pigmented and appear very different from the mass of mycelium. In C. olla they are also conspicuous but less pigmented, and in Crucibulum laeve they are frequently yellowish. These mycelial cords are formed of a rather loose aggregation of stout hyphae that lie close to one another and may even be twined about one another to some extent. The cords do not appear to have the morphological differentiation seen in socalled rhizomorphs such as those oiArmillaria mellea : indeed the hyphae do not even appear to be cemented in any way to one another and 52 / The bird's nest fungi

there is no outer bounding layer of modified hyphae. These mycelial cords may also be found in the natural environment. In Cyathus olla they can often be pulled up as long strong cords from the surface of an old rotting board and in Crucibulum laeve they are conspicuous on the substrate as prominent yellow cords (figure 21). Shortly after their first appearance, the mycelial cords come to bear numerous small mycelial knots that are about 0.25 mm in diameter when first noticed. These knots are the fundaments of the fruit bodies. When examined under the microscope, the knots at first have little regular organization, consisting merely of a mass of closely interwoven clampbearing hyphae having slightly swollen cells. Not every mycelial cord bears hyphal knots, at least not at first, nor does every knot continue to develop into a fruit body. The failure of some knots to become sporocarps on a laboratory medium may well be a matter of the availability of nutriment. Even in the natural environment one can find, among an abundance of fruiting bodies, a few hyphal knots which appear never to have developed further. Hyphal knots have never been seen to form at the very distal end of a hyphal cord; they arise back from the distal end at a distance of 2 cm or even more. E / O P E N I N G OF M A T U R E SPOROCARPS Because consideration of sporocarp development involves much detail finer than can be seen with the unaided eye, the internal morphogenetic aspects of fruit body maturation will be dealt with later in this chapter (5,1). At this point, the externally visible changes in fruit bodies will receive most attention. Some mycelial knots proceed to develop rapidly into recognizable fruit bodies in a matter of a few days under proper conditions. Young fruiting bodies are globose at first (figure 22), then somewhat pear-shaped and, as they approach maturity, they take on an

FIGURE 21 Mycelial cords that precede fruit body formation: a, Crucibulum laeve as seen in nature Xv b Cyathus stercoreus in flask culture, X2 '

53 / Heterokaryotic mycélium and fruit body formation

FIGURE 22 Young (a) and mature (b) fruit bodies of Cyathus olla developing on soil in greenhouse pots, X3. The furry appearance of young fruit bodies is well illustrated in a ; note well developed emplacement m upper left.

obconic form and begin to display, to the unaided eye, some of the characteristics of a particular genus and species. Eventually a number of fruit bodies have reached their full size. In some species, the shape is then strongly obconic and each sporocarp is expanded at the top (figure 23). The hyphal tufts, hairs, setae, and other components of the outer coat are still quite closely packed and the sporocarp as a whole appears very furry (figure 22). Further rapid expansion of the top region of the sporocarp, in the genus 54 / The bird's nest fungi

Cyathus, reveals the covering membrane - the epiphragm - which in some species is of uniform texture and white, yellowish, or tawny in colour, or, in other species, may be adorned with coloured tufts of hyphae. Actual rupture of the epiphragm, disclosing the peridioles lying within, is the result of a process of gelatinization of hyphae within the top part of the sporocarp. Such hyphae are the residue of internal changes leading to the formation of peridioles and attendant structures. The epiphragm can be seen to be curved out-

FIGURE 23 Cyathus stercoreus, newly opened fruit bodies: a, note epiphragm at rieht x , • h ,pKUcuSemp1,«mem,.OWeHe[,x3ic,,s,dua,8n,,enm88e,0ej:dr^;*^ 55 / Heterokaryotic mycélium and fruit body formation

,

FIGURE 24 Maturation of fruit bodies of Cyathus stercoreus showing pressure developed below epiphragm, X3¡ a, pressure distending epiphragm and drops being extruded; b, epiphragm rupturing naturally; c, epiphragm punctured by needle and liquid being released; d, young woolly fruit bodies

56 / The bird's nest fungi

ward at this time (figure 24) as a result of the pressure of liquid within the fungus cup. That an internal pressure exists can easily be demonstrated by piercing the taut epiphragm with a needle ; when this is done, a drop of transparent liquid (colourless or sometimes faintly yellow) will be seen to form above the rupture (figure 24) and to increase rapidly to a considerable size. Torn by internal pressure, the epiphragm withdraws from its centre towards the edge of the cup (figure 24) and finally only a few remnants of this membrane are seen around the internal rim of the fully open peridium. At first, the exposed pendióles lie in the remains of the clear liquid (figure 23), but the latter soon dries and the little splash gun is then ready to eject its first pendióles through the action of raindrops, as described in Chapter i. F / O R I E N T A T I O N OF S P O R O C A R P S Although no extensive experimental work has been published concerning the geotropism of young fruit bodies, many observations seem to indicate that they do respond to gravity as they develop. On small twigs that are free of the ground or obstacles, fruit bodies develop only on the upper side: I have never seen them develop otherwise, and in this example differences in light intensity can hardly be imagined to be a determining factor. In laboratory culture, if large numbers of agar slants bearing mycelium are allowed to fruit when the surfaces of the slants are randomly oriented, fruit bodies bend around (as much as the confinement of the tube allows) so that the fungus cups ultimately face upward. On one occasion, some young fruit bodies of Cyathus stercoreus growing on one side of a mass of horse manure were brought into the laboratory and inadvertently placed upside down in a moist chamber. The following morning it was observed that several sporocarps had made some effort to re-orientate themselves to an upright position (figure 25). At what period of their development and for how long fruit

FIGURE 25 Evidence for weak geotropic response in fruit bodies of Cyathus stercoreus : a specimen comprising two fully mature fruit bodies and one immature was placed in a moist chamber with the two mature fruit bodies in an inclined position ; the immature fruit body in maturing assumed an upright position. X4

bodies do respond geotropically are questions that remain to be answered. The phototropism of young fruiting bodies is more obvious. Always when cultures in the laboratory are unilaterally illuminated, they are seen to be bent strongly towards the source of the light (figure 26). Moreover, if the position of agar plates is subsequently reversed with regard to the light source, again it is seen that newly formed sporocarps become inclined towards the light. If the culture plate is repeatedly reversed in position, it eventually becomes obvious that no further change in position of mature sporocarps can take place. Probably by that time, growth in the lower part of the stalk has ceased. As is true for photoinduction, phototropic response is brought about mainly by light at the blue end of the spectrum.

57 / Heterokaryotic mycelium and fruit body formation

FIGURE 26 Phototropism in Cyathus stercoreus : fruit bodies developing on nutrient agar under unidirectional illumination are inclined towards the light source, X2. Photograph reproduced courtesy Dr Muriel Stringer

G / IRREGULAR FRUITING Some irregularities in sporocarp formation have been noted both in specimens collected from the natural environment and in laboratory-produced specimens. One of these has been described as twinning (Brodie, 1958). Naturally occurring examples of twinning have been described for Crucibulum laeve, Nidula niveo-tomentosa, Cyathus striatus, and C. stercoreus. Twinning is the formation of a double carpophore, composed of two contiguous carpophores with one part of the wall common to both cups, as seen in the specimen of Crucibulum laeve illustrated in figure 27. In other forms of twinning, the two carpophores are attached as one unit mainly at their bases (figure 27). The cause of the twinning abnormality is unknown. It may be supposed that some accident to the fruit body occurring when it was quite young or even earlier, in the primordium stage, could result in equal development from two points. In an effort to test this idea, young 58 / The bird's nest fungi

fruit bodies at various stages of development were cut part way through vertically with a sterilized razor. In other experiments one half of a young fruit body was excised and, in still others, fruit bodies were mutilated with a hot needle. Unfortunately these experiments failed ; even when young fruit bodies were so small that they could barely be handled, in all instances those subjected to microsurgery did not continue their development. Probably hyphae are too physiologically active and sensitive at this stage to survive even being broken. However, an interesting piece of research may still be possible on this problem. In some species, new fruit bodies can arise from within the remains of old. The process of regeneration, as it has been called (Brodie, 1958), has been observed in Crucibulum laeve, Nidula spp., and some species of Cyathus. A pad of mycelium at the base of the inside of the old fruit body apparently remains alive and, when conditions for renewed growth exist, a new fruit body is produced, at first completely encased within the old and eventually encased

FIGURE 27 Twinning and regeneration of fruit bodies : a, Nidula candida, on left a new fruit body developing from within an old one ; on right a partially twinned fruit body, X 4 ; b, Cyathus stercoreus, basally twinned fruit bodies, X3 ; c, Crucibulum laeve, twinned fruit bodies within each of which is developing a new fruit body, X5 ; d, Cyathus poeppigii, new fruits developed from within old ones, x$ ; e, C. poeppigii, basally twinned fruit bodies, X } . Photographs reproduced courtesy Svensk Bot. Tidskr., 1958,52: 378 (Pi. i, n) 59 / Heterokaryotic mycelium and fruit body formation

FIGURE 28 Aberrant fruit bodies of a (mutant?) strain of Cyathus poeppigii: a, aberrant strain (top) producing small uniperidiolar fruits while normal strain (bottom) is producing normal fruits at lower right x ! ; b, transverse section of abnormal fruit body containing a single sterile peridiole, x 20 • c abnormal fruit bodies (right) whole, with outer covering partly removed (left) and with globoid peridiole exposed (centre), x6. Photographs reproduced courtesy Am. J. Bot., 1955, 42: 170, 172, 175 60 / The bird's nest fungi

ê * FIGURE 29 Extreme variation in size, form, and colour of different collections of Cyathus stercoreus, Xi. The variation is partly of genetic origin and partly the result of environmental influence. Such variability emphasizes the need for caution in defining species.

Mutant fruit bodies (figure 28) were spherical and each contained a single peridiole devoid of funiculus and tunica. Spores produced by the aberrant fruit bodies failed to germinate and the possible course of inheritance could not be studied. In one experiment, mutant dikaryon mycelium was grown so as to come into contact with normal dikaryon in the hope that, if nuclei from the mutant migrated into 'normal' mycelium, aberrant fruit bodies might arise on the normal side. This did not occur; each dikaryon, when it fruited, produced its own type of fruit body (figure 28). Brodie (19553) speculated that the simple single-peridiole aberrant fruits might be related to some structurally simple little-known Gasteromycetes such as Protogaster. Perhaps of more value is the observation that aberrant fruit bodies of essentially the same morphology occur in two species that are closely related taxonomically, which suggests that the two taxa are sufficiently close genetically that the same kind of change (mutation?) was able to arise in mycelium of each species (see also Chapter 10, D and Chapter 12, c). H / V A R I A T I O N IN F O R M OF SPOROCARPS

mainly at the base (figure 27). New fruit bodies are known to be capable of being produced in this manner when the old sporocarp is as much as a year old. Perhaps the most striking irregularity in sporocarp morphology is the appearance of highly aberrant fruit bodies, which has been reported (Brodie, 19553) to occur in the closely related tropical species Cyathus poeppigii and Cyathus limbatus. In the former species, a peculiar sector of dikaryon mycelium in a nutrient agar plate was noticed and was transferred to fresh agar for further study. The sector mycelium (called 'mutant' for convenience although no genetic evidence of mutation was obtained) proceeded to fruit in due course and the fruit bodies developed were wholly unlike those produced by the parental mycelium.

Aside from the above deviations from the predominant form of carpophore, several species of the Nidulariaceae produce many different types of fruit bodies, frequently found in a very restricted area which precludes the possibility of considering them as ecotypes. This situation has been most fully studied in the coprophilous species Cyathus stercoreus. At times, collecting in one 'patch' of this species has yielded five or six (and in some instances more) morphologically distinct variants (figure 29). Individual fruit bodies varied greatly as to size, colour, and form, to the extent that many taxonomists would describe the several forms as species. This has, in fact, been done: as reported in Chapter 14, many names have been applied to the naturally occurring forms of Cyathus stercoreus, yet all of these possess

61 / Heterokaryotic mycelium and fruit body formation

Original wild-type fruit body

No. N3. mm I

Tissue culture No. N3.mm

Basidiospores from one peridiole SERIES OF HAPLOID MYCELIA

SEX GROUP AB

S w CQ

W PÍ P

ta u

1.8.9.10.11.12 15,16,19,20,23,28 285,30,31,32,35 36,40,42,45,46 48,49

SEX GROUP ab

SEX GROUP Ab

SEX GROUP aB

2,3,4,5,6, 7,14,17,17s 18 21,22,24,25,27^ 29,37,38,44,54,55

13, 39, 41 ' 41B, I 52

DIPLOID

13.40

33,

53, 56

MYCELIA

33.18

53.29 33.29

33.6 41B.285

53.2

FIGURE 30 Variation in fruit bodies of Cyathus stercoreus produced from dikaryon mycelia that resulted from unions of different homokaryon mycelia. Genetic recombination is the cause of such variation in fruit body form in this fungus. Reproduced by courtesy of Mycologia, 1948, 40: 622 62 / The bird's nest fungi

TABLE 3

Comparison of variant cultures of Cyathus Stercoreus and resultant sporocarps Fruit body type of mycelium planted in autumn 1949

Note on fruiting of cultures in garden, in late summer 1950

White, broad form

Six fresh sporocarps obtained, all white and broad

Narrow form with reddish cup

No sporocarps obtained

Small dark form

Two sporocarps obtained, larger than original

Wild-type parent of all above

Fourteen sporocarps obtained, like original

certain features in common, viz. smooth black pendióles, very large spores (30 ¡JL or more), and peridia which are hairy externally and non-plicate. That the variants of C. stercoreus found in nature are of genetic origin and represent the fruiting of recombinant genotypes was demonstrated by Brodie (i948b) who was able to achieve the fruiting of different heterokaryotic mycelia, each derived from the union of a different pair of compatible homokaryons (figure 30). It is recognized that, in addition, some variation in fruit body form may be caused by the action of light, as noted earlier in this chapter. Similar variation is observable in Cyathus striatus, C. olla, and a number of other species, but for these there has been no laboratory study to determine the cause of the variation. One experiment in this connection was performed in 1949 to determine whether or not mycelium of some of the most striking fruit body types of C. stercoreus obtained in the laboratory would be able to persist out of doors. For this purpose, the following strains were selected and planted in the autumn in a garden where C. stercoreus had been known to grow. Cultures of mycelium were 'planted' in

the garden by being covered with a sterilized mixture of equal parts of horse manure and soil. In Table 3 culture numbers (meaningless to the reader) are omitted. Wooden pot labels marked each planting site. In addition to the parental wild type, two other cultures persisted and fruited almost a year after planting. The new crop of the remarkable white strain consisted of fruit bodies closely resembling those obtained in the laboratory, whereas those of the small dark strain fruited poorly out of doors and were sufficiently unlike the original laboratory material to make it possible that they were wild types. The narrow red-cap form did not persist. Moreover, although the planted area was left undisturbed for another year, no more fruit bodies appeared. One can conclude that some morphologically distinct genotypes are sufficiently like the parental wild type physiologically to be able to persist in the natural environment. Another feature of the development of sporocarps worthy of note is the variation in the number of peridioles matured in each and in the degree to which the funiculus is developed. In Cyathus stercoreus large fruit bodies may contain up to twenty peridioles, while small ones may contain only three or four. Also, fairly frequently in this species, while the peridioles in the top half of the sporocarp may all possess fully developed funiculi, those of the lower half may bear abortive funiculi or none at all. A similar situation has been observed in an occasional sporocarp of C. poeppigii, C. Hmbatus, C. setosus, and a few others. I / D E T A I L S OF S P O R O C A R P DEVELOPMENT Details of the development of the fruiting bodies of Cyathus striatus were given long ago by Tulasne and Tulasne (1844). However, the most complete account of the developmental morphology of any of the Nidulariaceae is certainly that given by Leva Walker (1920), who

63 / Heterokaryotic mycelium and fruit body formation

glebal region

outer layer of peridium

peridiole primordium showing converging of hyphae

glebal region

peridiole primordium

undifferentiated region

FIGURE 31 Developmental morphology in Nidulariaceae (generalized without reference to particular species) : a, young fruit body with glebal region beginning to differentiate ; b, later stage showing pendióles beginning to form ; c, converging hyphae from which peridiole primordium was formed; d, nearly mature peridiole in which all main regions are well developed ; e, peridiole showing maturing funiculus and sheath ; f, cross section of mature fruit body; c, Xzoo;a, b, e, X3o;d, X35;í, X3O 64 / The bird's nest fungi

tunica

cortex

hymenial layer funiculus

parallel hyphae which produce funiculus future sheath

studied the three species C. striatus, C. olla, and Crucibulum laeve, examining sporocarps at all stages of development as they became available from cultures fruiting in the laboratory. The following account is based upon the observations of Walker for Cyathus olla and Tulasne for C. striatus. It may be taken as a sort of standard with which information concerning other genera may then be compared. As stated above (Chapter 5, D), the first appearance of fruit bodies on the mycelium is

in the form of small round or ovoid knots. At first they are white, but after three or four days they turn greyish or brown according to the species. The outer surface of the young sporocarp becomes woolly or rough, particularly in the apical region. Among common species, the woolly character of the young fruit body is least pronounced in C. olla and most pronounced in C. stercoreus. In the latter, the fruit bodies at this stage are quite cactus-like, with their covering of long tufted hairs (figures 22, 24). Internally, they are homogeneous,

65 / Heterokaryotic mycelium and fruit body formation

except that the hyphae of the apical region are more loosely arranged than those towards the base. i / The peridium Externally, the narrowed base of the young fruit body begins to show a brownish colouration indicating the formation of the two outermost layers of the peridium or wall of the fungus cup. The outer layer of the peridium develops from the loosely interwoven brownish hyphae on the outside of the fruit body. These hyphae undergo little modification as development proceeds. In section, it is possible to distinguish a gelatinization of hyphae in a zone extending in an arc from just below the less dense apical region of the fruit body (figure 3ia) downward to the base. This gelatinization results in a demarkation of a central glebal mass. The innermost layer of the peridium is made up of densely interwoven brown filaments, and at first it is quite thick. Between the outer and inner layers just described, there develops a third or middle layer which is colourless and is composed of loosely interwoven hyphae having rounded pseudoparenchymatous cells. Continued enlargement of the glebal region (figure 3ib), results in compression of the innermost layer until, in mature specimens, it is no thicker than the middle layer. Meanwhile, in the middle layer, absorption of the gelatinous matrix takes place from the top of the fruit body towards the base, a process which tends to bring the outer and innermost layers of the peridium together as the fruit body matures. Finally, in the basal or stipe region (figure 3ib), the disappearance of the gelatinous material does not bring about adhesion of the outer and innermost layers and so the stipe region becomes hollow (in Cyathus striatus). 2 I Development of pendióles The glebal region is differentiated from the 66 / The bird's nest fungi

peridium at an early stage by gelatinization of a zone of hyphae which become part of the inner wall of the peridium (figure 3ia). The gleba is at first composed of colourless, closely interwoven hyphae. As the pendióles develop, the hyphae forming the ground tissue between them undergo gelatinization, and, when the entire fruit body is mature, the peridioles lie in a homogeneous colourless mucilage resulting from the gradual breakdown of ground tissue. At the start of peridiole formation, there appear in the peripheral portions of the gleba points towards which hyphae converge, at first from the peridium inward towards the centre of the gleba, but finally from all sides (figure 3ic). Each of these masses of converging hyphae (the peridiole primordia) is surrounded by a zone of loosely interwoven hyphae with many intercellular spaces, which, in turn, is bounded externally by a slightly denser tissue. On the underside of each of the peridiole primordia, the zones described are interrupted by parallel hyphae in a line between the peridiole and the peridium. In this way are developed the primordia of the peridioles themselves and of the funiculi or cords by which they later become attached to the peridium. As Tulasne pointed out, the funiculus is actually not organized until late in the development of the fruit body. The three layers that bound the mature peridioles can be recognized before the funiculus has developed much beyond its primordial condition. For this reason, consideration of the developmental morphology of the funiculus will be deferred, for the moment. As both Tulasne and Walker observed, in Cyathus striatus, the first peridiole to be differentiated is towards the top of the fruit body and others follow in succession from the top to the bottom. In C. olla, Walker found the reverse order to hold, i.e., the peridioles develop successively from the base of the fruit body upward. In Crucibulum laeve they develop simultaneously in all parts of the cup. The peridiole primordia, as described above, then undergo the following changes. Each cir-

cíe of converging hyphae enlarges as a result of the formation of new hyphae (presumably by branching of those already present). This multiplication of hyphae occurs largely at the sides. A space filled with mucilage produced by gelatinization of hyphae of the ground tissue develops in the centre of the mass of converging hyphae. The cavity becomes oval in section (figure 3 id) as a result of the earlier unequal thickening of the walls of the pendióles, the upper and lower parts increasing in thickness and the sides remaining thinner. Furthermore, growth remains more active on the margins and the addition of new hyphae there results in the production of a flattened spheroidal body. At first the converging hyphae that line the central cavity of the peridiole are of equal diameter throughout. By the time the cavity has reached one-half its ultimate size, it is lined by a definite palisade of hyphae with enlarged ends (figure 31 d, e). Tulasne noticed that this palisade does not present an even surface but is composed of basidia and paraphyses of varying length, an observation confirmed by Walker. According to Walker's account, the hyphae forming this palisade are all binucleate when young. The paired nuclei then fuse and the young basidia are therefore uninucleate. When the peridiole is mature, the cavity is filled with binucleate basidiospores. Further detail concerning the cytology and mode of spore formation will be given in Chapter 6. The walls of the pendióles develop slowly. At an early stage of differentiation, the subhymenial tissue is characterized by large intercellular spaces and, external to this, is a denser layer which eventually develops into the thick inner wall of the peridiole (figure 3id, f). Gelatinization of ground tissue surrounding each peridiole now takes place. According to Walker's account, those hyphae nearest the denser filaments of the peridiole wall become gelatinized only slightly and, as the peridiole matures, remain to form the thin colourless layer covering the entire peridiole. This layer is called the tunica (figure 3id). Tulasne seems to suggest that the tunica layer is formed as an

extension of the upper or sheath part of the funiculus. The firm wall of the peridiole within the delicate tunica undergoes modification until two distinct layers are apparent at maturity: (1) the outer layer, or cortex, which is dense, opaque, brown or black, of equal thickness all around the peridiole and composed of short pseudoparenchymatous hyphae (figure 3id); (2) a greyish-white sclerotic layer of horny texture consisting of compactly interwoven hyphae, the walls of some of which are somewhat gelatinized. The hyphae of this layer are more loosely interwoven towards the hymenium, and the layer is not of equal thickness throughout, being thickest on the upper and lower surface and thinnest towards the rim of the peridiole. At maturity, the centre of the peridiole is occupied by a more or less homogeneous mass of spores that have long since been freed from their basidia and are imbedded in a gelatinous matrix. Martin (1927) showed that in Cyathus stercoreus the hyphae among which the basidiospores are interspersed act as 'nurse hyphae' and that, as they disintegrate, the basidiospores increase greatly in size after having been freed from their basidia. Thus, the nutrition comes from these nurse hyphae rather than through the basidia and sterigmata. 3 / formation of the funiculus Because the structure of the funiculus or cord by which the peridioles are attached to the inner wall of the peridium is so complex and bears such an important relationship to the dispersal of peridioles in nature, a detailed account of this subject will be given later (Chapter 7, B) . Further, the funiculus differs considerably in structure in different species and genera (Brodie, 1956). Therefore the following account is generalized to indicate merely how this structure arises as the young fruit body develops. As shown above, the funiculus arises from a

67 / Heterokaryotic mycelium and fruit body formation

group of parallel hyphae extending from the inner surface of the peridium to the peridiole primordium. Then actively growing hyphae appear at the inner surface of the peridium. These hyphae elongate rapidly to form a bundle of parallel filaments (figure 3ie). The hyphae surrounding this bundle become partially gelatinized and give rise to the outer covering or sheath of the mature funiculus (figure 3ie). The hyphae that constitute the central cord of the funiculus continue active growth and form a strongly coiled rope made up of parallel hyphae (figure 36). The central cord, in the mature funiculus, is attached to the peridiole by the parallel hyphae from which it arose, and at the base it is attached to the inner wall of the peridium by a mass of more delicate hyphae. Although she made no special remark about the origin of the central cord in Cyathus olla or C. striatus, for Crucibulum laeve Walker noted that the lower section of the funicular cord arises in the peridial region and grows upward towards the peridiole. This she determined by the astute observation that the clamp connections of the hyphae of the lower part of the cord are all turned in such a direction as to indicate that the hyphae must have grown upward from the peridium. 4 / Development and rupture of the epiphragm The peridium, whose formation has been described in the preceding pages, does not cover the fruit body at the top at least in Cyathus olla, although in C. striatus it extends part way across the top. As seen in figure 31, the mouth of the fruit body is covered by hyphae of the outermost layer of the fruit body, which, in this region, produce an abundance of long hairs. Beneath this outer coat lies the un differentiated tissue which originally filled the cup. Gelatinization of the ground tissue of the gleba begins at the base and progresses upward, the undifferentiated hyphae at the top undergoing gelatinization last.

68 / The bird's nest fungi

As lateral expansion of the upper part of the fruit body takes place, the tufts of brown hairs which were at first crowded towards the centre are spread apart, often in concentric circles, and there is exposed to view a thin tough white membrane, the epiphragm (figures 23, 24) covering the mouth of the fruit body like the head of a drum. Rupture of the epiphragm leaves the fruit body open at the top. When the fruit body opens, it is filled at first with mucilage (figures 23, 24), the persistence of which depends greatly upon weather conditions. In a freshly opened fruit body, the peridioles lie immersed in this mucilage. 5 / Development of the basal emplacement hyphae In all the common species of Cyathus, each fruit body is set upon and firmly attached to a solid mass of hyphae named the emplacement (figures 2,41). Tulasne noted its presence in C. striatus and Lloyd (1906) recognized it as a characteristic feature of many species. In C. striatus, in which it is most highly developed, it begins to take form shortly after the organization of the fruit body primordium. The brown hyphae at the base of the primordium spread out laterally and proliferate until a wide cushion or ball of closely packed hyphae is formed. In this species, the emplacement has reached its maximum width (8-12 mm), while the fruit body is still a mere 'button/ The hyphae may become intertwined with bits of substratum and a considerable amount of the final weight of the emplacement may be due to such inclusions. From observing the growth of numerous fruit bodies of C. stercoreus in laboratory culture, it has been found that the emplacement develops more tardily in this species. The young fruit body is frequently half grown before the emplacement hyphae spread from its base. Emplacements are also observed on fruit bodies of C. olla, but they are often below ground. A true emplacement seems to be lacking in Crucibulum laeve.

6 / Demonstration of the regions of growth and expansion of fruit bodies By marking young fruit bodies with India ink, Walker was able to determine the regions that expand most during growth. It appears that: (i) increase in length of a young fruit body from the time it is 3 mm until it is 10 mm in height is the result of growth chiefly in the apical region. (2) Later, lateral expansion is most rapid in this same apical region and results in spreading the tissues of the top of the fruit body; this lateral expansion, at least to some extent, assists in the rupture of the epiphragm. / / Special features of fruit body development in certain species The description of the development of the fruit body in Cyathus given in the preceding pages has been based largely upon the accounts of Tulasne and Walker. The respects in which the developmental morphology of other species and of other genera differ from this generalized picture may now be mentioned. (a) Cyathus striatus. One of the most characteristic features of the mature fruit body of C. striatus is that the inner surface of the upper part of the peridium above the pendióles is strongly fluted or striate (figures i, 41). Tulasne mentions the striae but says nothing about their origin. Walker gives only the following brief statement concerning them: As the fruit body reaches maturity, the three layers of the peridial wall extend practically across the top of the basidiocarp, quite in contrast with the condition in C. olla. In this upper portion the peridium becomes folded. At this time the pendióles reach practically to the top of the fruit body, as in younger stages ... At the time of expansion this folded upper part expands, forming the striate projections above the pendióles ...

Although Walker does not say so, one would assume that it is the folding of the two innermost layers of the peridium in the upper por-

tion that produces the striae or plications. Occasional specimens of C. striatus show some plication on the outside of the cup. In C. poeppigii the cup is strongly plicate externally as well as internally. (b) Crucibulum laeve. The peridium in Crucibulum is not composed of the three distinct layers which characterize Cyathus. When mature, the peridium in section appears homogeneous, except perhaps for slight differences in the colouration and density of the hyphae of which it is composed. When the fruit body is young, however, two zones can be distinguished: (i) an outer layer made up of loosely interwoven hyphae, which are thickwalled and brown and give rise to long hairs on the outside of the fruit body ; (2) an inner layer which is thick at first and is composed of somewhat gelatinized hyphae. As the glebal region develops, the inner layer becomes compressed and ends by being scarcely distinguishable as a separate layer in the mature fruit body. The epiphragm in this fungus is formed from the undifferentiated upper part of the fruit body and is covered with pointed hairs. Gradual upward gelatinization finally involves this epiphragm, and the fruit body opens. Walker states that there is no opening up of the upper portion of the fruit body by spreading such as occurs in the species of Cyathus. Another respect in which Crucibulum differs from Cyathus is in the development of pendióles, which takes place simultaneously throughout the fruit body of Crucibulum. (c) Nidularia. The fruit bodies oiNidularia are initially globose and there is no epiphragm, the peridium breaking irregularly at the top to open the fructification. The wall of the peridium is made up of one virtually homogeneous layer of hyphae. The manner of development of the peridioles is similar to the process in Crucibulum ; however, in Nidularia, peridioles form in the apical part of the fructification only, the basal part undergoing gelatinization. The first peridioles developed are towards the wall of the fruit body,

69 / Heterokaryotic mycelium and fruit body formation

those formed later arising further within the fruit body and nearer to its apex. The wall of the pendióle is complex and, according to R. Fries (1910), is composed of five layers. What significant morphological similarities may exist betwen Nidularia and Ni dula is a matter which has not been examined critically. From observation only, it may be worth noting that an occasional fruit body of Nidularia seems, superficially, to come close in appearance to the open cupulate type of fruit body of Nidula (e.g., Nidularia farota, figure 5oc). Nidularia australis in Lloyd's photograph (Lloyd, 1906, Fig. 8) also appears to approach, in gross morphology, the cupulate form of Ni du la. The only investigation of fruit body development in Nidula that has been made appears to be Overstreet's (1955). The main conclusions drawn from her study are the follow-

70 / The bird's nest fungi

ing: (i) in general, the developmental pattern in Nidula is similar to that of Crucibulum and of Cyathus, especially to Crucibulum ; (2) in late stages of development, much of the growth is basal in Nidula as it is in Crucibulum, whereas it is apical in Cyathus ; (3) the structures of the peridium wall and of the epiphragm are more closely related to those of Crucibulum than to those of Cyathus ; (4) pendióles are differentiated first, and develop sooner, in the upper portion of the gleba in Nidula ; (5) in a number of respects, the developmental morphology of Nidula macrocarpa is strikingly different from that of other members of the genus. Overstreet's study of the development of fruit bodies in Nidula led to the conclusion that 'the genus Nidula can be placed in a phylogenetic position intermediate between Nidularia and Crucibulum/

ó / Morphology and Behaviour of Nuclei

What was known of the behaviour of nuclei in the Nidulariaceae prior to 1953 was largely the result of the studies of R.E. Fries (1911) concerning Nidularia pisiformis (N. farcta). Fries's material was fixed in Flemming's Solution (strength not indicated) and stained with iron-haematoxylin. Safranin was also employed and gave better detail for some stages of nuclear division. According to Fries's account, two nuclei are regularly present in the young hyphae from which basidia develop in young fruit bodies. These nuclei are seldom more than 1-2 ¡JL in diameter. As the hyphae mature into young basidia, the two haploid nuclei fuse, producing a large fusion nucleus which expands and occupies most of the swollen apical part of the basidium (figure 32b). The fusion nucleus then undergoes a meiotic division with the spindle transverse to the length of the basidium (figure 32C, d). The two daughter nuclei next undergo a second division (mitotic) with spindles generally transversely oriented, and four nuclei therefore occupy the basidium as spores begin to form (figure 326). When the spores are of full size, a third (mitotic) nuclear division begins which is only completed in the spores themselves after the nuclear material has migrated through the narrow sterigma on which each spore is borne (figure 32Í, g); and each mature basidiospore is thus binucleate (figure 32h). Fries believed the first division of the 71 / Morphology and behaviour of nuclei

fusion nucleus to be reductional and the haploid number of chromosomes in this species to be three or four. Because of the lack of sharp differentiation of chromosomes, it was difficult for Fries to be positive about their number. Maire had commented previously on this same fungus in his report on the cytology of certain Basidiomycetes. The very regular spiral form of the chromatin thread in spireme noted by Maire was not found by Fries, who insisted that the spireme was very irregular. The nuclei illustrated by Maire in the four-nucleus stage of the basidium are much smaller than those observed by Fries for the comparable stage in his material. In her studies of Cyathus olla, C. striatus, and Crucibulum laeve, Walker (1920) demonstrated and illustrated the binucleate condition of young basidia, the single large fusion nucleus, and the regularly binucleate basidiospores in all three species. That the two basidiospore nuclei are two haploid sister nuclei may be inferred from Fries's work (1911) and from the fact that most species thus far studied have proven to be heterothallic. The only demonstration (of early date) of the nuclear condition in the mycelium is that of Miss Walker who showed that, in C. olla, nuclei are regularly disposed in pairs in the heterokaryotic clamp-bearing mycelium. For Cyathus stercoreus a detailed and interesting account of the appearance and be-

FIGURE 32 Nuclear events in basidia and spores of Nidularia farcta : a, young nuclei (haploid, n) before fusion ; b, fusion nucleus (zn) ; c, d, meiosis ; e, four nuclei produced by meiosis ; f, nuclei passing into spores ; g, mitotic nuclear division in young spores ; h, mature binucleate spores. X 2000. Redrawn from R.E. Fries, Zeitschr. f. Bot., 1911, m: 145-65

haviour of nuclei in basidiospores, and in haploid and diploid my celia of living material is available from the work of Miles (1953), who availed himself of the then new techniques of phase microscopy in an endeavour to follow the course of nuclear behaviour, especially during the process of dikaryotization. Although Miles was unable to follow nuclear migration during dikaryotization owing to the fact that nuclei disappear or are not resolved by the phase microscope during actual mitosis, his observations are worth recording in some detail because no other account of nuclear behaviour in living mycelium of this fungus is available, and because certain of these observations will undoubtedly be of value for future investigations. It has frequently been reported that basidiospores of the Nidulariaceae are regularly binucleate. However, Miles reported that in Cyathus stercoreus the uninucleate condi72 / The bird's nest fungi

tion prevails among young basidiospores (figure 33a). Spores were also observed to possess two, three, or even more nuclei, but such spores were older than uninucleate ones. In homokaryotic mycelium, actively growing hyphal tips are usually multinucleate (figure 33c), the number of nuclei varying with the length of the terminal hyphal unit. In older homokaryotic hyphal segments, nuclei are distributed one nucleus per cell in certain mycelia, but two nuclei per cell in others, an observation already made by Fulton (1950^. In heterokaryotic (dikaryotic) mycelia, nuclei are regularly disposed in pairs or dikaryons. The nuclei of a dikaryon have a strong tendency to remain at a constant distance from each other although they do move as a pair to different positions in a hypha at different times. Nuclear movement was frequently observed taking place in a direction opposite to the visible streaming of cytoplasm.

FIGURE 33 Appearance and behaviour of nuclei of Cyathus stercoreus as revealed by dark-phase contrast microscopy: a, basidiospore with single nucleus (note nucleolus), Xi/oo; b, the two nuclei of a dikaryon, X2ooo; c, monokaryotic hypha at tip showing multinucleate condition, Xi/oo; d, monokaryon hypha showing prominent mitochondria at arrows, X1700 ; e, nuclei of a dikaryon close to young clamp connection before nuclear division, Xi/oo; f, same clamp connection during division of nuclei when latter are not revealed by phase microscopy, Xijoo. Photographs reproduced courtesy Dr Philip Miles 73 / Morphology and behaviour of nuclei

FIGURE 34 Nuclear cycle and behaviour in Cyathus olla : a, nuclear pair in young basidium ; b, fusion nucleus in basidium; c, pachytene of meiosis showing bivalent chromosomes; d, diplotene showing an aberrant pairing; e, diakinesis showing twelve bivalents; f, metaphase n showing spindle; g, tetrad; h, mature binucleate basidiospores. All approximately X23OO. Reproduced courtesy Dr B.C. Lu and Can. J. Bot., 1964, 42 : 307-10, Pi. i, il, m 74 / The bird's nest fungi

Miles observed that nuclei of a dikaryon always move to a position directly opposite the lateral hyphal swelling that becomes the hook cell during the formation of clamp connections (figure 336). The nuclei of the dikaryon then suddenly disappear (figure 331) under phase observation (presumably when the nuclear membrane of each disintegrates) and do not reappear until mitosis has been completed. The time required for nuclear division was found to be six minutes, and the time that elapsed from the first evidence of the formation of a new clamp connection until complete fusion of the hook cell with the main hypha was 55 minutes. One of the most impressive features of living mycelium of Cyathus stercoreus, as seen under the phase microscope, is the conspicuous presence of numerous and (predominantly) long mitochondria (figure 33d). These are especially numerous in or near hyphal tips and they were seen by Miles to aggregate at the site of clamp connection formation where they display writhing movements just prior to division 75 / Morphology and behaviour of nuclei

of the nuclei. These mitochondria frequently assume positions strongly reminiscent of nuclear division figures and Miles was of the opinion that, had they been fixed and stained using a non-differentiating stain, they might easily have been mistaken for dividing nuclei. Further study of the large worm-like mitochondria of Cyathus should certainly be undertaken by more modern methods than those available to Miles. Phase microscopy of living material did not enable Miles to discern much structural detail of the nuclei. From his observations, supplemented by study of Feulgen-stained material, Miles reported (1953 p. 120): 'Nuclear structure in Cyathus stercoreus is similar to that reported by Savile [Am. J. Bot. 26: 585-609,1939] for spores of many of the rusts. When examined by dark-phase contrast, the metabolic nucleus is revealed as a dark spherical body surrounded by a light area. This is compared with the "expanded'' nucleus described by Savile ; the dark spherical body is the

endosphere and the light area is the ectosphere/ The development of an improved propionocarmine staining technique for fungal nuclei by B.C. Lu (1962) made possible the report by Lu and Brodie (1962) that the chromosomes of Cyathus stercoreus are relatively large in comparison with those of many fungi and that the chromosome number, n = 12, is larger than that which had been reported for most of the Gasteromycetes that have been examined. The latter report was followed by a paper by Lu and Brodie (1964) in which detailed observations were given of meiosis as it occurs in the young basidia of the common bird's nest fungus Cyathus olla. Using young fruit bodies, squash preparations of basidia were made and stained, following Lu's technique. Satisfactory staining, resolution, and separation of chromosomes resulted in the following being recorded. In Cyathus olla the ultimate cell of a sporogenous hypha contains two nuclei each of which displays a conspicuous nucleolus (figure 34a). As the cell develops into a basidium, the two nuclei fuse and undergo synapsis, the zygote nucleus filling the main body of the basidium (figure 34b). At early pachytene, bivalent chromosomes are thread-like, but by late pachytene they become shortened and thickened, allowing their bivalent nature to be discerned (figure 34c). At diplotene, loops may be seen in the bivalent chromosomes (figure 34d). The nucleolus is still present at this stage. At diakinesis, bivalent chromosomes are still shorter and become ring-shaped, possibly because of terminalization of chiasmata (figure 34e) ; the nucleolus disappears and at this stage twelve chromosomes can be resolved. At metaphase n the axes of both nuclei are parallel to one another and centrioles and spindles are demonstrable (figure 341). After the second division of meiosis a tetrad is formed in the basidium (figure 34g). As nuclear tetrads are formed, four basidiospores develop on each basidium and each young spore receives a single nucleus. The latter undergoes mitosis 76 / The bird's nest fungi

with the result that each mature spore is binucleate (figure 34h). Thus chromosome behaviour during meiosis in Cyathus olla appears to follow the pattern found in most organisms. Synapsis in this fungus is not entirely the same as has been described in the Ascomycetes, for apparently, in Cyathus, chromosomes do not contract into short rods before synapsis, which seems to take place at the end of the presynaptic process. A much fuller account of nuclear structure and chromosome cycles of Cyathus stercoreus was published by B.C. Lu (19643). Because this species could be fruited in laboratory culture, a continuous and abundant supply of young fruit bodies was available for fixation and staining. Lu noted that meiosis in young basidia most frequently occurred in young fruit bodies about three to four days from their inception, at which time they are about 5-7 mm X 10-12 mm in size. Fixation and staining methods were the same as those used in Lu's previous work on C. olla. Although clearer photographic evidence was obtained by Lu from material of C. stercoreus than from the material of C. olla, the conclusion as to the events of meiosis and mitosis was essentially the same. Lu reported that in C. stercoreus the fusion of the two nuclei of compatible mating-type in the developing basidium takes place (figure 353) at the end of the telophase of the presynaptic mitosis and that synapsis follows immediately after nuclear fusion. Meiosis and mitosis were considered to be essentially similar to those processes in most other organisms. In pachytene of the first division of meiosis the chromosomes and nucleolus are clearly defined, and a satellite of the nucleolar chromosome (figure 35b) is present. In metaphase i centrioles and spindles were demonstrable. At diakinesis chromosomes were sufficiently distinct to allow the chromosomes to be counted as n = 12 (figure 350,d). One additional point noted by Lu for this species was reported as follows : 'The presence of quadrivalents as well as secondary associations of

FIGURE 35 Nuclear cycle and behaviour in Cyathus stercoreus : a, presynaptic nude: in young basidium; b, Pachytene and satellite of nucler chromosome; c, d, photograph and drawing (c) of late pachytene with

twetalve chromosomes clearly resolved; e, twelve bivalent choromosos at pachytene cut from photograph; f' metabolic nuclei squeezed out of a mature spore. All X 2300, approximate photographs reproduced courtesy

Dr B.C. Lu and Chromosoma, 1964, 15: 170–84 77 / Morphology and behaviour of nuclei

like chromosomes suggests that Cyathus stercoreus may be a tetraploid species/ In a later publication, Lu (1964^ gave more complete evidence for the suggestion that Cyathus stercoreus may be a tetraploid species. He noted that some chromosomes of the haploid complement (n =12) resemble one another structurally in pairs. Figure 356 shows the twelve bivalents of the haploid complement in which will be noted the morphological similarity between chromosomes i and 2, and between chromosomes 7 and 8. Lu observed that these and other morphologically similar pairs exhibit, with high frequency, a tendency to form quadrivalents or to show secondary association at diplotene and diakinesis of meiosis, suggesting that the members of such associating pairs have some degree of homology and indicating that C. stercoreus may be a tetraploid species. It was suggested by Lu that the polyploidy of this species may have resulted from allotetraploidy involving species which evolved from a single ancestor. The extreme variation in the form and size of fruit bodies of C. stercoreus has been noted elsewhere (Chapter i, D; Chapter 5, H). It is possible that the species includes races, some of which are diploid and some tetraploid. Further investigations along this line might well throw much light upon problems of the variability of this species and others such as C. olla, where, again, the chromosome number is high. Regarding mitosis, which was studied chiefly in the dikaryotic mycelium, Lu clearly demonstrated the existence of chromosomes and mitotic machinery. He noted also that the mitotic divisions of the nuclei of a dikaryon are not exactly synchronous (as they have often been reported to be). The nucleus nearest the clamp connection always undergoes division slightly earlier than the other nucleus of the dikaryon. From the above information a fairly clear picture emerges of the nuclear events leading to the production of genetically haploid basidiospores in almost all species of the Nidulariaceae. Most species display the tetra78 / The bird's nest fungi

polar or four-mating-type sexual behaviour and their reported nuclear behaviour accords well with the genetic evidence regarding the interactions of monosporous my celia that have already been discussed (Chapter 4, c). Apparently the only known deviations from tetrapolar sexuality among the Nidulariaceae are the situations in Mycocalia denudata and M. duriaeana. Burnett and Boulter (1963) showed that M. denudata is heterothallic bipolar and also facultatively homo-heterothallic, while M. duriaeana is homothallic. The facultative homo-heterothallism of M. denudata results in basidiospores being produced within one and the same peridiole which are either homokaryotic or heterokaryotic in respect to mating-type factors. The condition is due to a dominant gene Pd which determines a precocious division of the four nuclei, formed by a normal meiosis, in each basidium. The eight nuclei so formed appear to migrate at random, in respect to the mating-type factors they carry, into the basidiospores which are each ultimately binucleate. A drawing showing the different distributions of the nuclei is given by Burnett and Boulter together with a diagram explaining the sexuality patterns. In the publications just reviewed, illustrations of metabolic nuclei either in monokaryon or dikaryon mycelium seldom reveal much detail. It will be recalled that Miles (1953) described the metabolic nucleus as seen under the phase microscope as consisting of a dark portion and a halo (figure 333), but he did not consider that the dark portion consisted only of the nucleolus. On the other hand, one of the clearest photographs published by Lu, showing two stained metabolic nuclei squeezed out from a basidiospore of Cyathus stercoreus, seems to indicate (figure 351) that the dark bodies of the nuclei are nucleoli. Lu comments: 'The structure of a "resting" nucleus is demonstrated clearly in Fig. 39 (loc. cit.) in which are shown two nuclei squeezed out of a spore. A "resting" nucleus consists of the nuclear membrane, the chromatin reticulum which is formed from the entanglement of chromosomes, and a nu-

cleolus. ' Further study may be necessary to reconcile the difference in appearance of the metabolic nucleus as seen under the phase microscope and as seen in stained preparations. Finally, when chromosome counts and detailed study of chromosome morphology, which are now available for only two species,

79 / Morphology and behaviour of nuclei

have been provided for other species, they will doubtless be a great aid in assessing the taxonomic relationships between species and between genera and in providing some insight into the course of evolution within the bird's nest fungi,

7 / The Fruit Body as a Spore Dispersal Mechanism

A / THE M E C H A N I S M It will be shown in this chapter that the sporocarps of Cyathus, the most highly developed genus of the Nidulariaceae, may be regarded, as Buller (1942) regarded them, as 'splash-cups' ; each sporocarp is a special structure of the right size, shape, position, and consistency to make use of the kinetic energy of large falling drops of rain in such a way that pendióles are splashed out of the cups and on to surrounding vegetation. In dealing with this subject, I am aware of the dangers of a philosophy of teleology. The fact is that the early investigators of the structure of the fruiting body, in particular the complex funiculus, failed to provide an explanation of spore dispersal in the Nidulariaceae. The explanation offered here does work, as shown repeatedly (Chapter 8). Moreover, it would be difficult to postulate any function for the funiculus other than the one offered herein, for no other concept would accord so well with observation and experiment. The functional form of the cup itself is emphasized by its occurrence, with demonstrated effectiveness, in other fungi, mosses, the liverwort Marchantía, and a variety of flowering plants (Chapter 10, c). We must now examine in detail certain aspects of sporocarp structure in Cyathus, Crucibulum, and Nidula in the light of the 80 / The bird's nest fungi

demonstrated function of sporocarps of these genera in dispersing the pendióles by rain. B / THE F U N I C U L U S A full account of the structure and function of the funiculus of the Nidulariaceae has been published (Brodie, 1956) and need not be repeated in toto. The details of the structure of the funiculus of the well-known species Cyathus striatus are taken from that publication with minor changes. i / The sheath or basal piece Broadly attached to the inner wall of the peridium is a tubular or somewhat conical mass of grey-brown hyphae (figure 36s). This structure varies between 0.8 and i.o mm in diameter and is slightly over i. o mm in length. At its upper end it flares outwards. In freshly opened fruit bodies in which the funiculus has not been disturbed, the sheath adheres loosely to the purse or upper part of the funiculus. The sheath is by no means a solid structure. It is essentially a tube made up of rather straight hyphae, fairly closely interwoven towards the outside and less so towards the centre. The central part is not entirely hollow but is traversed by a few loosely arranged hyphae (figure 36). About a third of the way from the top, the

FIGURE 36 Structure of funiculus and peridiole of Cyathus striatus : s, sheath or basal piece, x$o ; m, middle piece which is attached to the inner hyphae of the sheath, X5o; p, purse (also shown larger above), X3o; f, funicular cord, X3O (also shown X3oo at bottom of figure) ; h, adhesive hapteron, X3O (also shown X3oo below) ; t, tunica of peridiole and c cortex, X3o; at centre right note modified clamp connection. Courtesy Svensk. Bot. Tidskr., 1956, 50: 145 811 Fruit body as a spore dispersal mechanism

innermost hyphae of the sheath become continuous with the middle piece. Tulasne and Tulasne (1844) reported that the middle piece is attached directly to the wall of the peridium. Professor Buller and I both made careful dissections of the sheath and failed to corroborate Tulasne's statements in this respect. Details of the structure of the sheath are very difficult to see and, even when one is dissecting it under the binocular, the delicate hyphae are apt to become tangled. The attachment of the sheath to the middle piece is shown in figure 36. The hyphae of the sheath have considerable tensile strength for, if one pulls a peridiole away from the wall of the fruit body, the sheath seldom ruptures. 2 / The middle piece This part of the funiculus is not in view in freshly opened fruit bodies (figure 36, upper left), but it may be seen if a peridiole is pulled gently away from the wall of the body. It is a cord about 0.4 mm in diameter at a point midway between its two ends, and between o. 8 and 1.5 mm in length, depending upon how much it is extended by pulling. At its lower end, as noted, it passes into the sheath and becomes firmly attached to the latter. At its upper end, the middle piece (figure 36m) widens markedly where it is attached to the lower part of the purse. The middle piece is composed of slightly yellowish hyphae which are 4-5 JÜL in diameter and provided with numerous clamp connections. From the orientation of the clamp connections, it is possible to deduce that the middle piece resulted from upward growth of hyphae from the peridium when the fruit body was young. The hyphae are sinuous or often slightly coiled (figure 36m). This fact is undoubtedly connected with the observed elasticity of the middle piece. When a peridiole is pulled away from the cup gently without breaking the hyphae, the middle piece may be stretched, and

82 / The bird's nest fungi

it snaps back into its original position when the peridiole is released. 3 / The purse The uppermost part of the funiculus is a stout stem called the purse, about 1.5-2 mm long and 0.5 mm wide (figure 36p). The upper part is elongate, and slightly swollen in the middle. The lower part is short and bulbous and there may be a slight constriction between the parts. The purse is a kind of sac or envelope composed of hyphae which, in the upper part of the purse, lie mostly parallel to one another and, in the lower part, are loosely interwoven (figure 36, upper right). Under the high power of the microscope the lower part of the purse resembles loose crochet-work (figure 36, upper right). 4 / The funicular cord Undoubtedly the most interesting part of the funiculus is the funicular cord which is held within the purse. This structure was observed by Tulasne and illustrated faithfully by him, but he did not appreciate its functional significance. Although I have more than once described briefly how this cord may be pulled out of the purse and how it functions, a fuller account at this point is desirable. Let us suppose that, by means of small scissors, we have cut away from the peridiole wall a single peridiole, leaving the funiculus intact (figure 37). By placing this object on a glass slide, it is then possible to see the sheath, middle piece, and purse as described. If the entire funiculus be placed in water, no marked change is observed, except perhaps a slight swelling of the various parts if these were dry beforehand. If the peridiole is then grasped with one pair of forceps and the sheath pulled with another, the middle piece stretches (figure 37) and ultimately breaks. If the fungus is not too old, the break invariably occurs at the bulbous end of the purse. As noted above, the upper end of the middle piece

)•

FIGURE 37 Dissection of fruit body of Cyathus striatus carried out under water. Note explosive expansion of hapteron and pulling out of funicular cord, x6. Courtesy Natural History Magazine, 1952, 41: 403-7

is firmly attached to the lower end of the purse by a number of straight coarse hyphae. These usually serve to tear open the lower end of the purse and, when this happens, there follows a startling explosive swelling of the hyphae contained within the bulbous part of the purse (figure 37). It is sometimes possible to pull the middle piece free from the purse without rupturing the latter. It is then necessary to tear open the lower part of the purse to observe the explosion. This demonstrates that, although the purse may have been thoroughly wetted or even immersed in water, no explosive swelling of the purse contents is observed until the wall of the purse has been torn. The expanded tangle of hyphae which thus appears abruptly is the hapteron (figure 36h, 37). Its component hyphae are thick-walled, and are provided with modified clamp connections (figure 36). There are few free ends to be seen in the mass. The hyphae of the hapteron are much contorted and frequently bent at sharp angles, which suggests that intercalary growth of the hyphae took place after the bounding mem-

83 / Fruit body as a spore dispersal mechanism

brane of the purse had been formed. The hapteron hyphae are doubtless held within the purse under considerable pressure and their sudden expansion, when the purse is torn, may be due to the release of this pressure. Some additional features must contribute to the explosive expansion, however, for if an un wetted purse is ruptured under mineral oil, no explosive swelling takes place. Presumably oil would serve as well as water as a lubricant to allow the tangled hyphae to unravel. Since they do not unravel under oil, the normal operation of the hapteron apparently depends upon water being absorbed by the purse and hapteron. It is probable that the surfaces of the hapteron hyphae are covered with some hydrophilic colloid which not only causes them to swell, but also endows them with great adhesive properties, because the hapteron as a whole is highly adhesive, as will be explained later. When the purse has been ruptured in the manner described, one has only to take hold of the hapteron with forceps and lift the peridiole out of the water to see the funicular cord beautifully and spectacularly extended (figure 37).

If this is done successfully, the peridiole hangs suspended by its funicular cord, the length of which will vary, depending to some extent upon the way in which it is drawn out. Unless the funiculus is moist, as when fresh, or has been moistened for some hours if dry specimens are studied, the rupture of the funiculus may not occur as described. In very old material or in fresh material insufficiently moistened, a number of abnormalities may be observed, such as: (i) the middle piece may tear away from the sheath and therefore not rupture the purse; (2) the purse may break loose from the middle piece without being torn open. There is abundant evidence to show that when a peridiole is thrown from a fruit body by rain, it carries with it only the funicular cord terminated by the hapteron. Occasionally a few unravelled hyphae from the purse may remain attached to the cord, but certainly neither the intact purse nor intact sheath. Botanists may wish to carry out the fascinating dissection themselves or demonstrate it to students. For either purpose, freshly collected fruit bodies are preferable. Dried specimens should not be more than a year old and should be moistened on wet blotting paper for twenty-four hours before use. The funicular cord of an intact funiculus is enclosed within the purse, as can be seen by careful examination of the purse of living material (figure 36) and verified by study of stained sections of the funiculus (figure 31). From such observations it appears that the cord is closely packed within the purse by a zig-zag folding. The funicular cord of Cyathus striatus is a beautiful object formed of silky white hyphae (figures 36, 38b,d). Its length varies from 4 to 12 cm, depending upon the degree to which it is pulled out, and its width from 50 to 100 /¿. It is composed of a large number of hyphae spirally twisted about one another like the strands of a steel cable. The cable structure in part accounts for its amazing elasticity. Each hypha of the cable is 2-2.5 ^ ^n diameter and appears devoid or almost devoid of a lumen. Here and there modified clamp connections may be seen 84 / The bird's nest fungi

(figure 36). Probably as a result of the general process of thickening of the hypha walls, the clamp connections appear as mere knots or swellings and little of their original structure is evident. The hyphae are highly refractive as seen in a water mount under the microscope. The tensile strength of the cord is remarkable. When fresh and moist, it is capable of supporting a weight of 1.5-2.0 g. If dry, it can support as much as 3-5 g, providing the cord is not jerked by the weight. The weight of a fresh peridiole of C. striatus varies from 0.003 to 0.005 8- The specific gravity of a wet peridiole of this species is about i. 165. It is apparent that the funicular cord has more than enough strength to hold the peridiole during any phase of dispersal by raindrops and to act as a very strong attachment to herbage or other objects. Also, the cord has a capacity for extreme extension which is related to its cable structure. As Tulasne correctly reported, even if some of the hyphae of the cable break as the cord is pulled out beyond what would be the normal limits in nature, they do not all break simultaneously. Because they are spirally twisted, they tend to lock together. The cable may thus be frayed, but it long resists breaking. As to the fate of the purse when the funicular cord is pulled out, it usually seems to tear into small masses of hyphae which may adhere to the funicular cord on which it may occasionally be observed to form small lumps (figure 36, top centre). Some traces of the purse hyphae are often found at the upper end of the cord near the peridiole. A point of special importance to an understanding of the operation of the funiculus in nature is that, of the entire funicular cord, it is the hapteron or tail-end widened portion (figure 36) that is most highly adhesive. Occasionally slight adhesiveness of the cord above the hapteron is observed as well. If a freshly expanded hapteron is pulled across a dry glass slide, a trail of colourless mucilage is left behind. Also, if a fresh moist peridiole with its intact funicular cord extended be allowed to dry on a glass slide and then be shaken vigorously,

FIGURE 38 a, pendióle of Cyathus stercoreus germinating as a whole and producing dikaryon mycelium, X 4 . 5 ; b, pendióle of C. stercoreus entangled with epidermal hairs on strawberry leaf, X 5 ; c, Crucibulum laeve showing whitish pendióles and funiculus, X 5 ; d, pendióle of Cyathus striatus, the funicular cord wrapped around a woody stem, x$. Courtesy Svensk. Bot. Tidskr., 1956, 50: 155 85 / Fruit body as a spore dispersal mechanism

the pendióle and the cord become freed. The peridiole dangles by its cord, only the hapteron end of which remains attached to the slide.

main for consideration the details of the way in which the structure and operation of the funiculus contribute to the further spread of pendióles in nature after they have been splashed out of their cups. 5 / The function of the funiculus In Nidula, the peridiole is devoid of a The funiculus of other species of Cyathus funiculus, but is highly adhesive over its entire shows some deviation (Brodie, 1956) from that surface, and the mucilaginous coating enables of C. striatus just described. In the tropical C. it to adhere to foliage of other objects it may poeppigii, the funiculus is very similar to that strike. The form of the peridiole in Nidula and of the closely related C. striatus. In C. Crucibulum may aid adhesion by markedly increasing the area of contact with the target, stercoreus the sheath is more widely flared and also reducing the frontal area exposed to than in C. striatus and the funicular cord is wider; and in this species the funiculus occa- wind and rain. A similar form (on a smaller scale) is seen in various fungus spores which, if sionally is abortive, so that none appears on some pendióles. In C. olla the purse is wide pigmented, can be seen stuck in numbers to and the lower portion is not distinct from the leaves and culms under the stereo microscope upper part; also the hyphae of the funicular (see also 7, G). When the mucilage dries, the peridiole adheres firmly to its target and is not cord are more loosely spiralled than those of C. washed off by rain. We can only conjecture as striatus. The funiculus of Crucibulum is quite differ- to the subsequent fate of a peridiole of Nidula because too little is known about fungi of this ent from that of Cyathus. The purse consists not of an elongated sac but of a rounded knob genus in their natural surroundings. Foliage bearing adherent pendióles must 0.3-0.5 mm wide, attached to the underside of the peridiole (figure 38c). Attaching the purse frequently be eaten by herbivorous animals. If directly to the wall of the peridium is a stout the spores in the pendióles remained viable, and there is evidence (Brodie, 1948^ that in yellow-grey cord o.i mm wide and about 2.5 mm long. There are also other differences be- Cyathus they do, they might be deposited in tween the funiculi of Crucibulum and Cyathus the animals' faeces at great distances from the site of the parent fruit body. Pendióles adherfor which the reader is referred to the paper ing to foliage not eaten by animals could be cited above. quite widely dispersed when the leaves fall to There is no funiculus at all in the genera Nidula, Nidularia, and Mycocalia. Although the ground and are blown away by wind. In Cyathus, the peridioles do not have an traces of cords of partly gelatinized hyphae adhesive surface. It is by means of the funiculi may occasionally be found among the that peridioles of this genus adhere to vegetapendióles of Nidularia, these are not regularly connected to pendióles. Further, R.E. Fries's tion. Since peridioles attached to leaves and other plant parts can be found by anyone who account (1910) of development in Nidularia looks carefully, it is not teleology to regard the indicates that no structure comparable to a funiculus as a special attachment organ. funiculus is ever developed in this genus. The force inherent in a large water drop, Previous publications (Buller, 1942; Brodie, 1951) have demonstrated that the pendióles of perhaps 3 mm in diameter, falling with a terNidula, Crucibulum, and Cyathus are ejected minal velocity of approximately 7 metres per from the vase-shaped fruit bodies to a distance second is considerable. During actual experiof one to two metres by the splash created by ments in the laboratory, I have observed that a drop of water falling through a distance of heavy raindrops. The splash, however, is but three metres can throw a large heavy peridiole the initial phase of dissemination. There re86 / The bird's nest fungi

FIGURE 39 Two pendióles of Cyathus berkeleyanus attached by their funicular cords to petiole of Hibiscus. Photographed immediately after a heavy rainfall in Kingston, Jamaica. Xo.5, approx.

of Cyathus olla (weight 0.005 8) to a distance as great as two metres (Chapter 8). As a large drop falls into the rain-filled cup of Cyathus, the energy of the raindrop is transferred to a force acting parallel to the sides of the cup and is exerted upwards from the base of the cup towards the mouth in a piston-like action. (It is, in fact, a wave-front, which breaks up into droplets as it parts from the rim of the cup.) This thrust is ultimately exerted on a peridiole in the uppermost layer of the peridioles lying in the cup. The lenticular shape of the peridiole and its attachment to the funiculus by the centre of its lower side doubtless result in the peridiole receiving the upward thrust on its lower side - broadside, so to speak. The peridiole is then jerked away by the upward-driving water, and the strong middle piece tears open the lower part of the purse in the process. The peridiole thus begins its flight from the cup accompanied by some water, and with its funicular cord either extended or ready to be extended. 87 / Fruit body as a spore dispersal mechanism

The question of whether or not the funicular cord is pulled out as the peridiole leaves the fruit body cannot be answered definitely at this time. If the initial jerk that tears the purse also causes the cord to be extended, the peridiole may fly away from the fruit body with the cord trailing behind. This may occur; on the other hand, there is considerable evidence that the cord is extended only after the hapteron becomes attached to some object in the line of flight and while the peridiole is being carried on by its own momentum. When the flying peridiole strikes some object, the hapteron sticks fast to it, provided a good contact is made. The momentum of the peridiole causes it to continue in flight until it is checked by the elastic funicular cord. The peridiole is then jerked back by the cord and swings around the object to which the hapteron became attached. In this way, the funicular cord tends to wind itself around petioles (figure 39), stems, etc. If some unwound cord remains, the peridiole may dangle; otherwise it

may be brought into close contact with the stem. Examination of photographs of pendióles found attached to plants in nature shows that the funicular cord actually functions as has been indicated. A pendióle with its cord tightly wound around a stem is shown in figure 38d, and from figure 38b we can appreciate the extent to which a funicular cord may become entangled among the epidermal hairs of a leaf. The manner in which the cord serves to attach the peridiole reminds one of the action of the bolas used in South America to bring down running animals. Occasionally pendióles may land on foliage from above and stick flat to the upper surface of a leaf. When a peridiole has become firmly attached to some object by means of its hapteron and funicular cord, it is anchored there securely. If one allows a freshly discharged peridiole to dry for an hour or so on the object to which it has become attached, it cannot be shaken loose and can seldom be washed off even by prolonged exposure to the full force of cold water from a tap.

bound together in the intact wall they were pulling strongly against one another. The three layers differ considerably in their capacity to absorb water and the form and elasticity of the wall as a whole is the result of the forces operating within it. This elasticity is easily appreciated if one takes a fresh moist peridium, emptied of its pendióles, and compresses it firmly between thumb and finger ; when it is released it always springs back to its original shape. Moreover, when old dry fruiting bodies that may have been flattened in herbarium packets are moistened, they rapidly regain their original shape, provided they have not been treated chemically for preservation. Thus, in nature, the perfect shape of the fruit body of Cyathus, which is of great importance in its operation as a splash dispersal organ, is maintained by its elasticity. In Crucibulum and Nidula the fungus cups are also hygroscopic but the peridium walls in these genera are much thicker than in Cyathus - a feature which alone may be sufficient to prevent deformation by heavy rain.

C / THE WALL OF THE F R U I T B O D Y

D / S H A P E OF F R U I T B O D Y AND A N G L E OF ITS SIDES

Maintenance of the normal operation of the fruiting body (e.g., of Cyathus) is related to its elasticity, by virtue of which it does not become deformed by being battered by rain. Tulasne (1844) investigated the elastic character of the peridium and, according to him, it is the result of the hygroscopic nature of the three layers that make up the peridium wall. The observations of Tulasne and the delightful style in which they are reported make interesting reading recommended to all for profit and pleasure. In essence, Tulasne showed that because of the hygroscopic nature of the three separate layers, opposing tensions are set up within the peridium wall which gives to it great elasticity. When the outer and inner layers are separated from one another and floated free in water, they curve strongly and in opposite directions, indicating that when they were still 88 / The bird's nest fungi

The fruit bodies of Nidula, Crucibulum, and Cyathus have essentially the shape of cups or vases; those of Nidularia and Mycocalia are irregularly globose, but even in the latter genera there is an approximation to the cupulate form when the fruit bodies have cracked open at the top at maturity. This cupulate form is of great significance in the dispersal of pendióles by splash action, as will be shown in greater detail in Chapter 8. When large drops of water were allowed to fall into freshly opened fruit bodies of Nidularia, it was observed that pendióles were irregularly spattered around the fruit bodies, none having been ejected to a distance of more than four inches. Apparently the approximation to a cup shape of the fruit body of Nidularia (and presumably also of Mycocalia)

zontal. In C. olla, the cup flares out very widely at the top and makes an angle of about 60° or less with the horizontal in the flared part. In Chapter 8 experimental evidence is presented which indicates clearly that the slope of the upper third of the fruit body is important to its efficient operation as a spore-dispersing structure. The force of a falling raindrop is transferred to a thrust outwards along the side of the fruit body, and the slope of the plane over which this thrust operates is critical. FIGURE 40 High-speed photograph of the corona built up when a drop of liquid falls into the same liquid. Note the resemblance of the splash corona to the upper portion of the fruit body of Cyathus as shown in Figure 2, x/, approx. Reproduced courtesy Dr H.E. Edgerton

is not structurally adequate to produce the efficient splash that occurs in Crucibulum and Cyathus. Turning to Crucibulum, although the massive fruit body may be almost straight-sided on the outside, on the inside it is obconic. The inner wall is smooth and forms an angle of between 70 and 75° with the horizontal. The fruit bodies of Crucibulum and Nidula are efficient splash guns, although not so efficient as that of Cyathus (Chapter 8). It is evident that the cup shape and angle of the inner wall are both related to the splash action. In Cyathus itself the fruit body is funnelshaped (figures i, 2). Although considerable variation is found with respect to the shape of the cup, not only between different species of Cyathus but even within one species, one characteristic is remarkably constant, namely, the angle which the sides of the inner wall make with the horizontal. In Cyathus stercoreus, in which the cup does not flare out at the mouth, this angle is 70-75°. In C. striatus there are two regions to the cup, a lower region with sides making an angle of 80-85° with the horizontal and an upper flared region with sides making an angle of 60-70° with the hori89 / Fruit body as a spore dispersal mechanism

E / P L I C A T I O N S AND S E T A E

Certain species of Cyathus possess two additional morphological features which may have some significance in the operation of their fruit bodies in splash dispersal; these are (i) the plication or fluting of the peridium wall present in the well-known temperate region species C. striatus (figure i) and in such tropical species as C. poeppigii (figure 6oa,b); (2) the setae or bristles present around the mouth or lip of the fruiting body, of which the most striking example is the tropical C. setosus (figure 57b). In Cyathus striatus both morphological features are present. No experimental evidence is at present available concerning the possible role of plications and setae in the operation of the fruit body as a splash cup and the following points are merely conjecture offered to tempt the curious. First, regarding plication, it is of interest to note (see Chapter 8) that when a drop of liquid falls into a still mass of the same liquid there is built up quickly, from the formerly still liquid, a kind of crater or vase-like mass (figure 40) which bears a striking similarity in form to the fruit body of Cyathus (cf. figure 41). It may be noted that the angle of the sides of the splash crater approximates 70° to the horizontal l and i Actually, in photographs of splash action, it is the continuous rim of the crater or cup which approximates an angle of 60-70°. The peripheral columns into which the liquid of the crater merges form a small angle to the horizontal, which is close to 45° (Chapter 8).

FIGURE 41 Heavy basal emplacements of Cyathus striatus. Note striking plication of cups and prominent setae (especially at lower right). Xz.5

that droplets are thrown out at regular intervals around the crater just as they are (see Chapter 8) from the fruit body of Cyathus. It is suggested: (i) That the striae or flutings of the peridium may somehow provide for a more efficient conversion of the force of the raindrop (falling into the cup) into the outward thrust that accompanies splashing; the striae also might act as grooves along which the pendióle projectiles run prior to 'take-off/ (2) The setae could play a role in enlarging the effective orifice of the splash-cup or they might, again, have something to do with providing a stronger outward thrust to the projectiles or even with directing their flight.2 90 / The bird's nest fungi

F / P O S I T I O N OF F R U I T B O D Y

It has already been noted that young fruit bodies (e.g., of Cyathus) are both phototropic and geotropic and the belief has been expressed

2 It has also been suggested to me that setae may reduce surface tension drag at the instant of departure of a pendióle from the cup. Still another possible function of setae may be that they, like the downward-pointing hairs on the outer wall of the peridium, may discourage very small animals (notably springtails - Collembola) from climbing into the fruit body.

that both responses may be advantageous to bird's nest fungi in developing sporocarps placed so as to receive the full force of raindrops for pendióle ejection. One other feature of the sporocarp (present in the majority of species, but only weakly developed in a few) has to do with the maintenance of that position of the sporophore which is optimal for its operation. In considering the manner in which fruit bodies of the Nidulariaceae are anchored to the substratum, Crucibulum may be examined first. Here it is found that the cup is massive, its walls are very thick, and it is attached at its base to the substratum rather broadly (figure 53). Such a splash cup is able to withstand the battering it must receive from heavy showers without being deformed or dislodged. In Nidula, also, the cup is massive and broadly attached to the substratum. In a large proportion of the species of Cyathus, however, the cup is taller than it is in Crucibulum, is less massive, and is relatively narrow at its base. This feature makes the cups of Cyathus less able to withstand the battering of raindrops. Possibly for added stability, many species of Cyathus are provided with some kind of a mass of hyphae at the base, which Buller (unpublished) called the emplacement. This structure is most strongly developed in Cyathus striatus in which (figure 41) it is a broad solid ball of hyphae firmly attached to the stipe of the cup and often incorporating soil or other substrate material. If dry specimens such as those shown in figure 41 are placed on a flat surface and rocked, they resume their upright position because of the weight of the bottom portion and it is probable that the emplacement serves to enable the fungus cup to maintain the upright position essential to its operation. It has been observed that in species such as Cyathus striatus which regularly produce conspicuous emplacements, these emplacements are larger and firmer in structure when the fungus is fruiting on friable sandy loam than when on twigs or a similar substrate. There may, then, well be a correlation between sub91 / Fruit body as a spore dispersal mechanism

strate and emplacement size, but this has not been tested by actual experiment. G / S U R F A C E AND A R R A N G E M E N T OF P E R I D I O L E S In the genus Cyathus, the pendióles adhere to plants and other objects in nature by means of the hapteron or adhesive end of the funicular cord. In all species that have been examined, no other part of the pendióle or funiculus is strongly adhesive. It has been observed repeatedly that, in C. stercoreus, the surface of the peridiole when moist is slightly gelatinous. This film of mucilage is derived chiefly from gelatinization of the glebal tissue. In nature it does not serve to stick the pendióles firmly to plants, as may be demonstrated easily by removing pendióles carefully from a freshly opened fruit body and allowing them to dry on paper with the funiculus uppermost, so that it does not make contact with the paper. The peridioles stick slightly when dry, but they are easily dislodged by shaking and by washing with water, and it is apparent that the small amount of adhesive material on them would not fasten them securely in nature. Only the hapteron is sufficiently adhesive for this function. In Crucibulum, however, the peridioles are somewhat adhesive over their entire surface. Moistened peridioles will adhere to paper, when allowed to dry on it, even if they are placed on edge. If they are placed with either flat side in contact with the paper, or even with the smooth surface of clean glass, they adhere so firmly that they can barely be dislodged by shaking and only with difficulty by washing. Thus, in Crucibulum, the peridiole projected in nature may adhere to plants not only by its hapteron but also by virtue of the adhesive layer covering the entire peridiole. The cups of Nidula are filled with rather small peridioles lacking funiculi, and the peridioles are highly adhesive. In fresh specimens, the peridioles lie in a mass of mucilaginous material which serves to fasten them se-

curely to any object on which they may land in nature. Nidularia presents the maximum in the development of adhesive pendióles. Here again there is no funiculus and the pendióles are dependent upon their adhesive character for wider dissemination than is accomplished by the rather feeble splashing from the fruit body of Nidularia brought about by rain. The pendióles in this genus are small (about i mm in diameter) and have no depression on either side since they have no funiculi. If a pendióle is moistened and sectioned, it may be seen to be surrounded by a layer of jelly about 20-30 ¡JL thick. This jelly is not quite amorphous, but usually includes a few remnants of hyphae. It appears that the thick layer of jelly is derived from a special layer of hyphae surrounding the pendióle. This jelly swells greatly in water and causes the peridioles to adhere so firmly to any object upon which they may dry that they can be detached from it only by prying them off. The peridioles of Mycocalia are also imbedded in a gelatinous material as they lie in a freshly matured fruit body and, as in Nidularia, this jelly serves to cause them to adhere to vegetation. Regarding the features just described, the Nidulariaceae form, as it were, a series. In Mycocalia, Nidularia, and Nidula there is no funiculus and the peridioles are attached to herbage by their adhesive coats. The peridioles of Crucibulum, although provided with funiculi, are also somewhat adhesive, but the adhesive layer is less pronounced than in the aforementioned genera. In Cyathus, finally, the funiculus is a well-developed organ, the end of which is highly adhesive and in this genus the surface of the peridioles is not adhesive. In overall morphology there is a clearly graded series -Mycocalia, Nidularia, Nidula, Crucibulum, Cyathus - which probably reflects the evolutionary trend fairly closely. When one mechanism replaces another, the first one tends to disappear even if it becomes merely superfluous rather than deleterious. In 92 / The bird's nest fungi

Cyathus a sticky peridiole coat might be deleterious, apart from wasting substance ; for, if the peridiole stuck wherever it hit, it would defeat the operation of the bolas mechanism. We cannot, however, assume a strictly linear series; for Mycocalia and Nidularia, though otherwise primitive, must be considered specialized in peridiole adhesiveness, excelling Nidula (which presumably has an equal need for it). Tulasne and Tulasne (1844) pointed out that, in Cyathus striatus, the peridioles in the cup are oriented so as to lie inclined somewhat towards the centre of the fruit body and are seldom, if ever, horizontal. In this species there are from 12 to 15 peridioles in the cup, and they are usually arranged in four layers with three peridioles in each layer. Thus, if one looks directly down into a freshly opened cup, one sees a layer of three (sometimes four) peridioles (figure i, bottom centre). If these are removed, a second layer of three appears and these are alternate in position with the three that have been removed, and the same alteration occurs in the lower layers. That is, there is no overlapping of peridioles one above the other, but rather a fairly regular alternation in position. A similar arrangement is found in C. stercoreus where, however, it tends to be less regular, for the peridioles are larger and often appear somewhat crowded. In C. olla the peridioles are still more irregularly arranged although they lie alternately above one another, seldom directly above one another. In Crucibulum the peridioles are very numerous and crowded and there does not seem to be any constancy in the manner of their arrangement. Regarding the significance of the arrangement of the peridioles, little can be said at present. Actual experiment shows that peridioles are ordinarily discharged one at a time although this is not invariably true. The arrangement of peridioles in layers may explain this observation (Chapter 8).

8 / Experiments on Splash Dispersal of Pendióles

A / THE G E R M OF THE I D E A That falling raindrops might cause the pendióles of bird's nest fungi to be splashed out of the fruiting bodies was suggested by Ray as long ago as 1686. No experimental evidence to establish this fact, however, was given until Martin (1927) actually succeeded in splashing pendióles of Crucibulum laeve by dropping water into the cups in the laboratory. Martin's evidence also suggested that the pendióles, after ejection, may adhere to vegetation. Another glimmering of the idea that rain splash has some connection with the form and position of pendióles is found in the two editions of Withering's (1776,1792) A Botanical Arrangement of British Plants. Describing what apparently are Crucibulum laeve, Cyathus olla, andC. striatus, Withering noted (see Ed. 2, 1792, Vol. m, p. 446) for his Nidularia campanulata (= Cyathus olla): 'Capsules fixed by threads to the inside of the bell ... When these threads are fixed near the edge of the cup, the cases supported by them are found suspended on the outside.' An interesting note of early date (1830) appears in Withering's British Plants, 7th éd., Vol. 4, p. 443. Referring to Cyathus striatus (as Nidularia striata) is the following: Whether the spheroidal substances, the size of a turnip seed, within the campanulate receptacle, be 93 / Experiments on splash dispersal of peridioles

seeds, capsules, or lenticular bodies containing sporules, the author of 'The Journal of a Naturalist'1 remarks - 'In N. striatus I have drawn out this filament to nearly three inches in length. The thread appears designed to secure the vegetation of the seed by affording it the power of deriving nutriment from the parent plant during the period it is exerting its strength to vegetate in the earth. Heavy rains, I apprehend, fill the bells and float out the seeds in the spring months, the filaments then stretching to their full extent. In severe weather these bells are often emptied of their contents ...' The germ of the realization of the role of raindrops is thus old, but, even after Martin's experiments, more than a decade passed before evidence provided by Diehl (1941) and Dodge (1941) strengthened the view that rain splash was the clue to the old mystery. No thorough investigation of the problem, however, was made until it was begun by Professor A.H.R. Duller in mid-June 1941, and no clear and correct interpretation of the observed facts was published until Duller made the first announcement in May 1942 (Duller, 1942). At this point, I feel that the question of priority by date or by any other standard - is unprofitable pedanticism. As I have stated (Drodie, 1962): i Author anonymous, but ascribed by catalogues to John Leonard Knapp, 2nd ed. London, 1829

This was not a new idea, because many of the older and classical papers and text books indicated vaguely that certain gemmae and similar reproductive bodies are rain dispersed. A careful study of the literature, however, convinces one that what may have been a simple and obvious proposition was never unequivocally stated by the older botanists in the sense of any appreciation of the relationship between the cup shape and the efficiency of dispersal. We must certainly recognize our debt to Professor Duller for pointing out what was so obvious that most people missed it. B / THE E X P E R I M E N T S Now, more than thirty years after the publications of Martin, Diehl, Dodge, and Buller, it may appear unnecessary or even fatuous to record experiments conducted in 1941 by Professor Buller and myself, working together, and later ones conducted by me, experiments and observations that prove that the sporocarps of Cyathus, Crucibulum, and Nidula are remarkable splash-dispersal devices. No 'proof is needed for anyone who has found a number of fruit bodies of Cyathus and is willing to stand beside them in a pelting rainstorm and watch the exciting drama. Before actual experiments were begun, both Professor Buller and I did in fact watch peridioles of Cyathus olla being splash-ejected during a heavy rainstorm in Winnipeg on 15 September 1941. On many occasions since that time, I have watched the process during heavy showers in the West Indies and other places, and always with renewed interest and satisfaction in observing that the fruit bodies 'work' so neatly. As someone once said to me, 'Beautifully simple and simply beautiful/ The principal reason for recording the observations and experiments is that no other information of this sort has been published and that some conclusions to be drawn are germane to the contents of this chapter. Freshly opened fruit bodies only were employed in the following experiments. Various means of securing the sporocarps were used 94 / The bird's nest fungi

but, except where a large number were still firmly attached to a mass of substrate, it was found that the best procedure was to secure a single sporocarp to a thin slab of cork, using insect pins inserted at an angle of about 45° through the emplacement and into the cork. In this way the fungus cup was held upright and care was taken not to damage it in any way. The fungus thus attached to the cork slab rested on the floor of a room having a twelvefoot ceiling, along the top of which ran a number of service pipes to which a burette was clamped. Water drops fell free from the burette through a vertical distance of nine feet, so that each drop was approaching its terminal velocity2 by the time it struck the fungus cup on the floor below. The cork slab bearing the fungus was placed on a mechanical stage to allow ease of movement for adjusting its position so that drops would fall exactly into the centre of the peridium. Adjustment of the tap of the burette allowed the number of drops delivered per minute to be controlled, i.e., the dropping could be slowed to the point where consecutive observations were easily possible. In some experiments, it was desirable to be able to trace the splashing of water droplets ejected from the fungus as well as the position taken up by the ejected peridioles. In such ex2 Although it is unnecessary to enter into a detailed discussion of the physics of the falling of water drops, it may be relevant to note that Humphries (1940) gives a table (p. 280) of precipitation values which brings out the relationship between the diameter of the raindrop and the velocity of its fall in air as follows: diameter of drop (mm) 1.0,2.1,5.0; velocity of fall (metres per sec) 4, 6, 8. In experiments recorded in this chapter, water drops delivered by the burette were 4.7 mm in diameter (as calculated from the number of drops delivered per unit of volume) which, from Humphries's data, would give them a velocity of fall of slightly under 8 metres per sec. Such large drops are perhaps larger than those commonly encountered in rainfall, for Humphries gives the average drop diameter of 1.5 mm for 'heavy rain' and3.o mm for 'cloud burst/ Very large drops are, however, of frequent occurrence under the forest canopy when water drips from leaves.

TABLE 4

Cyathus olla. Splashing experiment no. i Diameter of drops, 4.7 mm; distance of fall, 279.5 cm (9 ft 2 in.) ; number of pendióles in cup, 20 Pendióles Drop discharged

Pendióles Drop discharged

periments, water in the burette was coloured with neutral red and the floor was covered with overlapping sheets of newsprint to a distance of ten feet on all sides of the fungus cup. i / Cyathus olla

From the experiments with Cyathus olla, whose results are shown in Tables 4-7, the following observations were made and conclusions drawn: (a) The fungus cup must be filled with water before pendióles are discharged by water drops. Presumably this moistens the funicular TOTAL 13 20 cord sufficiently to allow it to be broken by the Maximum horizontal distance splashed: for force of the drop. pendióles, 38 cm (i ft 3 in.) ; for water-drops, (b) Not every drop striking the cup causes i76cm (5 ft 0.5 in.) the ejection of peridioles. It appeared that drops striking exactly in the centre of the cup occasionally failed to eject peridioles, whereas those striking very slightly off-centre rarely failed to do so. TABLE 5 (c) Nearly 50 per cent of the drops discharged Cyathus olla. Splashing experiment no. 2 only one peridiole for each drop. Occasionally Diameter of drops, 4.7 mm; distance of fall, 279.5 cm (9 ft 2 in.) ; number of pendióles in cup, 15 two or even more peridioles were ejected together and these might land together or become separated in flight. Pendióles distance (d) A water drop landing dead-centre in the Drop Pendióles cup or very slightly to one side of centre prono. discharged cm ft in. duced a distinctive sound, a hollow 'plop/ 3 together, 4 in. whereas drops hitting nearer the periphery of 1 6 10 2.5 2 together, i in. the cup made only a slight splashing sound. *-5 i separate, 6 in. (e) To the extent that it could be followed by 1 2 ift the eye, a peridiole in flight appeared to de2 68.6 2 ft 3 in. 3 82.5 2 ft 8.5 in. scribe a curve. 1 43.2 i ft 5 in . 4 (f) The maximum horizontal distance to 0 — 5 which any single peridiole of Cyathus olla was — 6 O projected from its fruit body in these experitogether, 7 in. 17.8 7 3 — ments was 82.5 cm (2 ft 8.5 in.). The average 8-17 0 1 18 66.0 2 ft 2 in. distance to which thirty peridioles were — 19-20 0 splashed was 25.4 cm (10 in.). Peridioles of 21 78.7 2 ft. 7 in. 1 this species are large and rather heavy, the funiculus is more loosely constructed than in TOTAL 21 15 some other species, and the outer lip of the fruit body flares out widely. Any or all of these Maximum horizontal distance splashed: for features may account for the observation that pendióles, 82.5 cm (2 ft 8.5 in.) ; for water drops, 137 cm (4 ft 6 in.) peridioles of C. olla are not shot out to as great i 2 3 4 5 6 7

4 separately i only i only i only i only 2 together 2 together i separately

8 9 10 11 12 13

i only i only 2 separately i only i only i only

95 / Experiments on splash dispersal of peridioles

TABLE 6

Cyathus olla. Splashing experiment no. 3 Diameter of drops, 4.7 mm; distance of fall, 279.5 cm (9 ft 2 in.) ; number of pendióles in cup, 10 Horizontal distance from cup

Pendióles Drop discharged cm i

i

2

2

3-5 6 7

32- 2 62. 2 53- 3

ft in 1ft

1at

1at

5- 5 in

2 fto. 5 in. i ft 9 in .

O 1 1

8

o

9 10 11

i o i

12

2

13

1

TOTAL 13

10

8. 2 4 in. 16.5 6 •5 in

62.2 2 ft 4 in. 16.4 8 in. 7.6 (together) 3 in. 17.6 8.5 in.

Maximum horizontal distance splashed: for water drops, 122 cm (4 ft) ; for pendióles, 62 cm (2 ft 0.5 in.)

TABLE 7

Cyathus olla. Splashing experiment no. 4 Diameter of drops, 4.7 mm; distance of fall, 279.5 cm (9 ft 2 in.) ; number of peridioles in cup, 8

Drop

3 4 5 6 7

2, together i only i only i only i only i only i only

7

8

i 2

TOTAL

Peridioles discharged

Horizontal distance from cup cm 5-1

20.3 20.3 48.5 21.6

54.6 12.7

ft in. 2 in. 8 in. 8 in. i ft 7.5 in. 10.5 in. i ft 9.5 in. 5 in.

Horizontal distance splashed for three water drops farthest from the cup: 155 cm (5 ft i in.) ; 152 cm (5 ft) ; 115 cm (4 ft 9 in.) 96 / The bird's nest fungi

a distance as has been recorded for other species in the experiments that follow. 2 / Crucibulum laeve The records given in Tables 8 and 9 constitute but two examples of experiments conducted using Crucibulum. They were performed at Indiana University in 1947 and the manipulations were identical with those described under Cyathus olla. From experiments no. 5 and no. 6 it is evident that, in Crucibulum laeve, the peridioles, which are smaller than those of Cyathus olla, may be shot much farther. The maximum distance to which peridioles of Crucibulum have ever been observed (by the author) to be ejected in any actual experiment is 129.5 cm (4 ^ 3 in.), as indicated above. Tables 8 and 9 indicate clearly (and Tables 4-7 suggest) that the flight of peridioles becomes longer as the cup becomes less crowded with peridioles. When the cup is still rilled with peridioles, much of a raindrop's kinetic energy is probably absorbed through forming eddies thus reducing the thrust on any peridium that is in a position to be ejected. 3 / Cyathus stercoreus When abundant material had been obtained (at Indiana University, 1947) of this species with its large smooth peridioles, three splashing experiments were conducted using freshly opened peridioles. Water drops were allowed to fall ten feet and the distances to which peridioles had been ejected were measured at the conclusion of each experiment. The results in abbreviated form are shown in Table 10. In these experiments, most of the peridioles landed about two to three feet from the cup. A single peridiole was located at four feet in experiment 8. This seemed to be a record for distance. However, upon examining the laboratory carefully for stray peridioles, one was located adhering to the window pane, far to one side of the paper placed upon the floor to receive them. As this window was some

TABLE 8

TABLE 1O

Crucibulum laeve. Splashing experiment no, 5 Distance of fall of drops, 305 cm (10 ft) ; number of pendióles in cup, 21 ; number of pendióles discharged bv 8 consecutive drops, 11

Cyathus stercoreus. Summary of three experiments: distance of fall of drops, 305 cm (10 ft)

Maximum distance of any pendióle from cup

Horizontal distance from cup

Pendióles discharged

cm

ft in.

2, together i 2, together i i 2, together i i

15.2 17.8 45.8 48.3 76.2 76.2 86.3 88.8

6 in. 7 in. i ft 6 in. i ft 7 in. 2 ft 6 in. 2 ft 6 in. 2 ft loin. 2 ft 11 in.

Maximum horizontal distance to which pendióles were splashed, 89 cm (2 ft 11 in.). Ten peridioles were still left in the cup when the experiment was terminated.

Experiment No. of no. drops 7

8

8

20

9

10

No. of pendióles ejected cm 4 *4 8

Peridioles discharged 2, together i 3, together 3, together i i i i i

Horizontal distance from cup cm 1O.2

15.2 17.8 22.9 32.0 76.2 83.8 99.0 129.5

ft in. 4 in. 6 in. 7 in. 9 in. i ft i in. 2 ft 6 in. 2 ft 9 in. 3 ft 3 in. 4 ft 3 in.

Maximum horizontal distance to which peridioles were splashed, 129.5cm Í4 & 3 m -)- Fiye peridioles were still left in the cup when the experiment was terminated. distance from the floor the distance of the peridiole from its cup was measured by stretching a string from the peridiole to the cup. Allowing for a slight error resulting from tighten 97 / Experiments on splash dispersal of peridioles

78.7 2 ft 7 in. 238.8 7 ft 10 in. 111.7 3 ft 8 in.

TABLE 11

Cyathus striatus. Summary of three experiments: distance of fall of drops, 305 cm (10 ft)

Maximum distance of any pendióle from cup

TABLE 9

Crucibulum laeve . Splashing experiment n °-6 Distance of fall of drops, 305 cm (10 ft) ; numbe r °* peridioles in cup, 19 ; number of peridioles discharged by 8 consecutive drops, 14

ft in.

Experiment No. of no. drops

No. of peridioles ejected cm

10 n 12

8 n 8

10 14 8

ft in.

45.7 3 h 6 in. 170.2 5 ft 4 in. 12 9-5 4 it 3 in.

ing the string to straighten it, it was found that this particular pendióle had been thrown a horizontal distance of 7 ft 10 in. ! The specimen was shown to students and to colleagues as witnesses of the record given above. 4 / Cyathus striatus In 1949 numerous large specimens of this species were found on the campus of Indiana University and splash tests were made as before, the results of which are summarized in Table 11. Two points may be noted regarding the splash performance oí C. striatus. First, in the three tests, 27 peridioles were ejected singly by

32 consecutive drops; and, second, the maximum distance recorded in all three experiments was higher than for most experiments using C. olla or Crudbulum laeve. 5 / Nidula niveo-tomentosa Fresh specimens of this fungus were sent to the author in November 1949 by Mr Lindley Carson from Oregon. Experiments were conducted as before. Unfortunately the records of these have been lost and they can only be reported in general terms : pendióles tended to be splashed out in groups of two to four; most pendióles were projected about two feet from the fruit body and only two or three reached a distance of over three feet. From all the evidence presented, there can be no doubt that the pendióles of the bird's nest fungi are ejected from fruit bodies by the force of heavy raindrops. Peridioles may fly out singly or, occasionally, more than one may be thrown out by a single raindrop. The maximum distance to which any pendióle was observed to be thrown was 240 cm (7 ft 10 in.) (see expt. 8) ; but, considering all observations, it seems that peridioles of most species are usually thrown to a distance of 45-75 cm (ca. 1.5—2.5 ft). Moreover, of those species examined, it is apparent that the sporocarps of Cyathus striatus are the most effective as 'water guns/ 6 / Artificial cups During the course of these experiments, I was frequently impressed with the difference in the sound produced by a 'direct hit' splash and that produced when the incident drop fell slightly off centre. In a direct hit, a low-pitched 'plop' was heard; in an off-centre hit, a high-pitched splashing sound was heard. Moreover, in a direct hit, the initial 'plop' was followed quickly by the sound of a number of small splashed drops showering down on the surrounding paper. These observations, coupled with thoughts about the angle of the peridium wall, led to the concept that the physical nature 98 / The bird's nest fungi

of the splash action might vary depending upon the structure of the splash cup and upon the way in which the raindrop plummets into the cup. In an endeavour to gain some insight into this problem, the following study was made. Ten artificial fungus cups were made of modelling clay with approximately the dimensions of actual sporocarps of Cyathus. They were moulded in a series graded in the slope of their sides from almost flat to almost vertical, the length of the sides being nearly the same in all the model cups. The cups were filled with neutral red solution (in the flattest cups, only a large drop or two could be retained) and drops of neutral red solution were allowed to fall into each cup from a height of nine feet. Each cup was arranged on a large sheet of white filter paper in order that the staining of the paper by drops of dye splashed from the cups could be examined. The results of this experiment are striking and are presented in diagrammatic form in figure 42. For simplicity, the forms of all ten cups are not recorded. Instead three types only have been chosen since the results are well represented by so doing. Splashing from a flat or low-angle cup seldom projected any dye beyond an inch around the cup. Local splash was also characteristic of cups in that part of the series where the cup sides were nearly parallel. However, from cups whose walls made angles of from 60 to 75° with the horizontal, splashes resulted in the ejection of numerous small droplets of dye to distances up to four feet from the cups. Thus it appears that when a drop of water falls into a fruit body of Cyathus, the resolution of the force inherent in the falling drop is such as to cause small drops to be thrown out laterally to a considerable distance, and that this ejection of drops is more marked than it would be if the fungus cups had either flatter or steeper sides than they actually have. From the foregoing considerations it is reasonable to assume that the fruit bodies of common bird's nest fungi are of such size and shape as to secure the maximum lateral p rojee-

CUP TYPE

DIMENSIONS

SHAPE AND ANGLE OF SIDES

SPLASH AS SHOWN BY DYE

8 mm mouth 10 mm height 80°

II

8 mm mouth 10 mm height 65C

X>

P

III

18 mm mouth 4~6 mm height 15° ±

FIGURE 42 Diagram of results of experiment to demonstrate spread of liquid as an irregular splash or as discrete drops depending upon angle of sides of cup from which liquid was ejected by falling drops 99 / Experiments on splash dispersal of pendióles

tion of their pendióles as a result of the action of the outward thrust that accompanies splashing, Until records by means of high-speed photography are available, speculation regarding the fungus projectile serves only to arouse curiosity. From ballistic considerations, one would expect the maximum range of a projectile in the normal parabolic trajectory to come from a

100 / The bird's nest fungi

firing angle of 45° above horizontal. Bird's nest cups have side walls of 60-75° above horizontal ; however it is probable that surface tension between the projectile and lip of the cup may pull the angle of projection down to about 45°. Moreover, in some species of Cyathus, the lip is flared out or flattened so that the firing angle may actually be less than the angle of the side of the fruit body.

9 / Occurrence, Distribution, and Ecology

A / GENERAL The following records of the occurrence and distribution of bird's nest fungi will do more to raise questions than to answer them. The justification for including them lies, it is hoped, in their value as a possible stimulus for future investigations and in whatever interest may be aroused by the unusual circumstances under which these fungi are occasionally found in their natural abodes. A bird's nest fungus is like gold, which, as the early settlers used to say, 'is where you find it/ One can characterize only in general terms the sites in which the Nidulariaceae may be sought with some expectation of finding them. In temperate regions and in the tropics, they grow and fruit most frequently in moist, partly shaded sites. The sites are rarely in the most heavily vegetated or the most deeply shaded habitats; rather the fungi flourish along the edges of woods on trails or around lighted openings in the woods. Particularly in forests where most of the precipitation is in the form of mist and drizzle, large water drops effective in discharging pendióles result from the runoff from large leaves of the forest canopy. In dense cover, deep mosses and other vegetation may interfere with effective peridiole dispersal by large drops, whereas at the forest edge (e.g., by a trail) run-off drops fall vertically and peridiole dispersal is doubtless more effective. 101 / Occurrence, distribution, and ecology

In contrast, some grow in such places as pastures and grain fields where continuous moisture would seem to be precarious and shade is very local. A few species grow under moderate desert conditions and others grow in the subarctic where surrounding vegetation may be sparse. However, when the total information as to sites is reviewed it seems probable that these sites have in common chiefly physical (possibly some chemical) features of the microenvironment rather than of the macroenvironment in a broad and geographical sense. Thus in a generally flat, open woodland in temperate North America, one is more likely to find Cyathus striatus growing upon decaying twigs and leaf-mould in a moist depression of the ground than upon the same substrates in elevated areas of that woodland where the surrounding vegetation is essentially the same as in the depressed area. Here, moisture seems to be the important difference ; but in other instances it is difficult to believe that moisture is the prime factor of the environment determining the site of fruiting. B / OCCURRENCE i / Some common substrates (a) Horse dung and cow dung. Five species are known to grow upon old, rather dry dung of horses and cows, namely Cyathus stercoreus,

C. costatus, C. fimicola, C. pygmaeus, and Crudbulum laeve. Of these, Cyathus stercoreus, worldwide in distribution, frequently is seen in pastures growing upon masses of old cow or horse manure. It also is found frequently on the soil of gardens, plant nurseries, or greenhouses, to which manure has been added; there is no record of this fungus having been found on a substrate completely isolated from any contact with manure. C. costatus and C. fimicola are both found only in the tropics and their only known substrate is manure. Crudbulum laeve is primarily a lignicolous species, but it may sometimes fruit abundantly upon old cow dung or horse dung. Specimens of this species from these substrates do not appear to be taxonomically distinguishable from those that grow on wood or on plant fibres. Cejp and Palmer (1963) have recorded the occurrence of species of Mycocalia on the dung of rabbits and sheep. Cyathus gay anus has been recorded as growing on horse dung, but in my experience old wood is the commonest substrate for this species. If, in fact, bird's nest fungi do not grow (or at least do not fruit) upon other kinds of dung, it would be interesting to know why. One other point regarding the dung substrates; it seems extraordinary that there should be no records of the fruiting of bird's nest fungi on deer dung. In preagricultural times this must have been widely available, whereas the dung of domesticated animals would not have become available until a later date. The ecological implications of coprophily seem to be obvious. Pendióles discharged by rain-splash adhere to vegetation and a limited dispersal of the basidiospores away from the fruit bodies in terms of distance is thus effected. A much longer-range dispersal of spores is effected if cattle, horses, and other animals ingest vegetation to which peridioles are attached, for then the spores may be liberated in the animals' faeces at any distant point over which the animals roam. 102 / The bird's nest fungi

(b) Wood. A very large proportion of the species of the Nidulariaceae are found fruiting upon wood and some are never found on any other substrate. The wood substrate is almost invariably old but only partially decayed by other fungi. Bird's nest fungi never occur, as far as is known, on wood which is decomposed to the point where it is reduced to pulp or punk. On the other hand they are not usually found, in their natural environment, growing upon entirely sound wood such as freshly downed trees or fresh lumber; they have, however, been seen to fruit upon wooden pot labels in a greenhouse (figure 18). There is no information at present as to whether or not mycelium of any member of the Nidulariaceae must derive some essential nutriment from the wood upon which the fruit bodies are frequently produced. Indeed there is some evidence (Chapter 5, c) to indicate that wood commonly serves merely as the substrate (in a physical sense) for the development of fruit bodies. Examination of a list of the kinds of wood upon which bird's nest fungi have been found does not bring to light any clearly defined correlation between the fungi and their substrates. However, a careful search for correlation might be profitable. Two reasons for this suggestion are the following: (i) when one finds the common species Crudbulum laeve fruiting in great profusion, it is impressive to note that the sporocarps are very rarely found on gymnosperm wood although, in a small area, fruit bodies may be found attached to bits and pieces of many kinds of angiosperm wood ; (2) Cyathus africanus from Kilimanjaro is known only to fruit upon wood of the gymnosperm Cupressus lusitanica. The first observation suggests that the resinous materials present in many gymnosperm woods are inimical to the growth of mycelium and the formation of fruit bodies, but this idea has not been tested by experiment. The hard woody culms of bamboo also seldom appear to be the site of fruiting of bird's nest fungi under natural conditions in tropical forests: bamboo is very durable and perhaps its

smooth hard surface is unsuitable. In the author's herbarium there is a single collection of Cyathus setosus that had evidently been growing on bamboo, and one abundant collection of C. berkeleyanus was obtained from old wet bamboo logs used in the construction of a pigsty in Jamaica. However, as described later, bamboo commonly harbours bird's nest fungi when short pieces are used as pots for plant propagation in tropical nurseries. Here, they grow on the inner culm surface or the cut ends which are much less impervious than the glossy outer surface of intact bamboo culms. In the forest, one finds bird's nest fungi fruiting upon small twigs or branches, especially where they lie on damp ground and are partly imbedded in or surrounded by soil and such small green plants as low mosses. Where twigs and branches are bearing fungi well above the ground, the site is usually a pile of dead branches in a very moist and partly shaded place. In my experience, both in the tropics and in temperate regions, large logs have seldom yielded large numbers of fruiting bodies, and when they have it has been in very damp locations or where one end of the log projected into a body of water which kept the wood constantly moist. It is exceptional to find bark, especially if separate from the tree, as a site for abundant fruiting. Finely divided bits of wood such as sawdust or chips often harbour certain species; Crucibulum laeve sometimes grows upon sawdust spread over the soil, and it has also been found on chips around abandoned habitations. In Jamaica, Cyathus pallidus was found growing in abundance on chips around old log piles. It was noticed that the chips bearing bird's nest fungi were invariably those that had become partially imbedded in soil. Such chips were noted to be the only ones that did not become very dry during the heat of the day and it seems probable that they retained enough moisture to allow the fungi growing upon them to develop without interruption. The same observation has been made repeatedly in temperate regions: 103 / Occurrence, distribution, and ecology

in very exposed areas where specimens of the fungi are scarce, one can almost always find a few on chips of wood or bits of twigs that are partly imbedded in soil, even where none are found on wood that is free of the soil and which can dry out rapidly. Old boards seem to be a favourite fruiting site for such species as Crucibulum laeve and Cyathus striatus, especially where the boards lie in a damp shaded spot. The old-fashioned practice of retaining garden soil along the edge of a garden path by means of narrow boards often provides an ideal site for the fruiting of the above species and especially for Cyathus olla which seems to have a marked predilection for fruiting on old boards. In the West Indies, the author has found Cyathus poeppigii and C. setosus growing on old board edging in gardens. Driftwood that lies in low damp spots after the recession of the water that transported it frequently supports sporocarps of some members of the Nidulariaceae. Crucibulum laeve, sporadically, may be found on damp driftwood along rivers and streams and around the edge of lakes. Also, Nidularia is often found on driftwood and the little-known Cyathus helenae has been collected occasionally on this substrate. (c) Dead herbaceous stems. Dead stems, usually of herbaceous perennials if these are moderately woody, provide a substrate upon the bases of which bird's nest fungi often produce their fruit bodies. In this instance, the fungus cups develop just at ground level. In the prairie provinces of Canada, Cyathus olla not uncommonly fruits on dead stems of many garden plants such as Chrysanthemum, Lychnis, and others. In Idaho, collectors find Crucibulum laeve and Cyathus helenae growing on the dead basal parts of a variety of woody desert plants such as Artemisia. In British Columbia, Nidula niveo-tomentosa fruits frequently on the dead rachises of the fern Pteridium. And, in more general terms, bird's nest fungi are often found on dead plant trash especially when it is made up of partly

FIGURE 43'The Whole Story in a Nutshell.' Fruit bodies of Crucibulum laeve that have developed on old hosk of a nut. X 5 104 / The bird's nest fungi

rotted bits of herbaceous stems. Dead corn stalks form a common habitat for Crudbulum laeve in North America; thousands of fruit bodies are occasionally seen growing on old prostrate stalks in corn fields. Knowing this, the author was surprised at the complete absence of Nidulariaceae on dead stalks of another grass, sugar cane, in Jamaica and elsewhere in the West Indies. In continental Europe and in England, Cyathus striatus and C. olla often fruit upon or in close association with the dead culms of wheat and other grains - the origin of the old English name corn-bells for the bird's nest fungi. (d) Leaves. Bird's nest fungi apparently rarely fruit upon dead leaves unless these are mixed with small stems or soil. One exception only can be cited from my own experience. On the campus of Indiana University in September 1950, Crudbulum la eve was found growing in great abundance on a thick matting of leaves of Scots Pine under a pure stand of these trees. The occurrence was so unusual that care was taken to examine the pine needle mat carefully and it was observed that the mycelium and fruiting bodies were growing directly upon pine leaves which were not mixed with soil to any significant degree. (e) Plant fibres and fibre artifacts. Anyone in search of bird's nest fungi is very likely to find them if he or she will examine masses of plant fibre lying on the ground in very moist places. This fibre may be in loose masses as it is released from the parent tree, such as one sees so often on the forest floor in the tropics, or may be masses of fibre woven into mats or cloth or almost anything fashioned by man into useful articles. Coco, jute, and hemp fibre in the natural state or as artifacts are often covered with large numbers of bird's nest fungi, such as Cyathus olla or C. striatus in temperate regions and C. poeppigii or C. montagnei in the tropics. Examples of unusual finds on fibre matting and sacks are given below. There are numerous records in the literature of bird's nest fungi being found on 105 / Occurrence, distribution, and ecology

rotting cloth (e.g., Lloyd 1906). Although not all such records have been checked, personal experience leads to the belief that the cloth harbouring these fungi is always composed of plant fibre. Apparently bird's nest fungi have never been found growing upon materials of animal origin, such as wool and fur. (f) Nuts and other fruits. Woody fruits, and especially the shells of nuts, sometimes yield sporocarps of the Nidulariaceae. In temperate North America I have found only Crudbulum laeve growing on this substrate (figure 43), but in the tropics a number of species may be found on woody fruits. The only exception worthy of note is that neither the outer husk nor the inner hard coat of the coconut was found to harbour bird's nest fungi, although many other kinds of fungi commonly grow on old coconuts. (g) Soil. Although there are numerous published records of the occurrence of bird's nest fungi on soil, it is my observation that the mycelium is usually attached to sizeable bits of wood or stems included in the soil. These fungi apparently do not fruit on fine soil composed largely of mineral matter. 2 / Some red-letter finds A student of any group of plants or animals is delighted whenever a find is made involving an uncommon species or a spectacular quantity of any of the commoner species. Many of the author's botanically minded friends are unable to find bird's nest fungi even when given specific directions as to where to search and they are inclined to regard accounts of good finds as fish stories. A few examples only offish stories about Nidulariaceae are recounted herewith, accompanied by some photographic evidence to vouch for the fact that the fish did not escape. On two occasions that can be recollected from personal experience, bird's nest fungi appeared in enormous numbers and distributed over wide areas. The first was in Michigan in the autumn of 1929 when Crudbulum laeve

FIGURE 44 a, Cyathus montagne* on rottein jute sack beside stream in Guadeloupe, xo.5 ; b, C. berkeleyanus on bamboo pot in Hope Botanic Garden, Jamaica, xi 106 / The bird's nest fungi

was to be found on dead stalks of corn (Zea mays] in almost every corn field in the area. The second occasion of such wholesale development of sporocarps was in Bloomington, Indiana, in 1948 when my attention was drawn to a suburban one-acre garden plot on which manure had been spread in the spring. In late August, the garden was literally covered with fruit bodies of Cyathus stercoreus. On that occasion a count was made: in some parts of the garden as many as fifty fruit bodies per square foot had developed. On the campus of Indiana University during the late summer of 1949, Cyathus striatus, which is seldom abundant, developed in prodigious numbers, growing on old boards that formed the edge of an asphalt walk across part of the campus. For over a hundred yards on both sides of the asphalt, sporophores developed in almost contiguous fashion. An interesting feature of this phenomenon was the size of the fruit bodies of this species, which were the largest and broadest that I had ever seen. In Jamaica and elsewhere in the West Indies, bird's nest fungi (chiefly Cyathus berkeleyanus and C. limbatus) have been found growing on 4-inch bamboo pots, such as are used in the nurseries of many botanical gardens in the tropics. The fungi were found growing on more than half the pots examined, and commonly as many as twenty fruit bodies could be found on a single pot (figure 44b). On the last occasion of a visit to the bamboo pot sites in Jamaica it was found that nursery practice had changed; commercial plastic pots were beginning to supplant the old hand-cut bamboo ones. It was most interesting to observe that no fungi were found growing even on the soil in the plastic pots, whereas most bamboo pots yielded a few fruit bodies. From discussion with gardeners it was learned that exactly the same soil mixture went into the two kinds of pots and one might conclude that some characteristic of bamboo provided the proper physical conditions for fruiting. In July of 1969,1 observed Crucibulum laeve 107 / Occurrence, distribution, and ecology

growing on small bits of driftwood about 5-10 ft from the water's edge completely around Two Jack Lake near Banff, Alberta. Most fungus-bearing bits of driftwood (mostly poplar) carried a half dozen or more fruit bodies. At one point, eighty-five mature fruit bodies were taken from an area of one square foot and the fungi were present in almost equal density around the several miles of lake shore. So much for large numbers. For sheer density of fruit bodies per unit area of substratum two records from personal experience may be noted. Some years ago there was sent to me a large coco-fibre mat which had been lying on damp ground just outside a greenhouse on the campus of Duke University. The mat, which measured 4.5 ft x 6 ft was literally covered on its upper surface with large fully-developed sporocarps of Cyathus stercoreus, the fruit bodies standing so crowded in places that a finger could scarcely be put down on the mat anywhere without touching one of them (figure 45). I found a second example of a dense population of sporocarps in 1966. Two large old jute sugar sacks which lay at the edge of a stream close to St Claude on the West Indian island of Guadeloupe were almost covered with fruit bodies of the beautiful species Cyathus montagnei, growing so thickly that their edges were nearly all in contact (figure 443). Not as examples of the abundant occurrence of bird's nest fungi but rather as examples of finding fruit bodies where they would hardly be expected, a limited number of records may be noted. The first of these is the occurrence of Cyathus helenae on alpine scree (Brodie, 19663). As the fungi in this instance were actually attached to and doubtless derived nourishment from the roots and stems of dead alpine plants, their presence at an altitude of 7000 ft and on a scree (which seems an unlikely habitat for fungi) was surprising because such a habitat had not been recorded previously. On the subject of altitude, it may be noted that the author has in his herbarium a collection of Nidula emodensis collected by the Mount Everest climbers at an altitude of 13,000 ft on

FIGURE 45 Cyathus stercoreus fruiting on wet coco mat. Note crowding of fruit bodies, xo.25

108 / The bird's nest fungi

that mountain. No detailed information regarding this collection is available, but since that elevation in the Himalayas is at or even below tree line, it is probable that the site was not devoid of shrubs. The lower levels of the hills of the dry Peruvian coastal plain around Lima are almost devoid of vegetation. As one climbs the hills (e.g., near Attacongo) however, one reaches a level at which scant precipitation occurs in the form of dew. This level, called the loma, does support scant vegetation in the form of low isolated tussocks of woody plants. Because of the report that fungi sometimes are seen on the loma, I searched there in 1966 for Nidulariaceae with the almost certain expectation of finding none. It was therefore a great surprise to find well-developed typical specimens of Cyathus olla in this unlikely site. A few of the fungi were to be found with fair regularity on debris under many of the larger and denser plant tussocks. Still other examples from my personal experience of what (as far as macroenvironment is concerned) would be classified as xeric habitat are known which permit the development of sporocarps of the Nidulariaceae. At least four species, namely Crucibulum laeve, C. parvulum (Brodie, 19/oc), Cyathus pygmaeus, and C. helenae have all been found in fair abundance attached to old stems of such desert plants as Artemisia and Sarcobatus in very dry areas of Idaho where the rainfall is usually below 10 inches annually. On the exposed tops of buttes in the dry valley of the Red Deer River in Alberta, Canada, Crucibulum laeve and Cyathus helenae occur (although they are scarce) under the shade of prostrate vegetation such as Juniperus horizontalis. Many examples of bird's nest fungi becoming available precisely when they were required could be given. Some of these examples have already been recorded (Brodie, 1962^) and need not be included here. Besides, fish stories, like jokes of doubtful nature, should be recounted with circumspection.

109 / Occurrence, distribution, and ecology

C / D I S T R I B U T I O N AND E C O L O G Y It has been established that, despite some variation in the efficiency of the fruiting bodies of the Nidulariaceae among the different genera, in these fungi the first step in distribution depends upon the splashing action of raindrops (Chapters 7, 8), with a few minor but very interesting exceptions which will be considered later. Spore distribution by rain-splash, however much it may command interest and admiration, is nevertheless of only local importance in the overall geographic distribution of bird's nest fungi. Although a substantial body of information regarding rain-splash (short-range) dispersal is now available, little information exists as to the means of long-range dispersal of these fungi within a region or, more widely still, over and between continents. Information could be given summarizing the known distribution of Nidulariaceae as far as can be determined by published records and from the author's herbarium. However, except for a few interesting questions raised, little or no value can be derived from the records. It may be interesting to know that Ni dula niveo-tomentosa occurs in extreme and lonely isolation on the Blue Mountains in Jamaica, almost three thousand miles from the nearest spot where it is also known to occur commonly (in California). It would be interesting to know what mechanisms of distribution of this fungus could have been operative in producing such an apparent disjunction in distribution. The word apparent is important because it is always necessary to remember that distribution records reflect largely the wanderings of plant collectors and the accessibility of certain geographical areas. i / Known and possible means of wide distribution Something, perhaps quite a lot, can be learned by considering, first of all, what is known about and what may be usefully inferred from

the facts concerning long-range dispersal of bird's nest fungi. As there does not seem to be any basis for considering the following observations and records in any logical order, they are merely listed in sequence as they appear to be important and well founded or else plausible. We must first realize that there are three possible ways in which bird's nest fungi may become established in a new site to which they may have been transported. 1 / By means of living mycelium or living whole fruit bodies. Actively growing mycelium if transported from one place to another within or upon any of the substrates dealt with above (Chapter 10, B, i) and if accompanied by enough of the substrate to allow continued growth could effect introduction into a fresh site. There is little evidence from direct observation as to how long mycelium will survive the chief hazard of such transport, namely desiccation. Mycelium in culture tubes, in my experience, is occasionally found to be viable as much as a year after the culture was established and without retransfer to fresh medium having been made. Species differences and even strain differences however are such that no dogmatic statement about mycelial longevity can be made. Transfer of whole fruit bodies in nature is probably a less common occurrence than transfer of mycelium. Fruit bodies are probably most frequently transferred on driftwood, but again nothing can be said about their longevity. 2 / By means of peridioles which may germinate as a whole. The peridioles of most specimens of bird's nest fungi which I have examined have been found to be capable of growth when planted on nutrient agar (figure 38a), provided the specimens were not over two years old and had not been subjected to dry heat or to preservative fumigants. When a whole pendióle is planted on nutrient agar, hyphae of the tunica may begin to grow in a matter of hours. Failing this, the hyphae of the cortex may grow, and occasionally even hyphae from the inner portions of a peridiole. 110 / The bird's nest fungi

The mycelium produced by a peridiole germinating as a whole is dikaryotic and (normally) genetically identical with mycelium of the present fruit body. There is no evidence that basidiospores locked within a peridiole can germinate ; but even if they could, the dikaryotic mycelium formed from tunica or cortex hyphae would doubtless prevail by invading the available substrate before hyphae from basidiospores could do so. 3 / By means of basidiospores which have become freed from the enclosing peridiole cortex. Evidence is presented (Chapter 9, xa and Brodie, 194^, 1956 [p. 158]), which suggests that basidiospores are ultimately freed from the peridiole and from one another. Just how this liberation occurs is not known for certain at present. The cortex of a peridiole is a very firm coating which I have never seen split open of its own accord, though it is possible that it may eventually break down as the result of weathering. For coprophilous species, the peridiole wall may be digested in the alimentary canal of a herbivorous animal. Basidiospores thus set free may become scattered in the contents of the animal's digestive tract and finally deposited on the ground in the excrement. (a) Herbivorous animals. Because the fruit bodies of coprophilous species, as noted above, are developed on the dung of herbivores or upon soil into which manure has been incorporated, it is a reasonable assumption that horses and cattle are important agents in the longdistance transport of such species as Cyathus stercoreus. It has not, as yet, been proven by testing that the spores remain viable after being ingested along with peridiole-laden foliage. However, the fact that the bird's nest fungi appear on cow and horse manure, with no other known means of having been transported there, suggests strongly that spores do pass undamaged through the animals' alimentary tracts and germinate in the faeces. A further piece of suggestive evidence in this hypothesis is the fact that spores of C. stercoreus can withstand a temperature of at

least 40° c and indeed may not germinate at all unless heat is applied (Brodie, 19483^). Neither domestic horses nor cattle normally roam very far, but the author rests his case that they, and probably other herbivores, are the chief agents of dispersal in terms of a few miles for all coprophilous species and possibly also for some non-coprophilous ones. (b) Agricultural practices. At least two probable means of wide distribution of pendióles of the Nidulariaceae involve human agricultural and horticultural practices. First, that peridioles may be distributed as contaminants in seed packets has been observed a number of times. In 1944 a sample of seeds of meadow fescue grass (Festuca elatior) was sent to me by a laboratory for seed analysis and germination testing at Winnipeg, Canada. A number of small objects were found among the grass seeds and the laboratory had been unable to identify the contaminant as seeds known to the analysts. As may be guessed, the objects were pendióles of the bird's nest fungus Cyathus stercoreus. Pendióles must have been attached in large numbers to the fescue itself or to other plants in the field. When the crop was threshed, peridioles were broken away from the crop plants and thrown in with the seeds. Had that fescue seed, mixed with peridioles, been sown subsequently, it is very probable that the fungus would have been introduced into crop land at a place far distant from the point of origin of the peridioles. Second, there is considerable evidence that peridioles or mycelium, or both, may be transported on nursery stock. There are numerous examples throughout the literature of tropical species of Nidulariaceae appearing adventitiously in pots of greenhouse plants such as palms growing in temperate climates under glass (Lloyd, 1906). The most plausible explanation of the appearance of the exotic fungi seems to be that mycelia or peridioles were present on cuttings or whole plants (or possibly among seeds) imported from the tropics. That peridioles may be the means of such introductions is suggested by Dr Diehl's report (1941) 111 / Occurrence, distribution, and ecology

of finding Cyathus pallidus associated with Camellia plants in Florida. The origin of the Camellia cuttings referred to by Diehl is unknown, but his finding of this species of Cyathus, which constituted the first continental North American record of occurrence, is probably to be explained by the adhesion of peridioles to the leaves or stems of the Camellia plants. One more example of the possibility of long-range dispersal on nursery stock is provided by the finding of a rather rare form of Cyathus olla (forma anglicus) far removed from the only other known sites, Oregon and England (Brodie, 19523). The concluding paragraph in the reference cited is quoted in full: The Edmonton (Canada) plants were growing on rotting boards covered with fine twigs and leaves in Garneau, one of the oldest residential districts. More exotic trees and shrubs grow in this area than elsewhere in Edmonton, these having been introduced from England and continental Europe many years ago. It is likely that C. olla forma anglicus (which Lloyd believed to be 'solely English, for I have seen no specimens from any other locality' Lloyd, 1906) was introduced into Western Canada and Oregon on nursery stock.

(c) Rafting on wood. As for wood to which peridioles may become attached, the natural means of long-range dispersal is the transportation of wood by water. That driftwood is the substrate on which bird's nest fungi are frequently found has already been noted. Local dispersal of fungi around lakes is apparently of common occurrence. Dispersal over greater distances by streams and rivers is very probable. The author observed on many islands of the West Indies that branches, caught among rocks in mountain streams and kept moist by spray from water-falls, bore copiously species of Nidulariaceae that did not appear in surrounding areas away from the stream but which could be found in the forest at higher levels whence the stream had come. Finally, driftwood is carried for still greater distances by ocean currents. Whether or not peridioles

on driftwood could survive long exposure to salt water is another problem. On this point one personal observation may be relevant: in 1966 Cyathus berkeleyanus was found fruiting on driftwood sticks on the seashore near Trois Rivières on Guadeloupe. The sticks had been subjected to salt spray and fruit bodies were whitened with salt and were very salty to the taste. Whether or not the sporocarps contained germinable spores could not be determined at the time. As is true for seeds, so also human activity may make possible the long-range dispersal of pendióles attached to wood. Here, the extent to which the wood is kept dry and the length of time of transport would seem to be important factors. I can cite no evidence relevant to this speculation but it seems to have interesting possibilities. (d) Small terrestrial animals. Of small animals, such as insects, snails, and others, little can be said as to their possible role as translocators of peridioles of bird's nest fungi. I have often found fruit bodies of Cyathus partly chewed away and usually suspected that insects, particularly beetles, might have been the foragers; but not once have I seen beetles or other insects actually eating fruit bodies. Snails, however, are probably important culprits. In Fern Gully, Jamaica, the remains of a large number of sporocarps of a species of Cyathus were observed to have been eaten away to the extent that little but the basal parts remained. Snails were not actually seen eating the fungi but many were present on the wood that bore the fungi and on the surrounding ground. We may suspect small animals of being important distributors of Nidulariaceae, but direct observation and perhaps experiment are needed for confirmation. (e) Birds. Still another possibility exists that merits mention even if only in the hope of stimulating observation. Several botanists have suggested to me that birds could possibly distribute peridioles by eating them as they would seeds. If peridioles eaten by birds are capable of passing undamaged through the 112 / The bird's nest fungi

birds' digestive apparatus a very important means of distribution would exist, for migratory birds fly far and might transfer fungi across large water and mountain barriers. 2 / Association with other plants The question is often asked 'Are bird's nest fungi ever or regularly associated with other living plants?' The answer at present must be vague or even negative, but a few observations may be interesting and provocative. In temperate North America and in Europe, when I was looking for Nidulariaceae, it frequently happened that the first 'scent' was the finding of puffballs, which are nearly all larger than fruit bodies of the bird's nest fungi and more easily seen. Closer search in the location of puff balls yielded the bird's nest fungi with a fair degree of regularity. Although the association is by no means certain, it would not be surprising to find that two groups of closely related fungi require the same conditions for vegetative growth and fruiting. This apparent association with puffballs was not observed in the tropics - which poses still more unanswered questions. An interesting association in which the degree of correlation (in my experience) is fairly high is that between Nidula niveo-tomentosa and the bracken fern Pteridium latiusculum on the Canadian West Coast. The Nidula fruits regularly in the immediate vicinity of the fern, although the fruit bodies form not only upon old fern rachis but also on bits of hardwood and softwood. The significance of this association, even if it is not infallible, is unknown. It could be due merely to similarity in the environmental requirements of the fungus and the fern. At one time an effort was made to try to disclose any possibility of a mycorrhizal association. Fern prothallia were grown in pure culture and mycelium of Nidula was introduced into the latter. No mycorrhizal association could be detected but it is still possible that it exists and further investigation might be profitable. Two examples can be given of fungi occa-

FIGURE 46 Hypocrea latizonata growing as a parasite on fruit bodies of Cyathus striatus. The parasite forms conspicuous broad white or cream-coloured bands around upper part of fruit bodies. X2

sionally found growing upon carpophores of the Nidulariaceae. The first of these, reported by Lohman (1938), is the parasitism of Cyathus striatus by the ascomycete Hypocrea latizonata. Parasitized Cyathus fruit bodies are made conspicuous by the presence of a broad whitish band of the ascomycete stroma. The parasitism results in considerable abnormal development of the carpophores of the bird's nest fungus. I have never observed this parasitism in nature but have seen specimens of C. striatus showing it. These were collected and sent to me by the late Dr G. W. Martin in Iowa in 1941 (figure 46). The second example concerns the occurrence of and effect upon fruit bodies of Cyathus stercoreus of species oiFusarium; here there is no evidence as to whether or not the Fusarium is parasitic upon the Cyathus. In July 1941, Professor Duller drew my attention to some 113 / Occurrence, distribution, and ecology

specimens of C. stercoreus which had been collected at Louisiana State University. The pendióles of the Cyathus were bright brick red in colour and, upon examination, were found to be covered with spores and mycelium of a species of Fusarium. The latter was not identified as to species. However, a note (unpublished) of Buller's records the following: 'Mr George Nyland transferred the Fusarium to other pendióles in other cups and got it to grow. ' In September of the same year, I found specimens of Cyathus striatus in Winnipeg, Canada, in which peridioles were the same colour as those described above; again Fusarium spores were found on the red peridioles but failed to germinate on nutrient medium. In general, one can conclude that fruit bodies of the Nidulariaceae are seldom parasitized by other fungi or even have other fungi merely growing upon them.

3 / Ecology, other considerations (a) Dispersal without rain. Undoubtedly the most interesting and important aspect of the ecology of the Nidulariaceae is the distribution of their spores through the operation of their fruit bodies as rain-splash dispersal mechanisms. Most of these fungi fruit where, at least occasionally and even in desert areas, rain at one time or another discharges the pendióles. I have observed, especially in the tropics, that the dripping of water from the leaves of tall large-leaved plants is just as effective as rain in the operation of splash dispersal. In the West Indies it was observed that water drops falling from large leaves discharged peridioles even when there was no appreciable rain falling. Run-off drops are also important dispersal agents for bird's nest fungi in coastal fog forests. There is one example, however, from the author's experience that needs special attention. As noted above, Cyathus olla was found fruiting abundantly on the loma on the dry coastal plain of Peru. Rain almost never falls in this area and it seems unlikely that peridioles of C. olla are dispersed by rain there, or at least rarely ; the only precipitation that occurs is in the form of dew and, as there are no large-leaved canopy plants on the loma, very large drops of water are unlikely ever to fall upon bird's nest fungi. Under these circumstances some other agency must have distributed spores or mycelium from one flowering-plant tussock to others. Goats were observed feeding around the plant tussocks and it is possible that the feeding activities of these animals or perhaps of small seed-hoarding rodents must, in some way, have effected the spread of the fungi. (b) Soil conditions. The only serious consideration of the ecology and distribution of any group of the Nidulariaceae that has been published is the work of J.T. Palmer (1958b) concerning species of the genus Mycocalia, especially those found growing in the British Isles. Palmer reported that two species, M. denudata and M. minutissima, were found growing only 114 / The bird's nest fungi

under moderately to higher acid conditions. M. denudata fruited mostly upon old birch wood and upon dead parts of Juncus effusus, whereas M. minutissima fruited upon bark of pine. In contrast, M. duriaeana was found only on calcareous dunes fruiting upon branches of pine and poplar and stems of grasses and moss sporophytes. Regarding M. minutissima, Palmer reported: 'The finding of scattered peridioles and developing peridia on submerged leaves of Juncus effusus opens the question as to whether this species might not also occur in purely aquatic conditions/ Two species of Mycocalia, M. denudata and M. duriaeana, were found by Palmer fruiting on the dung of animals, both on rabbit dung and M. denudata also upon sheep dung. Except for the above, no deliberate attempt has been made to study the ecology of the Nidulariaceae except in relation to spore dispersal; therefore our knowledge is not yet at a point where serious discussion of their ecology is very profitable. For example, no one has tested the acidity of the substrates upon which these fungi grow in nature, and so the experiments of Garnett (1958), which showed that fruiting in culture of Cyathus stercoreus is greatly delayed or even inhibited at pH above 6.0, cannot at present be correlated with the behaviour of this species in nature. And, in any case, physiological measurements under the artificial conditions of laboratory culture are applicable to the conditions found in natural environments only with great caution. The bird's nest fungi do not seem to differ greatly, in physical and biochemical requirements for mycelial growth and fruiting, from other macrofungi ; and it would be difficult, and unwise, to attempt to make a special case for them in the matter of ecology. Bearing this in mind, the following pages merely report certain observations and some experiments that appear pertinent to ecological considerations. (c) Longevity and survival. It has been found that the basidiospores of some species of the Nidulariaceae, if still enclosed within their peridioles, may be induced to germinate in the

laboratory when the pendióles have been kept in herbarium packets under dry conditions for as long as four years. In contrast, the spores from fresh fruit bodies frequently cannot be induced to germinate in the laboratory (Brodie, i96zb). Spores six months or more old germinate slowly and in low percentage; however, the mycelium derived from such spores has not been noted to be different from that derived from fresh spores. Nothing is known about the viability of spores or pendióles (in their natural environment) that are more than four years old. There seems to be little evidence concerning the survival in nature of the vegetative mycelium as such except what might be inferred from the survival and revival of the fruiting bodies as will be noted. Laboratory cultures two years old have occasionally revived but only when they had not become completely dry. As might be expected, mycelium of certain species appears to survive neglect better than others. It has been observed that mycelium of Cyathus olla, C. pygmaeus, and C. striatus survived longer as old partly dried cultures than did mycelium of other species such as Cyathus triplex, C. setosus, Crucibulum laeve, and Nidula spp. There is abundant evidence that the fruit bodies of certain species of the Nidulariaceae can survive the drought and cold of the winter in temperate North America (Brodie, 1958). This survival consists of the renewed growth of fruit bodies a year or more old and has been observed for the following species: Crucibulum laeve, Nidula candida, N. niveotomentosa, Cyathus striatus, and C. stercoreus. In the tropics renewed growth has also been observed in such species as Cyathus poeppigii. New fruit bodies develop from a mass of hyphae situated on the inner surface of the bottom of old fruit bodies. The evidence available (Brodie, 1958) indicates that fruit bodies at least a year old can produce new cups the following year. Regarding the renewal of growth of fruit bodies whose development is repeatedly inter115 / Occurrence, distribution, and ecology

rupted by periods of drought, only one personal observation can be recorded. In August 1969 I found a number of sporocarps of Cyathus olla growing on an old hardwood stump on the river bank at Edmonton, Canada, during a dry period. Six of these were small and in different stages of maturity. On three successive occasions the site was revisited after heavy rainfall and it was noted that all six young sporocarps had progressed further towards maturity with each rainfall, although none had opened. On the fourth visit, after an unbroken drought of three weeks, the sporocarps had stopped maturing. There is, therefore, probably a limit to the extent to which immature sporocarps can be interrupted in their development and yet mature fully. The capacity of various kinds of wood to retain sufficient moisture to allow continuous development of sporocarps growing upon it is doubtless also important in this connection, as was indicated in the discussion of substrates (Chapter 9, B, i b). It is also true that, at times, fruit bodies of some species of bird's nest fungi may be extremely abundant and no trace of them remains in the same site a year later. In July 1969 Crudbulum laeve was fruiting on almost every dead hardwood chip and twig on the top of Tunnel Mountain at Banff in Alberta. In 1970 not a single specimen was found in the same location in July and later. It is difficult to explain the total disappearance of this species, especially when it is known that old dry specimens are commonly persistent. (d) Atmospheric humidity. As to the effect of atmospheric humidity (independent of free rainfall) on the appearance and distribution of bird's nest fungi, here again it is difficult to correlate laboratory experiment and observation with experience in the field. Fruit bodies have not been observed to develop in uncovered culture plates when the surrounding atmospheric humidity is low (about 25% R.H.) . They do develop profusely, however, in covered culture plates where the humidity is constantly high and probably near saturation.

One might therefore expect sporocarps to develop and be commonly found in nature in regions of constant high humidity such as at the cloud level in the tropics. It was for these reasons a great surprise to me to discover that sporocarps of the Nidulariaceae could not be found in abundance (and rarely at all) at the cloud level in the West Indian Islands. However, one had only to descend a few hundred feet below cloud level or go above it to find them. One would naturally conclude that the relative constant humidity at the cloud level is not conducive to fruit body development and that marked fluctuation in humidity is necessary. This, however, does not accord with laboratory observation and we are left with an unsolved problem. (e) Chemical nature of substrate. Although the nutritional requirements of mycelium of bird's nest fungi is fairly well established for some species (Brodie, 1962^, little is known of the nutritional requirements of fruiting. S.-h. Lu (1973) showed that if the element calcium is absent from the nutrient medium, a high proportion of the fruit bodies develop abnormally (figure 15). Except for this observation (which has not yet been pursued in detail), all efforts to relate fruiting in the Nidulariaceae to the chemical composition of the nutrients supplied have been disappointing. From the work of Garnett (1958) and Miles (1953) it appears that, provided the nutritional requirements of the mycelium are met, the addition of chemical substances has not, in any conclusive experiment, determined whether or not mycelium can be induced to fruit. Garnett (1958) even showed that there is nothing in the filtered residue of nutrient medium which enhances or inhibits fruiting. Of physical environmental factors, however, it is known (Chapter 5) that light and temperature do play some role in varying degrees in determining fruiting. In short, it appears at present that the physical nature of the substrate (and of the environment) is more important than the chemical nature in determining where and when a bird's 116 / The bird's nest fungi

nest fungus will fruit, although this statement is open to question especially for coprophilous species. That the physical nature (texture, etc.) of the substrate is of prime importance to fruiting has been demonstrated by facts reviewed above (Chapter 5, c). (f) Development and orientation of fruit bodies. That the sporocarps of the bird's nest fungi are, early in their development, phototropic and probably geotropic is a fact of ecological importance. Because of their positive response to light they tend to bend away from very local overhanging objects and thus to mature where raindrops can easily fall into them. Because of their geotropism they mature only on the upper side of the substrate where they can be hit by raindrops. Even where Cyathus striatus or C. pallidus were found growing in great numbers on brush piles, I have never seen one sporocarp developing on the lower surface or even the side of the substrate. 4 / Geographical distribution Tables have been presented by various authors (e.g. White, 1902) showing the distribution of species of the Nidulariaceae in different parts of the world. It would serve no purpose to repeat or summarize these for, obviously, more intensive collecting in areas already explored and exploration of still other areas could easily result in quite different distribution records. A few notes on geographical distribution may be of some value however. These notes are based upon published records, examination of large herbaria, and records provided by my own herbarium. Some species are circumpolar in distribution. The best example of this is Crucibulum laeve which is known from nearly every temperate country in the world but has apparently only rarely been found in the tropics. Cyathus striatus is likewise a widely spread species of the temperate world. Its morphological counterpart, C. poeppigii, is a widely spread tropical species which is rarely found in subtropical and never in temperate climes.

There is no doubt that far more species of the Nidulariaceae occur in the tropics than in temperate countries. Of those species considered valid in this monograph, 9 have been recorded in Europe but only 4 or 5 of those are of common occurrence. In Canada and the United States, only 20 species are known, only 8 of common occurrence. In the West Indies, on the other hand, about 25 species have been recorded, most of which are widely distributed, and 11 species are known from the Hawaiian Islands. Much greater speciation has apparently occurred in the tropics than in the temperate zone, although at present no explanation of this difference can be offered. Of course, many kinds of exotic plants have been introduced into the West Indies and the Hawaiian Islands and many species of Nidulariaceae may have been introduced with these plants or with plant products. The genus Ni dula presents an interesting picture. Two species N. candida and N. niveo-tomentosa inhabit only the western mountains of North America (but not only at high altitudes) from British Columbia to California and have never been recorded east of the eastern slopes of these mountains (at that latitude) with the single exception of the occurrence of N. niveo-tomentosa on the Blue Mountains in Jamaica; this extraordinary isolation from the main body of the population has no explanation at present. Nidula macrocarpa is only known to occur in Chile. The apparent rarity or absence of certain species in some areas probably, in most instances, reflects only chance. Lack of collectors or lack of success of collectors cannot however explain the rarity of Cyathus stercoreus in Europe where mycologists have been active for centuries. This species has seldom been found in continental Europe, although it is widespread throughout the rest of the temperate world and is found also in the tropics. Stranger still, C. stercoreus has, to my knowledge, never been found in England. Knowing this fact, I attempted, in 1952, to introduce this coprophilous species by planting cultures at 117 / Occurrence, distribution, and ecology

Rothamsted Experimental Station in Hertfordshire. Mycelium of a genetically marked strain of dikaryotic mycelium was grown in sterile horse manure and soil in a greenhouse and, when the cultures were fruiting abundantly, they were removed from their pots and planted in six separate locations including pasture, woodland, and gardens. Examination was made of the marked sites for several years following the plantings. All the original cultures disappeared and C. stercoreus did not appear subsequently at or close to the planting sites. No explanation can be given for the failure of the fungus to survive. Species that may possibly be endemic can be judged so only on the basis of present knowledge. It is probable that Cyathus novae-zeelandiae is an endemic New Zealand species, for there are no records of its occurrence elsewhere, and it is a distinctive species unlikely to be overlooked or misidentified. Cyathus crassimurus is known only from the island of Hawaii but there is no reason to doubt that it will eventually be found elsewhere, because there is nothing unique in its type locality. One example can be given of a species that may indeed be endemic to a rather limited area and for which the difference between its habitat and that of the surrounding region may very well constitute a barrier ensuring isolation. This is the recently described Cyathus annulatus, known to occur only in the Cypress Hills of south-eastern Alberta in Canada. This distinctive species (figure 61 f) was found only in moist groves in the Cypress Hills. This mesic region consists of an elevated area which, climatically and floristically, is sharply separated from the surrounding xeric plains, which may well constitute a barrier to the spread of C. annulatus away from its presently known habitat (Brodie, 19703). Regarding morphologically distinctive forms of a species that occur exclusively in a given region or predominate there - forms that might be regarded as geographical races - no clear-cut instance can be given. Lloyd (1906)

believed that European specimens of Cyathus striatus are morphologically distinguishable from those of North America. It is my experience, however, that every morphological variant of this fungus that occurs in Europe also occurs in North America and that it is impossible to make the distinction suggested by Lloyd. This species is extremely variable; at the one extreme are tall dark types and at the other extreme small pale types. Neither of these, (nor any of the numerous intermediates) appears to be so restricted in distribution that distinguishable geographical races can be recognized. Lloyd also considered that a peculiar large form of C. olla which has been named C. olla forma anglicus was confined to England and might be taken as a geographically restricted race. However, it is now known (Brodie, i^o/d) that the same large form also grows in Oregon and in Western Canada, al-

118 / The bird's nest fungi

though it may have been introduced into these areas by man (Chapter 9, c, i (b)). There is no reason to assume that taxonomic geographical races do not exist but, on the basis of morphology of fruit bodies, it does not appear possible to recognize them at present. It has been my experience that strains of any one species such as Cyathus stercoreus, derived from fruit bodies from separate regions, do differ in physiological characteristics, such as ability to fruit in laboratory culture, optimum temperature for growth, nutritional requirements, etc., which indicates that isolates of different provenance are indeed physiologically distinct races. Geographically separated races may also differ in their alíeles of the genes which determine sexual behaviour, as has been demonstrated in Crucibulum laeve and Cyathus striatus (N. Fries, 1940,1943) and in Cyathus olla (Brodie, 196yd).

10 / Miscellaneous Observations and Notes

Long acquaintance with any group of organisms often results in a miscellany of knowledge not particularly pertinent to the main research objectives. What seems to be the most significant of such material is dealt with hereunder. A / I N G E S T I O N OF B I R D ' S NEST FUNGI BY HUMANS

I have no record of fruit bodies of the Nidulariaceae ever having been consumed as food for humans. The fruit bodies are not sufficiently large, fleshy, or odorous to be of interest to humans as food, although apparently snails and insects occasionally consume fresh sporocarps (Chapter 9, c, i(d)). The question has been raised occasionally as to whether or not the Nidulariaceae would be poisonous if eaten by humans, but no positive answer can be given. If these fungi possess poisonous alkaloids or other poisonous substances it has not been demonstrated, or at least not published. That bird's nest fungi have been and are eaten by humans for purposes other than nutrition is, however, an interesting and intriguing probability which may well be worthy of investigation. Two species have been reported as being involved and there is a strong suggestion that they are believed to act as an

119 / Miscellaneous observations and notes

aphrodisiac or to stimulate fertility, or perhaps both. 1 / Cyathus limbatus In 1965, some specimens of this fungus were sent to me from Harvard University with the request for identification. Interest had been aroused in the specimens from Colombia because it had been reported to the collector that the fungus is eaten by native peoples in that country 'para tener familia ' (as the matter was relayed to me), i.e. 'to have a family/ However, if any serious study of this supposed virtue of C. limbatus has been made, I am unaware of it. Having in mind the report concerning C. limbatus, I repeatedly made enquiries, particularly throughout the West Indies, in the hope of bringing to light any use of bird's nest fungi by native peoples for medicinal or other purposes. The results were negative except for the single instance that follows. 2 / Cyathus microsporus In 1966, Père le Gallo, then living in Vieux Fort, Guadeloupe, told me about having read (in some notes on the fungi of Guadeloupe written by R.P. Duss (1903)) that a species of bird's nest fungus, identified by Duss as C. microsporus, was formerly used by the native

peoples of Guadeloupe as an aphrodisiac. The fungi were dried and ground to a powder. What the effects, if any, of these fungi may be upon human physiology are unknown; it is also unknown what chemical substances may be present in members of the Nidulariaceae that might produce the effects ascribed to their consumption. Moreover, if the doctrine of signatures is involved here, it would be interesting to know what aspect of the form of a bird's nest fungus could suggest its use as an aphrodisiac.

B / S O M E M E T A B O L I T E S OF CYATHUS i / The Cyathin complex Although chemical information concerning metabolites of members of the Nidulariaceae is unfortunately not available in relation to the problem posed in the above paragraphs, some highly interesting facts have very recently come to light concerning the chemical nature and possible practical value of a series of compounds produced particularly by the mycelium of Cyathus helenae and to some extent by that of C. striatus, C. limbatus, and C. poeppigii. Some, but not all, of these metabolites represent chemical substances which appear not to have been previously recognized by chemists; the metabolites, moreover, have been demonstrated to possess antimicrobial properties. In either or both of the foregoing aspects, their metabolites may prove to be one of the most distinctive characteristics of the bird's nest fungi. Because biochemical knowledge comes quickly to the fore in present-day biology, it is entirely possible that bird's nest fungi may yet achieve renown. The complex of chemical substances which are produced by Cyathus helenae and have antibiotic properties have been collectively named cyathin (Allbutt et al., 1971). Wilkins (1954) reported that bacteriostasis is produced by mycelium of Cyathus striatus, 120 / The bird's nest fungi

but no further investigation of the activity of the fungus was published. In 1966, I named a previously unrecognized species of bird's nest fungus Cyathus helenae (Brodie, i966a). Olchowecki in 1967 studied this species in culture and observed that its mycelium inhibited the growth of bacteria with which some culture plates had accidentally become contaminated. Olchowecki showed that the fungal antibiotic, which was named 'cyathin,' inhibited the growth of a number of bacteria commonly used as test objects and also reported weak antibiotic activity of C. striatus, C. limbatus, and C. poeppigii. Immediately following Olchowecki's work, B .N. Johri at the University of Alberta undertook a detailed study of the biological activity of cyathin. John's results were incorporated into a doctoral thesis presented to the University of Alberta (1969). At this point, a number of chemists headed by Dr W. A. Ayer agreed to undertake to characterize cyathin and its components chemically. When homokaryotic mycelia of Cyathus helenae are grown in liquid medium, most cultures soon begin to produce dark-pigmented exudates, in some instances the nutrient medium becoming almost black. This dark exúdate contains the complex of chemical substances collectively named cyathin. The highest yields of the exúdate are obtained when the fungus is grown at room temperature for 25 days on a chemically defined liquid medium in static culture. It has been shown (Allbutt et al., 1971) that the cyathin complex, to a greater or lesser degree, depending upon the species involved, inhibits the growth of actinomycetes, bacteria (Gram-positive as well as Gram-negative), and some fungi, including dermatophytes. Of all organisms screened for sensitivity to cyathin, Agrobacterium tumefaciens, which is the cause of crown gall disease of many plants of economic importance, was found to be the most sensitive. Penicillin-resistant strains of Staphylococcus aureus were also reported to be sensitive to cyathin.

In the first report, Allbutt et al. (1971) indicated that the cyathin complex consists of seven different chemical compounds, namely: 2,4,5-trihydroxybenzaldehyde (C7H6O4); cyathin A3 (C20H3oO3); cyathin A C 4 2oH3o°4)'' allocyathin A4(C20H30O4); cyathin B3 (C20H28O3) ; cyathin B4 (C20H28O4) ; cyathin C5(C20H26O5). Aside from 2,4,5trihydroxybenzaldehyde, these compounds appear not to have been previously described. Moreover, it appears that even the one relatively simple compound of the complex, viz. 2,4,5-trihydroxybenzaldehyde has not previously been obtained from living plant material. In a later publication (Ayer and Taube, 1972), cyathin A3 and allocyathin B3 were shown to be representatives of a new type of diterpenoid, the carbon skeleton of these compounds being different from those of any diterpenoids known previously. The substance previously named cyathin A3 (see above paragraph) was shown to be a mixture of a major component, C20H30O3 (cyathin A3), and a minor component, C20H28O3 (allocyathin B3, not previously reported). Almost simultaneously with the paper by Allbutt et al. (1971), Johri and Brodie (1971^ reported studies of the physiology of production of the cyathin complex by Cyathus helenae. The main new points made by these authors are as follows: The concentration of sugar in either medium (semisynthetic or chemically defined) determined the yield of cyathin. Of micronutrients tested, zinc had a stimulatory effect on the production of cyathin. A pH range of 4.5 to 5.5 and temperature of 22° c were the best combination for maximum yield of cyathin. Of the seven vitamins tested, only riboflavin influenced cyathin production. A study of various nitrogen sources showed that C. helenae grew better on organic nitrogen ; nitrates enhanced cyathin production considerably without an appreciable change in the growth of the fungus. Dextrose, fructose, maltose, and starch all proved to be good sources of carbon for the production of cyathin. 121 / Miscellaneous observations and notes

One of the unusual features of the production of cyathin by C. helenae is the fact that the complex of metabolites is produced only by homokaryotic my celia; no antibiotic property of heterokaryotic mycelia of this species has as yet been reported. It is surprising to find that a battery of complex organic compounds such as those of the cyathin series can be synthesized by haploid individuals without the full gene complement of any particular isolate of the species, but that the synthesis as far as is known at present, does not take place in the heterokaryotic phase of the fungus with the full gene complement. Perhaps in the presence of the latter, metabolism follows other pathways. In any case an interesting biochemical genetic problem remains to be solved. It seems probable that the production of antibiotics confers a biological advantage on the producer in suppressing the growth of other organisms. In this instance it is interesting to speculate why such an advantage, if it exists, should be confined to the homokaryotic phase of the fungus, especially since reproduction (i.e., sporulation) occurs only in the dikaryotic phase. 2 / Comparison of the indolics of Cyathus spp. When it had been shown (Johri, 1969) that the chromatograms of the cyathin complex produced by mycelium of each of the three species, Cyathus helenae, C. striatus, and C. africanus appeared very similar and yet showed some differences, Johri and Brodie (19713) made an effort to find still another group of secondary metabolites which might possibly be used to indicate similarities or differences among the three species. The species chosen for this study were available and studied as homokaryotic mycelia. Cyathus helenae and C. striatus are closely related morphologically and genetically (Olchowecki and Brodie, 1968), whereas C. africanus is morphologically not closely related to either of these species. A preliminary survey was made of the

indolic substances produced by the mycelium of each species and found in the nutrient medium. All three species produced metabolites which, on chromatograms, showed several spots characteristic of the Índole ring. The chromatographic pattern was different for each species although similarity was observed in the chromatograms obtained from mycelium of C. helenae and C. striatus. Cyathus africanus differed from the other two species not only in the pattern of the indolic spots but also in the intensity and size of the spots. The indolic substances were not specifically identified, but the position of major spots on chromatograms corresponded closely to indole-3-acetic acid, Índole acetonitrite, and tryptophane. These results indicate that the species relationships suggested by morphological and other evidence are borne out by a comparison of the indolic metabolites of the three species and suggest that a detailed survey of the genus Cyathus might yield interesting chemotaxonomic information.

C / RAIN-SPLASH DISPERSAL IN OTHER PLANTS Discussion in detail of the many other kinds of cupulate plant structures which are now known to operate by means of rain-splash in the dispersal of spores, sperms, gemmae, seeds, pollen grains, and other reproductive units is not primarily germane to this book. Nevertheless, because the recognition of 'splashcups' in the plant world is one of the most widely applicable concepts that has been developed, chiefly as the result of studies of the bird's nest fungi, a brief account of this subject is given here. The very simplicity and obviousness of this concept may be the reason for its neglect by a considerable proportion of the modern text books of botany. For brevity, only a summary of this topic is given, taken

122 / The bird's nest fungi

mainly from an earlier review (Brodie, 1957). 1 I Raindrops bring about the dispersal of gametes, spores, seeds, and other kinds of reproductive units in a great variety of plants. Over thirty rain-operated dispersal mechanisms are recognized, scattered throughout almost all groups of plants. 2 / Special organs called 'splash-cups' are produced by fungi, lichens, liverworts, mosses, and seed plants. A typical splash-cup is 5-8 mm wide at the mouth, is vase-shaped or crucible-shaped, has sides which make an angle of 60 to 70° with the horizontal and matures in a vertical position. Raindrops splash pendióles from the fruit bodies of the fungus Cyathus, spores from the fruit bodies of other fungi, sperms from the cupulate male plant rosettes of the moss Polytrichum and of other mosses, and seeds from the open seed capsules of such angiosperms as Portulaca, Chrysosplenium, andMzfei/a. 3 / In certain flowering plants, such as Salvia lyrata, rain operates a 'springboard' device. The calyx tube of Salvia produces a kind of cup which catches raindrops. The blow received by the cup depresses the calyx tube and, when the latter springs back into normal position, nutlets are thrown from the calyx tube. The plantlets of Kalanchoe tubiflora are dispersed by a similar springboard action. The bulbils of Lycopodium selago and L. luddulum are also known to be dispersed by a rain-operated mechanism of the springboard type. 4 / The discharge of spores from puffballs is brought about by the impact of water drops with the papery top of the endoperidium. 5 / The pollination of some cup-shaped flowers such as those of certain species of Ranunculus, Caltha, Narthecium, and Chrysosplenium is brought about by the splashing action of raindrops. 6 / Many kinds of curved fungus spores, not ordinarily dispersed during dry weather, may be carried by minute water droplets which strike a spore-laden surface and bounce away taking spores with them.

D / O T H E R CUPULATE FRUIT BODIES AND SOME THOUGHTS ON EVOLUTION Because there seems to be no very direct evidence pertaining to the possible origin of the cupulate fruit body form which is predominant in the Nidulariaceae, speculation is probably of little value. Some casual observations may be of interest. Not all the cupulate sporocarps of fungi operate as rain-splash dispersal mechanisms. Of those that do (Brodie, 1957) tne resemblance to the splash organ of Cyathus is remarkable. Remarkable also is the close approximation of the form of the podetia of certain lichens (e.g., Cladonia pyxidata) to that of Cyathus striatus. Lichen podetia are able, however, to scatter their soredia not only by rain-splash but also through wind action. Among the cupulate ascomycetes, spores receive the initial thrust for dispersal through the explosion of asci and apparently no one has yet shown that the cup form of ascomycete fruit bodies has any connection with dispersal of their spores by rainsplash. It is probably coincidental that some basidiomycetous fungi other than the Nidulariaceae occasionally develop, or show the tendency to develop, fruit bodies that are cupulate and obconic. I have often been intrigued by the resemblance of some of such fruit bodies to species of Cyathus or Crudbulum. A few curios of this sort are illustrated in figure 47, and one has only to glance through photographs of tropical species, especially of Thelephora and Stereum, to find many examples of Cyathus-like cups. However, if any of the latter operate as splashdispersal mechanisms, this has not been demonstrated. One of the examples given in figure 47 is certainly a splash-cup, however, as I discovered some years ago (Brodie, 1951^. That portion of the fruit body of Polyporus conchifer which develops first, usually in late

123 / Miscellaneous observations and notes

summer or early autumn, has the form of a cup so reminiscent of the fruit body of Crudbulum that specimens have frequently been so labelled (figure 47c). Rod-shaped, asexually produced oidia are developed within the polypore cups and the oidia are dispersed by rain-splash. When this asexual stage has passed, the then empty cups oí Polyporus conchifer continue to expand horizontally from one side to produce a shelf-like portion, on the under side of which sexually produced basidiospores are ultimately developed in the manner typical of the family Polyporaceae to which P. conchifer belongs. This evidence alone leads me to suggest that all vase-shaped or cup-shaped fruit bodies in the fungi (as well as the fruiting structures of similar form belonging to other groups of plants) should be studied carefully as to the manner in which their spores or other reproductive units are dispersed in nature. And I predict that when such studies have been made, many new and interesting dispersal mechanisms will have been brought to light. Regarding evolution and phylogeny in the Nidulariaceae, there is as yet no evidence to indicate clearly which genera and species are to be regarded as primitive and which as advanced; at least there is no evidence that could not be refuted by an alternative interpretation. Leaving aside the problems of the phylogenetic origin of the series of fungi known as the Gasteromycetes and the phylogenetic relationships of the several orders within that series, one aspect of the morphology of the puffballs and their allies that interests mycologists is what appears to be a tendency for the gleba or fertile portion of the basidiocarp to become dissected by sterile plates or sheets of tissue often, although perhaps not correctly, referred to as the trama. A series of genera and species can be selected to illustrate a supposed progressive dissection of the gleba into units which may ultimately play the role of propagules or reproductive units. Gàumann in his textbook Comparative Morphology of Fungi (1928) went so far as to apply

FIGURE 47 Fruit bodies of some fungi sometimes mistaken for Nidulariaceae: a, Stereum sp. from Jamaica, Xi.5; b, Polyporus sp. from Jamaica, Xi.5; c, Polyporus conchifer, North America, Xi

this concept to the Nidulariaceae, of which he that Nidularia (and My cocolía) are the simpwrote: 'The members of this family are charac- lest and least efficient splash-cups ; that Ni du la terized by gleba chambers formed only in small could be considered a step forward, followed by Crucibulum ; that Cyathus may represent the numbers, surrounded by a special sclerenculmination of the series in that it is the most chymatous wall and hence disseminated as a structurally complex of the genera and that its unit. The tendency for the trama to split splash-cups are the most efficient as dispersal (which we found in Rhizopogon luteolus and in devices. Pisolithus) culminates in this family/ Second, it was suggested that progressive The possible steps in the evolution of the stages in the development of the funiculus can most efficient type of fruit body insofar as be postulated: thus, no funiculus is present splash dissemination of pendióles is concerned in Nidularia, Mycocalia, and Nidula ; the was discussed in a publication by myself funiculus is of simple construction in Cruci(Brodie, 1956). First, regarding the shape and general con- bulum ; the funiculus of Cyathus is the most complex (and efficient) of the series ; even struction of the fruit body, it was suggested 124/The bird's nest fungi

within Cyathus some species have a more loosely organized funiculus than others. Efforts to use mycelial characteristics to show possible phylogenetic relationships have not as yet been sufficiently extensive to permit important conclusions. Perhaps when chemo-

125 / Miscellaneous observations and notes

taxonomic studies such as those of Johri and Brodie (i97ia) have been extended, some other bases for estimating the degree of relationship between the genera and species of the Nidulariaceae may be forthcoming,

11 / Review of Taxonomic Characters

A/ INTRODUCTION Although my acquaintance with the Nidulariaceae has been a fairly long one and even although I am responsible for the names of a few of them, the following account of their taxonomy is presented mainly with the belief that it would be a service to mycologists to gather all descriptions into one place. While so many species are still represented in herbaria only by such scanty collections, the attempt to assess the evidence is difficult and the result should not be regarded as ultimately authoritative. The problems involved in classifying and identifying fungi of the family Nidulariaceae are typical of those involving the taxonomy of other groups of organisms. Very old descriptions are often worded in terms which may long ago have been suitable for distinguishing some bird's nest fungi from other kinds of fungi, but are of little use at present in the exact definition of taxa within the Nidulariaceae. To point out the limitations of certain old descriptions does not presume to suggest a derogatory attitude towards them; indeed, many have a virtue which has been all but lost in our time, namely, the virtue of beautiful words. Consider, for example, how appropriate is this phrase describing the fruit body surface of the beautiful fungus Cyathus olla : 'there is a gentle asperity to the touch, on the outside' (James 126 / The bird's nest fungi

Bolton, 'An History of Fungusses growing about Halifax/ B. White & Son, London, 1788, p. 102). How much greater honour is done to the fungus by Bolton's phrase than by our present-day words 'rough' or 'hispid'! Some old type material is faded, worn, and fragmentary and sometimes no spores are present in type specimens. Usually no one feature alone can be used to define a species. Perhaps the most troublesome problem is the extreme variability in the gross morphology of the fruit bodies of certain species - variability in size, colour, and other features. Fortunately, the genera of the Nidulariaceae are well defined and can be distinguished to a large extent on the basis of what can be discerned with the unaided eye, supplemented by what is revealed by a hand lens. Even so, many specimens have been found in herbaria labelled Crucibulum, which are, in fact, Cyathus, and vice versa. The external form of Crucibulum only very rarely resembles that of Cyathus, as in Crucibulum cyathiforme (figure 53c); and, even in that example, examination of the funiculus immediately distinguishes the two genera as does also the structure of the peridium wall. Old specimens of Ni dula spp. are frequently labelled Crucibulum because of some gross resemblance, although a funiculus is present in Crucibulum but is lacking in Ni dula. Some comments about the structural fea-

tures of the fruit bodies of the Nidulariaceae that have been used to distinguish genera and species will be found in the monographs of Tulasne (1844), Lloyd (1906), White (1902), Cunningham (1924), and others. These features are described in some detail here to facilitate comment upon their respective values taxonomically and to point out certain difficulties that arise in their use. B/MACROSCOPIC CHARACTERISTICS 1 / Shape Since the fruit bodies of Cyathus are all basically infundibuliform, description of shape alone is seldom of much value in species differentiation in that genus except in the sense that the student may acquire by experience the realization that most specimens of C. stercoreus, for example, have the form of a slender inverted cone (figure 23), whereas most specimens of C. poeppigii or C. limbatus have the form of a broad inverted cone (figure 60), or that most specimens of C. pallidus have the form of a chemist's porcelain crucible with curved sides (figure 56). However, different fruit bodies of the variable species C. stercoreus may have any of the variations (figure 29) of an inverted cone just described. Slight but possibly significant differences of shape are difficult to describe precisely and in the majority of publications the differences as described are difficult to visualize. For 15 species of Cyathus described in Lloyd's (1906) monograph, the following terms are used with the frequency noted: campanulate, 5 ; obconic, 3 ; conic narrow, 2 ; conic bell-shaped, 2 ; conic cup-shaped, i ; cylindrical cup-shaped, i ; urn-shaped, i. Shape is of prime value in the recognition of one species of Crucibulum (figure 53c) and of one species of Nidula (figure 52C).

2 / Size Descriptions usually give the dimensions of 127 / Review of taxonomic characters

the fruit bodies in terms of width at the top (or mouth) and total height. There are two difficulties encountered in using published dimensions to apply to a specimen in hand. One is that there is much variation in size displayed by almost any species; specimens of Cyathus stercorus vary from 3 mm in height to as much as 12 mm (figure 29). A second is that it is rarely stated in descriptions whether the height given is measured from the lowest part of the inverted cone only or whether the total height includes the basal hyphae called the emplacement. One has only to glance at a photograph (figure 41) showing a large emplacement such as that present on the fruit body of Cyathus striatus (or of many other species) to realize that the height measurement would vary greatly depending upon whether or not the emplacement were included. In my experience it is usually obvious that the height given refers only to the peridium proper and does not include the emplacement. 3 / Colour If the colour given in a description is a reasonably standard term or if some standard colour nomenclature is used, colour is of some value as an aid to defining species; but here again there are difficulties. One assumes that many of the older descriptions were based on dry herbarium material and one compares the term (say 'light brown') to the colour of dry specimens which are being studied. If this assumption is not valid, then a description could be useless in the matter of colour. For example, many collections of the tropical species Cyathus berkeleyanus appear a dark ochrebrown when freshly gathered; upon drying, the same specimens appear light buff. Moreover there are some species in which different fruit bodies display a very great range of colour. Most specimens of C. stercoreus are golden brown or tawny, but very lightcoloured forms exist as well as some that are close to black. Fresh specimens of C. berkeleyanus are usually tawny or ochre brown but there are also very pale forms. C. pallidus is

usually a pale straw colour, but some specimens are of a much darker shade. Thus descriptions involving form, dimensions, and colour cannot be taken too literally, but must be interpreted with caution. This is not to say that these characters may not sometimes be important; in fact, in some instances they may be what Lloyd (1906) often called 'leading' characters: for example, the tall slender cyathus-like form of Crucibulum cyathiforme (figure 53c) is unique, and that character alone enables the species to be recognized; because Cyathus pygmaeus has fruit bodies of such small size, it could not easily be confused with other North American species ; no species oiNidula except N. macrocarpa has a tawny colour. 4 / Outer covering of peridium The outer surface of the peridium wall as seen by the unaided eye or with the aid of a hand lens presents different aspects, depending upon the diameter of the hyphae that compose the surface and the various ways in which the hyphae may be aggregated. If the surface is uniform and relatively smooth, suggesting a fine woven cloth or perhaps velvet, it is commonly referred to as the tomentum, e.g., Nidula, Crucibulum, Cyathus earlei (figure 54). The longer, coarser hyphae in some species may be somewhat irregularly aggregated to"sform a kind of shaggy pile to which such terms as woolly, shaggy (figure 23), hirsute, etc. have been applied. In addition to having a hirsute outer covering, some species display a coating of conspicuous 'hairs' made up of the aggregation of numerous coarse hyphae. These aggregations may appear as tufts (figure 55) or, in the extreme instance, as long clustered hairs which usually are bent towards the base of the fruit body (figure 56). All these features are of considerable value in characterizing species ; in a few instances they are good, readily used specific characters, but in others they serve only to delimit groups of species. 128 / The bird's nest fungi

5 / Plication The walls of the peridium in Cyathus may appear, either externally or internally (or both), to be grooved, fluted, or plicate. The termstriate (e.g., figures i, 2) has often been employed to describe this condition, but is less accurate than plicate because the grooves are actually relatively wide folds in the wall like corrugated roofing rather than lines cut into it ; i.e., the wall is zigzag in cross section. Plication and lack of it are valuable criteria for separating groups of species. However, a word of caution is in order ; some species are only faintly plicate and it is sometimes difficult to decide which fork of the species key to follow. Faint plication is sometimes present in dried fruit bodies, the result merely of some shrinkage during drying. Those species that are correctly assigned to a plicate group show the character best when moistened. A few tropical species are so woolly externally that the plication may be hidden unless some of the external hyphae are rubbed away with a needle. Moreover, the condition which is the opposite of plicate has been frequently described by the word smooth. Almost no peridium is in fact perfectly smooth even on the inner surface and it must be realized that 'smooth' in older descriptions usually means 'not plicate/ Some species, notably of Cyathus, may also bear transverse ridges, particularly on the inside of the cup, but these vary in the degree of distinctness in different specimens so much that the character has little diagnostic value. 6 / Setae When the lip of the fruit body of almost any of the cupulate bird's nest fungi is examined under a stereoscopic microscope it may be seen almost always to have some tufts of hyphae projecting beyond the main mass. Such tufts follow almost inevitably from the organization of the peridium wall from closely interwoven hyphae. Sometimes this appearance is described in terms such as Tip minutely fimbriate.' Such a description of any species is of little value, although it might be relevant in a

comparison with another species the lip or mouth of which is very different. In a fair number of species, e.g., Cyathus setosus (figure 57), C. striatus (figures i, 2), and others, hyphae at the sporocarp lip are aggregated into very conspicuous bristles called setae. Even when setae are short, there is no doubt about their presence when the fungus is examined even with a 10 x hand lens. Setae are durable: even in old battered specimens, a few can still be found if they were part of the fresh specimens. Setae are therefore valuable structures in the definition of many species although they play a lesser role in key construction because groups of species are more surely segregated on other bases. 7 / Emplacement The solid mass of hyphae at the base of the fruit body, the emplacement, is important to the operation of the fruit body in splash dispersal (Chapter 7). Because it is so conspicuous in some species, one might expect it also to have value in species delimitation, but this is doubtful. In Cyathus striatus the emplacement is usually of such solid construction (figure 41) that the attachment of the stipe of the fungus cup to the emplacement is seldom broken when specimens are pulled away from their substrate. The colour and wide-spreading form of the emplacements of some tropical species (e.g., Cyathusafricanus (figure 55) are probably useful taxonomic features, but it is my experience that emplacements of the latter, rather frail type are seldom to be found still attached to the dry herbarium specimens which the taxonomist examines. Lloyd pointed out that three species of Cyathus have been described in which the emplacement is the chief diagnostic character, two of which are merely forms of C. stercoreus having very conspicuous emplacements. C/MICROSCOPIC CHARACTERISTICS The structures that must be examined with the 129 / Review of taxonomic characters

microscope, and which have been most extensively employed to define species, are parts of the peridiole. Pendióles must be soaked in water for several hours before they can be sectioned. Free-hand sections cut in pith are perfectly satisfactory. One precaution worth mentioning is that a tunica, if present, may easily become separated from the remainder of the peridiole section if the latter is handled roughly. Before deciding that no tunica is present, therefore, one should examine the sections carefully while they are still in water in the sectioning dish. The loss of different parts of sections can be overcome if a freezing microtome is used, but this procedure is less convenient for the examination of an occasional specimen. After sections have been studied microscopically and spore measurements recorded, the sections may be mounted in glycerine-jelly for future reference. i / Tunica The outermost layer of the peridiole may consist of a thin layer of colourless, grey, or brownish hyphae called the tunica (figure 483). In Crucibulum the tunica is quite thick and may even be discerned in the dry state if flaked off with a needle. In Cyathus it is often difficult to decide whether or not a tunica is present because it is thin in most species and may easily be lost in sectioning. A very evident tunica is present in Cyathus striatus, and an unusually thick one in Cyathus crassimurus. No tunica at all is found in certain species, including C. stercoreus. A point of possible confusion is that the term tunica was applied by Tulasne (1844) to the hard cortical layer or cortex, which is described below, whereas Lloyd (1906) and subsequent workers differentiated between the delicate and often evanescent tunica, as described above, and the dark hard cortex (figure 48). The word tunica has been used in the description of species oiNidula. According to the report of Overstreet (1955), a tunica, distinct from the hard peridiole wall (the latter com-

FIGURE 48 Tunica and cortex of pendióles : a, Cyathus pallidus showing one-layered cortex and tunica, X5o (detail on right X5oo) ; b, two-layered cortex of C. stercoreus and no tunica, X5o; c, photo of section of pendióle of C. africanus showing one-layered cortex, remnants of tunica and inner sporogenous zone, X 65. Figures a andb, courtesy Trans. Brit. Mycol. Soc., 1954, 37: 152; Figurée, courtesy of Can. J. Bot. 1967, 45'• l654 130 / The bird's nest fungi

posed of dark-coloured indurated hyphae) is present in three species of Nidula but is absent in N. emodensis, in which the outermost layer of the peridiole is composed of rigid, interwoven, coloured hyphae bearing short spiny branches. Overstreet indicated that what she called the tunica in N. candida is composed of two distinct layers. The relationship of tunica to cortex in Nidula must be studied carefully before it can be stated categorically that these structures are strictly comparable to those described by the same terms in species of Cyathus. In any case, the species oí Nidula are readily separated by other characters.

certain species the cortex may give the impression of being two-layered because it is not of the homogeneous structure found in the onelayered type. Descriptions may note, as for C. triplex (e.g., Lloyd [1906], p. 23) 'Cortex thick, evidently double, but subhomogenous I have observed that persons not experienced in identifying bird's nest fungi very frequently assign a species to the two-layered cortex groups because a single cortex may, in a section, be bounded externally by a tunica, if the latter is tough and thick enough to remain intact during sectioning. An example of the latter situation is illustrated in figure 48a.

2 / Cortex Lying immediately beneath the tunica, or forming the outer layer of the peridiole if no tunica is present, is a hard dark-coloured coat of compacted hyphae called the cortex. The cortex is only rarely composed of an entirely uniform mass of hyphae, although at first glance it may sometimes appear to be. Three types of cortex can be recognized: (a) One-layered cortex. The cortex consists of a single layer of closely compacted darkcoloured hyphae (figure 483). In descriptions this is usually referred to as cortex simplex. (b) Two-layered cortex. The cortex consists of two distinct dark layers of compacted hyphae, separated by a band of lightercoloured and loosely interwoven hyphae (figure 48b). Actually this cortex (referred to in descriptions as cortex duplex) is composed of three layers. There is much variation in different species in the width and distinctness of the middle loose layer. In some species, e.g., C. stercoreus, the middle layer is uniform and easily discerned (figure 48b). In a few species, e.g., C. gracilis, the middle layer is so loose in organization that it may allow the two dark layers to separate when sections are cut. (c) Cortex of intermediate type. This type of cortex has apparently seldom been employed in descriptions, which usually refer to the first or second type. What is meant here is that in 131 / Review of taxonomic characters

3 / Spores The use of spore measurements to characterize species in the Nidulariaceae is fraught with several difficulties. Lloyd (1906) wrote in his monograph: The general size and shape of spores characterize species, but the particular size is of no value whatever, and much latitude must be given to all spore measurements. Spores not only vary in size in the same collection, but in the same peridiole, and I have noted two spores side by side differing more than ten mic. in length/ The situation is, however, less depressing than suggested by Lloyd's statements, which were intended as a warning. Extreme variation in spore size is characteristic of certain largespored species such as Cyathus stercoreus, C. poeppigii, and some others and results, as noted previously, from the fact that basidiospores of these species may increase greatly in size after they have become detached from their basidia. In these species one must note the size of the largest spores and of the smallest and form some idea (preferably by measuring a large number) of the average size. If this is done, little difficulty is usually encountered in fitting specimens to a description when all other characteristics are duly considered. Among the small-spored species such as C. pallïdus no great range in spore size is encoun-

tered in any one collection. However, spores of one collection may be reasonably uniform among themselves, but those of other collections (of different provenance) may often be smaller or larger on the average. I have found that some deviation from published spore size is less important in species determination than is the total concept derived from the consideration of all the species characteristics. The tropical species C. poeppigii, for example, is readily recognized by the aggregate of its morphological characteristics, but races of this species from the West Indies almost invariably have spores that are longer and wider than spores of races from the Pacific islands. Some of the problems that beset the taxonomist regarding spore size were noted by Palmer (i96ib) who reexamined the Nidulariaceae in Persoon's herbarium. Of this, one example will suffice. Concerning C. microsporus, Palmer wrote: Cyathus microsporus was characterized by the Tulasnes (1844) as having small spores. However, according to the type these are no smaller than those recorded for Cyathus pallidus Berk, and Curt., said to differ by the light-coloured, fragile fruit bodies with long, rigid hairs and, of course, the larger spores. Lloyd gave Cyathus hookeri Berk, as having spores 6 X 8 ja although later (Lloyd, 1915) he thought that it should be considered a synonym of C. microsporus.

Spore size is, of course, an asset in the construction of keys which differentiate between

132 / The bird's nest fungi

groups of species, especially if the size differences are marked. Spore shape (figure 8) is of considerable value in species identification. The spores of Cyathus striatus are distinctly ellipsoidal, i.e., one dimension is markedly less than the other. Spores of C. stercoreus are globoid, often perfectly spherical. However, in the group of tropical species including C. pallidus, spore shape is seldom of any value diagnostically since there is too much variation in any one species to make meaningful a comparison of spore shape with that of another species. 4 / Microscopy of peridium wall Details of the structure of the wall of the peridium have only rarely been used as species characters. Nidula emodensis has peculiar spiny, branched hyphae present in the outer peridium wall. Cyathus crassimurus has peridium walls which are twice to three times as thick as those of its nearest relatives (Brodie, i97ib). The peridium wall of Crucibulum cyathiforme is only about half as thick as that of C. laeve. As noted in Chapter 7, Palmer (i96ia) erected the genus Mycocalia on the basis of differences in the hyphal elements of the peridium wall from those of the wall of Nidularia. Except for these few examples, little attention has been paid by taxonomists to the microscopy of the peridium wall, and possibly new taxonomic criteria might be found by further study.

12 / The Genera Nidularia and Mycocalia

A / D I S T I N C T I O N B E T W E E N THE TWO G E N E R A The bird's nest fungi which, until recently, have been properly assigned to the genus Nidularia are now divided between two genera, Nidularia and Mycocalia. The latter was established by Palmer (1961^. Despite legalistic difficulties, Palmer proposed conservation of the genus name Nidularia of Fries and he distinguished Nidularia from Mycocalia principally on the following basis: Peridium robust, tufted when young, cream to cinnamon in colour; peridium including tinted, rigid, spinose aseptate hyphae which continue into long simple threads (figure ^b, c) Nidularia Peridium thin, usually white, ephemeral; peridium formed of hyaline, branched, septate hyphae bearing clamp connections (figure 493) Mycocalia Cejp and Palmer (1963) recognized two species of Nidularia, a possible third species, and five species of Mycocalia. I have made no special study of the fungi belonging to these two genera beyond examining as many types as were to be found in the fungus herbaria in Kew and Paris. For the most part, the following descriptions and notes about the species take advantage of the studies of Cejp and Palmer and of the conclusions reached by them. The fungi of these two genera occur mostly in north temperate regions and have not been 133 / The genera Nidularia and Mycocalia

commonly reported by mycologists except for the recent work of Palmer (1958^). The sporocarps develop mostly in very moist locations. For example, the reasonably large fruit bodies of Nidularia pulvinata, in my experience in Canada, are known only from moist mixed hardwood forests (e.g., in Ontario) and from the moist west coast (e.g., British Columbia), where they are fairly common on driftwood. None have so far been recorded on the dry prairie or parkland areas of Canada. Several species oí Mycocalia appear to occupy a distinctive ecological niche and are found growing upon various plant materials in wet areas, especially among rushes such as Juncus effusus and commonly upon debris of that plant itself. M. duriaeana appears to have adapted itself to arid areas such as dunes. Palmer (1958^) commented 'A pertinent point is why these species have been found so infrequently ... by such careful collectors with broad horizons as Berkeley and Cooke? Perhaps it was the fact that moorlands, bogs and sand-dunes were considered to be poor habitats in which to search for fungi/ Details of distribution and habitat are given in the descriptions of the individual species that follow. B / THE G E N U S N I D U L A R I A Nidularia Fries, Symb. Gast. fase. 1:2,1817, nom. cons, (non Nidularia Bull, ex St. Hil. 1805, nom. rejic)

- Nidularia Fr., Sects. Scutulae et Granularía Tul., Ann. Sci. nat. sér. 3, i, Bot. 98, 1844

- Granularía Roth ex Nées p.p., Hor. phys. Berol. collect., 6 (non Sow., 1815) Etym. L. nidula (diminutive), a little nest ; thus 'like a little nest' Type species: Nidularia far eta (Roth, ex Pers.) Fr.

(1823)

Plants subglobose, the peridium wall of one layer and without an epiphragm, not opening regularly or forming a perfect cup, but breaking up irregularly and leaving the pendióles in an exposed pile on the substratum. Pendióles grey-brown to red-brown, shining, not attached by funiculi to the peridium wall but imbedded in a mucus when fresh, the mucus drying down and sticking the pendióles together. The above description, adapted from Coker and Couch (1928), adequately defines the genus before Palmer's description of Mycocalia. To the above there must now be added the following paraphrase of the description of Nidularia as given in Cejp and Palmer's monograph (1963, p. 115). Fruit bodies clustered or single, containing many peridioles, rounded in shape and somewhat contracted. Peridium thick and partly persistent, on the outside granular to hairy and formed of tinted, rigid, spiny, branched hyphae which elongate into simple threads ; sometimes hyaline hyphae are also present which are branched, septate, and bear clamp-connections.

i / Nidularia pulvinata (Schw.) Fr., Syst. Myc. 2: 301, 1823 Cyathus pulvinatus Schw. Syn. Fung. Carolensis Superioris; Schr. Nat. Ges. Leipzig i: 51, 1822 Granularía pulvinata (Schw.) Kuntze, Rev. Gen. Pi. 2: 855, 1891

Etym. L. pulvinata, cushion-shaped or convex Figure 5oa, b 134 / The bird's nest fungi

C(yathus) sparsus orbicularis pulvinatus clausus testaceus pulvere tectus, capsulis nigr o-ciñereis compressis majusculis. In arboribus maxime putridis. Magnitudinem unciae dimidie nel unciae quadrantis adipiscitur. Superficie pulvere copioso badio inquinanate tegitur aetate in flocculos quasi congesto. Capsulae difformes, majusculae, numerosae, primum albae, aetate cinereo-nigrae (von Schweinitz). In view of the fact that the original description does not contribute much to the present-day concept of this species (except in the phrase referring to the peridioles as 'cinereo-nigrae' and that, regarding the type 'none of the original material remains except some wood on which it grew' (White, 1902, p. 274), the description given below is based upon the material seen by the present author, taking into account Palmer's view of the correct disposition of North American material. Peridia globose or subglobose, sessile, 2-10 mm high and wide, golden-brown to reddishbrown, mostly deep cinnamon-brown fading to dull grey-buff, at first floccose or powdery, becoming smoother in age; inner surface shiny, smooth, brown; peridioles numerous, irregularly shaped, rarely wrinkled when dry, greyish-brown, barely i mm in diameter; cortex of pendióle of much-branched, thickwalled, densely-fused, inseparable hyphae; spores hyaline 6-10 jut x 4-7 JUL. Frequently the peridium is tubercular from pressure on the peridioles. Palmer (in litt.) states that the dark colour of peridioles of this species is associated with the densely fused hyphae in contrast with other species e.g., Mycocalia denudata in which loosely interwoven hyphae impart a light colour to the peridioles. This fungus is widely distributed but rarely abundant in North America (Type locality, South Carolina, USA) where it is found on a variety of kinds of old wood, occasionally on driftwood. According to Palmer (i96oa), both this species and N. far eta (and a possible third species) occur in North America. Lloyd and others considered North American material

FIGURE 49 Hyphal elements in Mycocalia and Nidularia : a, hyphae from peridium oiMycocalia denudata ; b, Nidularia farcta, skeletal hyphae from peridial wall (left) and hyphae from interior of peridial wall (a, b, Xi5o, approx.) ; c, d, N. farcta skeletal hyphae from peridial wall showing spinous nature (x75, approx.). Redrawn from Taxon, 1961, 10: 57, by permission of Taxon 135 / The genera Nidularia and Mycocalia

FIGURE 50 Nidularia pulvinata : a, b, showing clearly the large numbers of small gel-covered pendióles, X3 ; c, specimen of Nidularia far eta that shows a remarkable resemblance to Nidula in external form, X}

not distinguishable from European and included this and the following species under the name N. pisiformis. Although Lloyd did note differences among his specimens, he did not believe that it is possible to distinguish more than one species. The characters used by Cejp and Palmer (1963) to distinguish N. pulvinata from European material (which they referred to N. far eta) are italicized in the description given above. Palmer also reported N. pulvinata as occurring in Brazil but I have not seen the material (collection in the Lloyd Herbarium according to Palmer, in litt.). 136 / The bird's nest fungi

2 / Nidularia farcta (Roth, ex Pers.) Fr., Syst. Myc. 2: 301, 1823 Cyathus farctus Pers., Syn. Fung. p. 239, 1801 Cyathus globosus Ehr., Syl. Myc. Ber. 28, 1818 Nidularia globosa (Ehr.) Fr., Syst. Myc. 2: 302, 1823 Nidularia australis Tul. Ann. Sci. Nat. 3(1): 93, 1844 Nidularia pisiformis Tul., Ann. Sci. Nat. 3 (i) : 100, 1844 Nidularia confluens Fr., Syst. Myc. 2: 301, 1823 Nidularia microspora Vel., Nov. Mycol. 169,1939

Etym. L. farcta, stuffed, solid Figure 500 This species (Type locality, Bremen, Europe) is widely distributed in Europe from as far north as northernmost Norway (Eckblad, 19/ia) and is apparently the only European species known, growing upon partially rotted wood. Palmer also reports it from North America (i96ob) and, with reservations, refers to collections of this species from Chile and Tasmania. The description given above for N. pulvinata applies equally well to N. farcta except for the following points. The pendióles of the latter are reddish-brown, lenticular, wrinkled when dry and have a cortex composed of thick-walled, separable hyphae which are sparsely branched. It may be precarious to separate the two above entities on the basis of the colour of the pendióles; in the material that has been seen, both European and American, there is considerable variation in the shade of brown displayed by the pendióles. The nature of the hyphae of the cortex may prove to be a more reliable criterion for separation. Until further study can be made, the author feels that mycologists must accept Palmer's distinction between these two species, based as it is upon detailed study.

3 / Nidularia sp. Palmer (i96ob) and Cejp and Palmer (1963) believe that a third species exists in North America. The latter is (according to Palmer, in litt.) 'macroscopically similar to N. farcta but differs in the cortical hyphae of the peridioles being much branched, which is intermediate between those of N. farcta with simple very sparsely-branched hyphae and N. pulvinata with much-branched densely interwoven hyphae/ Palmer (in litt.) informs the author that he has refrained from describing this species until more fresh material can be studied. C / THE G E N U S M Y C O C A L I A Mycocalia J.T. Palmer, Taxon 10: 58, 1961 137 / The genera Nidularia and Mycocalia

- Nidularia Fr., Symb. Cast, i: 2, 1817 - Nidularia Fr., Sectio n, Sorosia (Clausae) Tul. Ann. Sci. Nat. Ser. 3,1: 98, 1844 - Granularía Roth ex Nées, Hor. Phys. Berol. collect 6, 1820, pp., non Sow. 1815 Etym. Gr. mykes, fungus, and kalia, bird's nest Type species: Mycocalia denudata (Fr.) J.T. Palmer Fructifications gregarious to solitary, multi- to uni-peridiolar. Peridium thin, evanescent; with hyaline, branched, and septate hyphae bearing clamp-connections. Peridioles free, lenticular, spores hyaline. Metamorphosed basidia often present. KEY TO SPECIES OF MYCOCALIA i

Cortex of much branched, tapering, thickwalled hyphae with main stem up to 20 /m thick; peridioles yellowish-brown to brown; spores ca. 9 X 5 ¡JL (tropical species) M. reticulata(p. 139)

i

Cortex of uniformly interwoven to compacted hyphae, typically producing a labyrinthiform pattern 2 Cortex i-layered, with pale yellowish-brown, ventricose spores ca. 13 X 5 /¿, consistently with one peridiole ca. 500 ¡m diam. M. sphagneti (p. 140)

2

2

Cortex 2-layered, with hyaline ovoid spores

3

3

3

Metamorphosed basidia ovate to globose with a truncate base, often with a large oil globule ; spores ca. 5 X 3.5 JUL; consistently with one peridiole, ca. 22 JJL diam. M. minutissima (p. 138) Metamorphosed basidia ellipsoid to pyriform, basidiospores ca. 7 X 5 /¿; rounded fruit bodies with one to many peridioles over 250 /¿ in diameter (often only loose peridioles remaining) 4

4 Peridioles yellowish to tan, becoming conspicuously biconcave when dry ; exocortex loosely interwoven and endocortex more closely compacted M. denudata (p. 138) 4 Peridioles dark-red to blackish, rarely becoming biconcave; exocortex closely compacted and endocortex a thin layer M. duriaeana (p. 138)

i / Mycocalia denudata (Fr.) J.T. Palmer, Taxon. 10: 58,1961 Nidularia denudata Fr., Symb. Cast, i: 2, 1817 Cyathus denudatus (Fr.) Sprengel, Syst. veg. ed. 16, 4: 416, 1827 Granularía denudata (Fr.) O. Kuntze, Rev. gen. plant, 2: 853, 1891 Nidularia fusispora Massée, Bull. Misc. Inf. 1849: 125, 1898 Nidularia arundinacea Velenovsky, Nov. mycol. p. 169, 1939 Etym. L. denudata, stripped or bared Fruit bodies globose, up to 1.5 mm wide, often confluent, with frail ephemeral peridia which are white (often slightly yellowish), thin, and smooth. Peridia composed of hyaline, branched, septate hyphae bearing clamp connections. Upper surface of the peridium dry but often bearing droplets of liquid resulting from the development of the fungus. Pendióles (300-400 jit) very numerous, free in a hyaline gelatinous mass, when fresh and moist disk-shaped, when dry coin-shaped (biconcave) ; smooth and bare, yellow-ochre. The cortex has two layers ; the outer (exocortex) is loosely woven and of a yellow colour, the inner (endocortex) is thicker and red-brown. Under the microscope the layers of the cortex appear to have a labyrinthiform design. There are two kinds of spores: (a) Basidiospores (7.5 X 5 JLL) ovoid, hyaline, and (b) metamorphosed basidia (12 X 7 jit), pear-shaped to ellipsoidal or globose, with a thick wall (Cejp and Palmer, 1963). The outstanding features of this species are: the small size, the white peridia with branched, septate, clamp-bearing hyphae, and the yellowish to chestnut-brown pendióles with a double cortex. The metamorphosed basidia, which resemble large spores, were first described by Olive (1946) for Mycocalia duriaeana. M. denudata was first described by Fries from Sweden. It is widely distributed in northern Europe, including Great Britain, and has been reported from Australia, Chile, Tasmania, and British Columbia (Canada). The fungus grows mainly on old wood, and on

138 / The bird's nest fungi

stems of rushes (¡uncus spp.) usually in acidic areas. 2 / Mycocalia duriaeana (Tul.) J.T. Palmer, Taxon. 10: 58, 1961 Nidularia duriaeana Tul., Ann. Sci. Nat. Bot. (3) i : 99-100, 1844 Granularía duriaeana (Tul.) O. Kuntze, Revis, gen. plant. 2: 855, 1891 Granularía castanea (Ell. et Ev. in Herb.) ex White, Bull. Torrey Bot. Club 29: 276-7, 1902 Nidularia castanea (White) Saccardo, Syll. fung. 17, 216, 1905 Etym. Named in honour of Durieu de Maisonneuve, a French botanist In Palmer's opinion (1957, i96ib), 1963 this species is very similar to M. denudata, above, but is distinguished by the following: the peridioles are dark blood red to almost black; the outer layer of the cortex is firmer and thicker than the inner ; the centre of the peridiole seldom becomes sunken when dry ; the peridioles are usually smaller and the basidiospores broader than in M. denudata. Peridioles are about 300 /x in diameter and 150 JUL thick. Basidiospores measure 7 X 5 . 5 ^ . The species was described from Algeria and is also known to occur in Czechoslovakia, Holland, Great Britain, the east coast of North America, and Tasmania. The fungus grows on dead wood, rotting canvas, etc. In England, Palmer found this species only on alkaline dunes on culms of Ammophila, on rabbit dung, and on dead twigs and pine needles.

3 / Mycocaliaminutissima (J.T. Palmer) J.T. Palmer, Taxon. 10: 58,1961 Nidularia minutissima J.T. Palmer, Naturalist (London), Jan.-Mar. 4,1957 Etym. L. minutissimus for very small 'Sporocarps gregarious or solitary, white, uniperidiolar. Peridium very thin, evanescent, with branched hyaline hyphae. Peridioles free, lenticular with two-layered cortex, dark brick-red or deep yellow, bi-concave when dry, about 210 JLL in diam-

éter. Basidia clávate, 2-spored to 4-spored, or metamorphosed. Basidiospores hyaline, elliptical, about 6 X 4 f i . Metamorphosed basidia hyaline, spatulate to globose or irregular, frequently containing large drops, about 11.5 X 8.5 JUL' (Palmer, 1957).

To the above description may be added a few points from Palmer's discussion of the species: (i) peridium looser and more net-like than that oí Mycocalia denudata ; (2) fruit bodies do not coalesce; (3) fresh peridium always moist in appearance ; (4) fruit bodies develop in a little drop of mucilage, in contrast to the four other species, the peridial walls of which are always dry. The type collection came from Lancashire in England and grew on the stem bases of the rush ¡uncus effusus in a damp field. Palmer has also recorded its occurrence in England on grass debris, dead leaves oiBetula, on moss and on a decorticated branch of Finns sylvestris ; in Europe it has been recognized from Czechoslovakia and Germany. Palmer notes (1958^ p. 61) 'indications are that N. (= Mycocalia) minutissima requires slightly more acid conditions than N. (— M) denudata. A very moist environment appears to be essential, and the peridia seem to develop in a thin film of moisture on the Juncus root stocks and leaf sheaths. The finding of scattered pendióles and developing peridia on submerged leaves of /. effusus opens the question as to whether this species might not also occur in purely aquatic conditions/ Careful comparison of Palmer's descriptions of the entire pendióles (only) of M. minutissima with those of M. denudata leads the author to the conclusion that they are extremely difficult to distinguish. The whole fruit bodies of M. minutissima are about 210 /¿ in diameter whereas those of M. denudata are 300-400 /¿, a point of distinction which may be helpful. The entire fruit body of Mycocalia minutissima comprises a peridiole covered by a thin evanescent peridium. Pendióles of the two species apparently differ somewhat in size, those of M. minutissima being somewhat 139 / The genera Nidularia and Mycocalia

smaller. In their key, Cejp and Palmer (1963) separate the latter species from M. denudata using, as the primary basis of divarication, the form of the metamorphosed basidia; those of M. minutissima are given as 'ovate to globose with a truncate base'; those of M. denudata as 'ellipsoid to pyriform.' In view of the fact that uni-peridiolar peridia are found in M. denudata and other species of the genus (Palmer, 1963) and that Brodie (19553) recorded a strain of Cyathus poeppigii which produced only 'aberrant' uniperidiolar fruit bodies (figure 28), (see also Chapter 5, G), it is possible that the fungus named M. minutissima consists of single pendióles of some other fungus or is an aberrant uniperidiolar strain of some species which is normally multiperidiolar. Only much more study of this unique fungus, including an examination of the morphogenesis of the fruit bodies, will reveal its relationship to other species and, indeed, to other genera. 4 / Mycocalia reticulata (Petch) J.T. Palmer, Taxon. 10: 58, 1961 Nidularia reticulata Petch, Ann. roy. bot. Gard. Perideniya, 7: 60, 1919 Etym. L. reticulatus from the reticulate peridiole surface presumably Fruit bodies small, white when young, becoming pale brownish, subglobose, up to 2 mm in diameter (or up to 4 mm by anastomosis). Peridium, when dry, a thin transparent sheath, becoming gelatinous and pallid when soaked. Pendióles lenticular, unattached, 0.45-0.55 mm in diameter and 0.2 mm thick, bright yellowish-brown when soaked; peridiole surface strongly reticulate with a broad translucent margin. Outer layer of peridiole wall bearing stout brown antler-like hyphae. The basidia bear spores in somewhat irregular fashion. The spores are hyaline, cylindrical, thick-walled, without apiculus, 8.5-9.5 x 4-5~5-5 M-

The above description by Martin (1939) is given because it was based on abundant fresh material. Cejp and Palmer (1963) give 9 x 5 JUL

as the basidiospore measurements and 10 X 8.5 JÜL for metamorphosed basidia. This is apparently a tropical and subtropical species which was first described in 1919 by Fetch from Ceylon. Palmer reports (1963) that it is known also from France (where it grew, apparently adventitiously in a greenhouse), from Panama, and from the United States (Baton Rouge and Bogalusa, Louisiana). 5 / Mycocalia sphagneti].T. Palmer, in Cejp 6 Palmer, Ceská Mykologie, 17: 122,1963 Etym. L. sphagnetum, of a sphagnum bog association Fruit body solitary to social, never coalescing, containing a single pendióle which develops either horizontally or vertically. Peridium white, woolly at first, dry, later smooth, formed of hyaline, branched, thin-walled hyphae, about 3 ¡JL thick with clamp connection, closely adhering to the peridiole (hence the presence of a gelatinous matrix has not been ascertained), becoming arachnoid as the peridiole matures and finally disappearing, sometimes leaving a column of white hyphae supporting the peridiole. Peridiole about 450—685 ¡JL diam. X 100-300 jit thick (very variable in size), free, at first white, becoming dark blood-red to almost black, usually biconcave while fresh with the centre of the disk darker than the rim; drying darker, smooth, or with only a few wrinkles. Cortex one-walled, constructed of closely compacted hyphae producing a labyrinthiform pattern. Basidiospores about 13 X 5.5 ¡JL, pale yellowish-brown, smooth, ventricose with occasional small oil globules. Structures resembling metamorphosed basidia (about 12 x 10 jit, hyaline, ellipsoid to pyriform, usually with a truncate base) have been observed. Basidia about 25 x 8 jit, hyaline, clávate, with 4 (often only 2) spores apically on short sterigmata.

Palmer's description is quoted above. Palmer notes in addition that M. sphagneti differs from other species of Mycocalia in 'the onewalled cortex and the large yellowish-brown typically ventricose spores/ The type material from Derbyshire, Eng140 / The bird's nest fungi

land, fruited on dead axes of Juncus effusus in a sphagnum bog and the species is apparently known only from England. This uniperidiolar fungus presents the same kind of problem raised above in connection with M. minutissima. I prefer to withhold judgment on these two species until a detailed study has been made of the morphogenesis of the fruit bodies. Palmer notes (in litt.) 'This species has fruited in culture and produces typical fruit bodies. I did contemplate separating this species as a separate genus but, after sounding opinion, I decided to place it in Mycocalia/

D / N O T E S ON D O U B T F U L S P E C I E S OF N I D U L A R I A 11 Nidularia heribaudii = Cyathus olla The type of this species, described by Patouillard (Lloyd, 1906), is very probably only a small fragment of a specimen of Cyathus olla in which the funiculi are abortive or at least not recognizable. I examined the type in Paris in 1952 and was of the opinion that it is C. olla, and Palmer (personal communication) has confirmed this opinion. 2 / Nidularia australis = N. /arcfa Tulasne described this species from Chile. Martin (1939) commented that this species 'both in Tulasne's drawing and in Lloyd's photograph shows strong resemblance to Nidula/ My note (1952) on the type in Paris is 'cupulate, red-brown to cinnamon, velvety rugulose. Not all specimens look like Lloyd's Fig. 8 but it is distinct and seems almost like another genus/ Palmer (personal communication) is of the opinion that it is identical with N. farcta which is known to occur also in Tasmania. A specimen of N. farcta bearing considerable resemblance to a Nidula is illustrated in figure 5oc.

3 / Nidularia campor Spegazzini in Bol. Acad. Nov. Sci. Cordoba 25, 31,1921

4 / Nidularia (?) bonariensis Spegazzini in Fun8- Arg. m / 1 5°/ x^97

Material under this name is unknown to the author and also to Palmer (personal communication).

This 'species' is likewise unknown to the author and to Palmer,

141 / The genera Nidularia and Mycocalia

13 / The GeneraNidula and Crucibulum

A / NIDULA Nidula White, Bull. Torr. Bot. Club 49: 271,1902. Lectotype: Nidula candida (Peck) White, Bull. Torr. Bot. Club 29: 271, 1902 - Nidularia candida Peck. Reg. Kept. 45: 24, 1893 Etym. L. maula, a little nest 'Peridium composed of a single homogeneous, but layered membrane which is at first continuous over the mouth much as in Crucibulum ; sporangioles very numerous, at first immersed in a glutinous substance, very closely packed, entirely filling the central cavity and in no way attached to the peridium wall; no filaments intermixed with the spores/

White's description of the genus as given above must be expanded if the reader is to be able to visualize the genus and the four beautiful fungi at present known to belong to it. The shape of the fruit bodies of three of the four species is that of a mug, i.e., a cup with almost vertical sides (figure 51), the lip of which is flared or bent outwards. In colour the cups are white, grey, or buff in three species, tawny in N. macrocarpa. The fruit bodies are massive, thick-walled (with the exception of one species) and are covered externally by a shaggy or woolly tomentum. White described the peridium wall as homogeneous. Actually the wall is homogeneous (or nearly so) in only one species, N. macrocarpa ; in the other three 142 / The bird's nest fungi

species it consists of at least two layers, possibly three (Overstreet, 1955). Peridioles are small, numerous, reddish-brown, or greybrown. They are gelatinous when moist and are devoid of a funiculus. White's description indicates that no filaments are intermixed with the basidiospores ; however, Overstreet (1955) found hyphae abundantly interspersed with spores and presented photographs to illustrate the observation. KEY T O S P E C I E S O F N I D U L A

i

Fruit body large (8-15 mm high) Fruit body pale grey or grey-brown, tomentum shaggy; pendióles 1.5-2 mm wide N. candida (p. 145) 2 Fruit body tawny to cinnamon in colour, tomentum not shaggy ; pendióles o. 8-1 mm wide N. macrocarpa (p. 145) i Fruit body small (4-6 mm high), white to pale yellow or light brown, tomentose to shaggy (smooth in age) 3 Sides of fruit body almost parallel in lower part ; peridioles 0.5-1 mm wide, spores 5-9 jU long N. niveo-tomentosa (p. 143) 3 Sides of fruit body strongly inclined, fruit body crucible shaped; peridioles 1.5 mm wide, spores 3-4 x 4-5 jit N. emodensis (p. 145) 2

i / Nidula niveo-tomentosa (Henn.) Lloyd, Myc. Writ. 3 : 455,1910 Cyathus niveo-tomentosa Henn. ; Hedwigia, 37 : 274,1898

FIGURE 51 Nidula niveo-tomentosa, X4« Photo reproduced courtesy of Mycologia, 1951, 43: 334

Nidula microcarpa Peck ex White; Bull. Torrey Bot. Club 29: 272,1902 N. microcarpa var. rugispora White; Bull. Torrey Bot. Club 29: 272, 1902 Etym. L., niveo snowy and tomentosa, tomentose Figure 51

This is one of the most beautiful of all bird's nest fungi (figure 51) when its snow-white furry cups are seen, as they are often, growing among mosses. Young fruit bodies (mostly 4-6 mm high) open widely, the mouths of the cups being surrounded by a fringe of glistening white hyphae, and within the cup numerous small mahogany-brown pendióles lie imbedded in a shiny, glutinous matrix (figure 51). Pendióles are 0.5 toi mm wide and spores are 6-9 X 5-6 ¡JL, elliptical to subglobose. Older fruit bodies at times tinged with yellow and brown, sometimes 143 / The genera Nidula and Crucibulum

becoming so thin and smooth that they have frequently been mistaken for old specimens of Crucibulum. The species is quite abundant in western North America from British Columbia southward as far as California (Type locality : California, Potter Valley). In South America it is known from Venezuela, Chile, and Argentina. It occurs also in Japan, New Zealand, and on the Blue Mountain in Jamaica.

Lloyd at one time distinguished this species from N. emodensis (from the Indian Himalayas) but later decided that the two species are the same. Cunningham accepted the synonymy when writing of the New Zealand Nidulariaceae (Cunningham, 1924). However, my examination of the type of N. emodensis at Kew and isotypes in Paris led to the realization

that the latter species is more massive than N. niveo-tomentosa, much darker in colour and externally more felty. In British Columbia N. niveo-tomentosa grows almost invariably in close association with bracken fern (Chapter 9, c, 2). In culture, this species displays several morphological features which distinguish it sharply from the other common North American species N. candida (Brodie, i95ic). 2 / Nidula candida (Peck) White, Bull. Torr. Bot. Club 29: 271, 1902 Nidularia candida Peck, Reg. Kept. 45: 24,1893 Etym. L. candidus, pure, shining, white Figure 5za Usually considerably larger (1-1.5 cm high) than N. niveo-tomentosa, rougher and more shaggy in appearance and, when fresh, grey in colour or even light wood-brown, and having a wide-flaring mouth. The pendióles are large (1.5-3 mm—c^- Nniveo-tomentosa), grey or light brown with a thin tunica. Spores are 8-10 X 4-6 ¡JL, ellipsoid.

N. candida also ranges along the west coast of North America from Alaska to Oregon (the type from Olympia, Wash.). It is apparently abundant in Oregon at altitudes up to 3000 feet. In British Columbia it occurs in much the same sites as the previous species but is seldom as abundant. It was recorded for New Zealand by Cunningham in 1924 and has since again been recorded from that country. The author has not seen the latter collections.1 Old specimens may appear thin-walled and bleached, and, if devoid of pendióles, are often misidentified by the inexperienced. In culture, this species shows numerous i I have recently examined a single collection from Caracas, Venezuela, that appears to be N. candida. The specimens have large, flat pendióles and the spores measure 6 x 7-9 ju; the specimens are unlike N. candida, however, in that the pendióles are dark brown and the inner surface of the peridia is brown. The collection is too scanty to allow me to be dogmatic about the existence of N. candida so remote from its known range.

144 / The bird's nest fungi

morphological differences from cultures of N. niveo-tomentosa. Comparison between the two species as to morphology and characteristics in culture is given in detail in a paper by Brodie (i95ic). 3 / Nidula emodensis (Berk.) Lloyd, Lloyd, Myc. Writ. 2, The Nidulariaceae, p. 12, 1906 Cyathus emodensis Berk., Hooker's J. Bot. 6: p. 204,1854 Crucibulum emodense Berk., Hdbk. N.Z. Fl. p. 621, 1867 Etym. L. emodensis, of the Himalayas (from Emodus or Emodi Montes, the classical name) Figure 52C Peridium 4-6 mm high, 4-5 mm wide at mouth, tapering to truncate base; exterior dingy grey, becoming darker, covered with closely oppressed tomentum ; interior smooth, shining, dingy white becoming darker ; mouth recurved in old specimens ; pendióles numerous, reddish-brown to black, 0.5-1 mm diam., rugulose; spores narrowly elliptical, or obovate or pyriform ; apex rounded, base acuminate, 6-9 x 4-6 ¡JL [paraphrased from Cunningham (1924)].

This species resembles Nidula niveo-tomentosa but, as noted above, I feel that until more material becomes available the species should be retained. It was originally collected by Hooker in the Indian Himalayas. I have specimens from Ceylon and one from a 1953 collection made by the Mount Everest Expedition of that year (at 13,500 ft). The species has also been reported from China, Japan, Australia, and New Zealand. All specimens seen by the author (including the type and isotypes) are smaller than most of those of N. niveo-tomentosa, have walls thicker and more felty, and are buff-grey to rusty-brown in colour. The sides of the fruit body are quite strongly inclined so that it has the shape of a crucible (figure 52), whereas the sides of the fruit body of N. niveo-tomentosa are almost parallel in the lower part. The reddish-brown pendióles are 1.5 mm in diam-

FIGURE 52 Nidula spp., X4: a, N. candida ; b, N. macrocarpa ; c, N. emodensis ; d, N. m'ueo tomentosa

éter and contain spores which, in the type (at Kew), measure 4-5 x 3-4 /¿. The altitude at which the 1953 collection was obtained is a record as far as the author is aware, although altitude per se probably has little, if any, significance in the distribution of bird's nest fungi. 4 / Nidula macrocarpa Lloyd, Lloyd, Myc. Writ. 5: 731, 1917 Etym. L. macrocarpa, having a large fruit body Figure 5zb 145 / The genera Nidula and Crucibulum

Cups about a cm high, 6-7 mm thick at the summit, at first appressed tomentose, becoming smooth and brown when old. Pendióles a scant mm, brown, smooth. Spores abundant, hyaline, smooth, 12-16 X 5-6 IJL. (In Lloyd's description, as paraphrased, apparently 'thick' in the first line should be 'wide'.)

This unique species is quite unlike the other three members oí Nidula in several respects. It was described from a collection made by M. Espinosa in Chile. I studied the type material some years ago and have examined three other collections, all from Chile. The species has not

FIGURE 53 Crucibulum: a, C. laeve on old rope, X o. 75 ; b, C. parvulum, note magnification, xio;c, (left) C. cyathiforme and (right) C. /aeue for comparison, X3.5

146 / The bird's nest fungi

been recorded from any other part of the world. In all these specimens, the fruit bodies are tall and vase-like with the mouth flaring outward strongly but abruptly near the lip (figure 52b). The wall of the fruit body is thin and brittle and the whole fruit body is of a deep fawn (often slightly red-brown) colour quite unlike that of any other species of Nidula. Pendióles are smooth and brown, i mm or less in diameter and covered with a tunica. Spores are variable in size, mostly 5-10 i¿ x 3-5 /¿. These characteristics make N. macrocarpa unique in the genus Nidula. However, fruit bodies of this species are also structurally quite different from others. Overstreet (1955) pointed out that the peridium wall in N. macrocarpa is homogeneous or one-layered, in contrast to the multi-layered condition of other species and that the two-layered epiphragm of N. macrocarpa bears peculiar hyphal coils not present in other species. It is clear that N. macrocarpa is well separated taxonomically from the other Nidula species. Study of the morphology of fresh material would be interesting not only in relation to other species but in relation to other genera, especially Crucibulum. B / THE G E N U S C R U C I B U L U M Crucibulum Tulasne, Ann. Sci. Nat. Ser. iii, Vol. i: 89,1844 Etym. L. (Medieval) Crucibulum, crucible, melting pot Type species: Crucibulum laeve (Huds.) ex Relh. Kambly ; Kambly and Lee, Univ. of Iowa Stud. Nat. Hist. 17 (4): 167, 1936 Tulasne's rather lengthy description of the genus may be reduced to the following essentials. Fruit body short cylindrical, sessile (figures 21, 38c, 43, 53) ; peridium of a single layer, thick, the exterior velvety or woolly when young, becoming smooth with age ; interior pale, smooth, the epiphragm covering the mouth, disappearing at maturity and exposing the numerous pendióles ; pendióles disk-

147 / The genera Nidula and Crucibulum

shaped, pale, covered by thick tunica and attached to the peridiole wall by a simple funiculus, the upper end of the funicular cord being attached to a conspicuous nipple-like protuberance on the under surface of the peridiole.

A single character would serve to separate the genus from all others in the Nidulariaceae. Lloyd's description of this feature is as follows : 'The funiculus is asimple, thin cord, capable of long extension when wet. It is attached to a little nipple-like protuberance on the peridiole/ Old specimens of Nidula or even of small species of Cyathus are frequently mistaken for Crucibulum but, if pendióles are present, one glance at the little button-like purse at the apex of the funicular cord (figure 38c) is sufficient to identify Crucibulum ; no purse in Cyathus is remotely like it and there is no funiculus in Nidula. One other character of Crucibulum should be emphasized. The pendióles are provided with a thick and persistent tunica, from which they appear white or light buff when fresh and conspicuously white in age (figures 38c, 53b) ; such light-coloured pendióles are not found in other genera with which Crucibulum could be confused. Crucibulum has long been considered to be a monotypic genus. Although many 'species' have been described, Lloyd (1906) and others have taken the position that, because there are so many variants towards and away from typical C. laeve (known in older literature as C. vulgare), the designation of these variants as species is not justifiable. Where intermediate forms of C. laeve exist in similar habitats and close proximity, I likewise have considered taxonomic separation unwise. However, recently two kinds of Crucibulum have received attention which fit neither Tulasne's original description of C. laeve nor the various expanded versions of that description that have been commonly used. On the basis of morphological distinctness, difference in habitat

and distribution, and (for one species) genetic isolation, I have added C. parvulum (figure 53b) and C. cyathiforme (figure 53c) to the genus (Brodie, 19/oc, i97ic). Crucibulum is separated from Cyathus by the thick peridium wall consisting of a single layer of hyphae as well as the funiculus of simple structure; from Ni dula it is separated by the presence of a funiculus. KEY T O S P E C I E S O F C R U C I B U L U M

i

Walls of fruit body thick, shape mainly cylindrical with sides essentially parallel, base not slender, colour when young some shade of yellow or brown, tomentum velvety or woolly C. laeve (p. 148)

i

Walls of fruit body thin, shape obconic, colour not yellowish, tomentum very fine-textured, not woolly 2

Fruit body very small (2-4 mm high), wide-flaring, grey, peridioles small (0.15-1.25 mm) C. parvulum (p. 149)

2

Fruit body larger (to 8 mm high), narrow and tapering to narrow base, pinkish ; peridioles 2.5 mm C. cyathiforme (p. 149)

i / Crucibulum laeve (Huds. ex Relh) Kambly, Kambly and Lee, Univ. of Iowa Stud. Nat. Hist. 17(4): 167, 1936 Cyathus laevis DC., Fl. Fr. 2: 269,1805 Crucibulum vulgare Tul., Ann. Sci. Nat. 3 (i): 90, 1844 Etym. L. laeve, smooth Figures 2ia, 38c, 43, 53

In his monograph of the Nidulariaceae, Tulasne (1844) listed fifteen names as synonyms of the one he used, viz. C. vulgare Tul. It would serve no purpose to re-record all this synonymy, for Tulasne himself commented upon it as did Lloyd (1906) much later. The synonymy and dates may also be found in Kambly and Lee (1936). The main point to note is that Tulasne transferred to Crucibulum the species for which he used the name vulgare. 148 / The bird's nest fungi

According to present usage, that name is not valid and the earlier name la eve must be substituted. C. laeve was the name first validly applied to the fungus by De Candolle who, in turn, had based his species upon Nidularia laevis in Bulliard's Histoire des Champignons de la France (Paris, 1791). The following is a free translation of the description given by Tulasne. Crucibulum with thickwalled alutaceous-yellow peridia, sub-smooth on the outside, on the inside very smooth, shiny; mouth entirely smooth; peridioles pale ochraceous, later becoming white ; spores minute, ovate. Fungus 5-8 mm high, almost as wide at the mouth, peridioles frequently 1.4 mm, occasionally between 1.6 to 2 mm in diameter, 0.4 mm thick, uniform; spores elliptical, 8.8 X 4.4 ¡JL.

Lloyd adopted a description similar to Tulasne's but in addition commented upon the variability of the fungus ; he noted also that the fruit bodies tend to become smooth and white in age and that peridioles are variable in size, even in the same cup. Lloyd also mentioned the light-coloured tunica, which is usually white in old specimens. These and subsequent descriptions consistently emphasize the following characteristics oí C. laeve: fruit body subglobose to short cylindrical (figures 38c, 43, 53), thick-walled, tawny yellow, when young; exterior surface velvety and epiphragm coarsely tomentose. C. laeve is a species of circumpolar distribution: it has been recorded from almost every country of Europe and from the Canary Islands ; in North America it ranges from Alaska to Mexico; in South America collections are known from Chile and Terra del Fuego ; it is known also from Japan, Australia, and New Zealand. C. laeve is almost exclusively a temperate-zone species and has not been found in the tropics proper. It is not surprising that a species which has such a wide distribution and is found in such a variety of habitats should exhibit great variation in size, form, colour, and texture. Small pale forms are most fre-

quently found in the far north and in xeric habitats. Large, strongly coloured forms seem to predominate in warm regions of high rainfall (e.g., Indiana). However, there is no clear-cut regional distribution of any one form. In the mountains of British Columbia and Alberta, for example, I have collected at least six quite different phases of C. laeve, no one of which occurred predominantly in any particular area or was confined to a particular habitat. C. laeve fruits on stems, twigs, old nut shells, chips, old matting, andón manure, but, in my experience, never on soil or on large logs. This species is one of the few Nidulariaceae that show twinning (figure 2/c) and growth of new fruit bodies from within the old (Chapter 5, G). 2 / Crucibulum parvulum Brodie, Can. J. Bot. 48: 848, 1970 Etym. : L. parvulum, very small Figure 53b Peridia very small, 1.5-3 mm wide at the mouth, 2-4 mm high, white to grey or pale buff, never yellow, strongly obconic and tapering to a narrow base ; wall of peridia thin (150-180 ¡JL at lip, ca. 300 /¿ near base) and brittle, on the outside covered with a fine-textured tufted tomentum, inside smooth shiny, white to pale buff; lip of peridium essentially smooth except for remnants of delicate epiphragm. Pendióles very small, variable 0.5-1.25 mm wide and bearing conspicuous white or pale buff tunica. Spores variable, elliptical, mostly 4-5 x 7-8 JJL.

The species fruits on old or dead roots of Juniperus horizontalis and stems oí Artemisia spp. in Alberta, Canada, both dry-land plants. It has also been collected in dry areas of Idaho, fruiting upon dead stems of Tetradymia and Atriplex.

149 / The genera Nidula and Crucibulum

This very small Crucibulum is distinctive in its size, greyish colour, thin-walled peridia, delicate tomentum, and small pendióles. That it is not only morphologically a distinct species but is also genetically isolated was shown by Brodie (i97oc), who reported that monospore mycelia failed to show any fertility when they were paired with monospore mycelia of C. laeve. 3 / Crucibulum cyathiforme Brodie, Can. J. Bot. 49: 2009, 1971 Etym. L. resembling Cyathus in form Figure 53 c Peridium obconic and tapering to a narrow base, 3- 6 mm wide at mouth, 5-8 mm high, light pink cinnamon to pale buff; wall of peridium thin (0.2-0.3 mm )/ °n the outside covered with a fine-textured tomentum, inside pale buff, smooth; lip of peridium smooth except for remains of delicate epiphragm. Peridioles 2.5 mm diam., circular to ellipsoidal and bearing a thin greyish tunica. Spores variable, ellipsoidal, 11-17 x 6.5-8 ¡JL, slightly or (rarely) strongly curved (figure 8c).

This unusual species is distinguished from C. laeve and C. parvulum by its larger and curved basidiospores, its striking Cyathus-like shape and its pinkish colour. As noted under C. laeve, it is rare to find Crucibulum in the tropics. This species is known at present only from Colombia, where it grows at an altitude of close to 7000 ft, so that its habitat can scarcely be considered 'tropical/ No other bird's nest fungus is known to me to possess curved basidiospores. Note - Crucibulum albosaccum Lloyd = Cyathus olla Pers (Chapter 14).

14 / The Genus Cyathus

A / THE G E N U S Cyathus Haller, Stirp. Helvet., 3, 127, 1768; ex Pers. Syn. Meth. Fung., 236, 1801 - Cyathia P. Br., Civ. and Nat. Hist. Jamaica, 78, 1756 Etym. L. Cyathus from Gr. Kvàdoç, a cup Lectotype: Cyathus striatus (Huds.) ex Pers., Syn. Meth. Fung., 237, 1801 Peridium microscopically composed of three distinct layers, macroscopically at first closed by a thin whitish epiphragm, which dehisces by an irregular rupture. Pendióles lenticular, dark-coloured, in some species covered by a thin tunica, attached to the inner wall of the peridium by a funiculus of complex structure. Basidiospores hyaline, variable in size and shape.

From the two other genera of the Nidulariaceae in which the fruit bodies are cupulate or vaseshaped, Cyathus is distinguished as follows: from Crucibulum in the distinctly threelayered wall and the complex funiculus ; from Nidula in the presence of the funiculus. Cyathus is of worldwide distribution, but is apparently rare in the arctic and subarctic. North America (excluding Mexico) is not rich in species, there being five in Canada and six in the United States. Of the commonest species, only three occur in Europe. Most of the species are native to warm climates, where they also

150 / The bird's nest fungi

occur most profusely. More than 75 species have been described, but many species names are regarded as synonyms, as will be noted under the description of species. Tulasne (1844) divided Cyathus into two sections: Eucyathus, having fruit bodies that are plicate on the inside; Olla, having fruit bodies that are not plicate inside. Lloyd (1906) divided the species into five groups (subsections), two of Eucyathus and three of Olla. I have adopted a grouping somewhat different from Lloyd's to accommodate species that have been named since Lloyd's monograph was published. The grouping is based on apparent similarity of species with regard to the morphological characters presently in taxonomic use, and it is recognized that a different grouping might result were some other characters to be emphasized that might, in fact, be better indices to true phylogenetic relationships. It does not seem possible at present to construct groups whose characteristics accommodate in all respects all the species believed to belong in those groups. B / THE G R O U P S OF C Y A T H U S i / Theo//a group: peridia flaring out widely in the upper third, not plicate; comparatively smooth-textured, the tomentum consisting of fine appressed hairs and usually devoid of long

conspicuous hairs. Outer peridium wall not strikingly different from inner wall. Spores mostly ovate, thin-walled. Tunica thin.

tomentum hyphae aggregated into nodular or pyramidal tufts. Spores ellipsoidal, moderately thick-walled. Tunica thin.

n / The pallidus group: peridia mostly light coloured, not flaring out abruptly at the mouth, not plicate; basic texture of outer peridium wall fine, woolly, but bearing conspicuous long downward-pointing hairs. Inner peridium wall smooth. Spores mostly ovate, thin-walled. Tunica thin.

v / The stercoreus group: peridia not plicate, external hyphae of peridiole wall bearing abundant hairs of irregular length giving a shaggy or woolly appearance. Pendióles black or dark, devoid of tunica. Spores large, subglobose.

m / The triplex group: peridia mostly dark coloured, sometimes having faint plication, visible only internally; outer surface with spreading, sometimes tufted hairs. Inner surface smooth, silvery white. Cortex onelayered, subhomogeneous, or distinctly twolayered. Spores mostly ellipsoidal, thickwalled. Tunica thin or absent. rv / The gracilis group: peridia not plicate,

vi / The poeppigii group: peridia distinctly plicate externally and internally ; outer peridium wall hirsute to shaggy, strong brown. Peridioles dark to black. Spores large, globose, or ellipsoidal. Tunica none. vu / The striatus group: peridia distinctly plicate internally, sometimes obscurely so externally; outer peridium wall hirsute to shaggy. Spores mostly elliptical. Tunica present.

C / C Y A T H U S S P E C I E S AND S Y N O N Y M S LISTED ALPHABETICALLY Species and varieties considered valid are marked * Two species described while this book was in proof are marked t affinis (stercoreus), p. 168 *africanus, p. 159 ambiguus (poeppigii), p. 171 *anglicus (f.). (olla), p. 155 *annulatus, p. 179 atrofuscus (Crucib. laeve), p. 148 *badius, p. 160 baileyi (stercoreus}, p. 168 *berkeleyanus, p. 178 boninensis (stercoreus), p. 168 brazlaviensis (v.). (stercoreus), p. 168 *bulleri, p. 179 byssisedus (montagnei), p. 176 *canna, p. 158 *cheliensis, p. 173 *chevalieri, p. 177 *colensoi, p. 156 complanatus (olla), p. 154 *confusus, p. 160 151 / The genus Cyathus

*costatus, p. 172 *crassimurus, p. 167 *crispus-t, p. 181 dasypus (olla), p. 155 deformis (Crucib. laeve), p. 148 desertorum (colensoi), p. 156 dimorphus (stercoreus), p. 168 dura (olla), p. 155 *earlei, p. 156 elegans (stercoreus), p. 168 *ellipsoideusjr, p. 181 *elmeri, p. 167 fascicularis (olla), p. 155 *fimicola, p. 169 *gayanus, p. 172 * gracilis, p. 164 *helenae, p. 175 hirsuta (striatus), p. 173 *hookeri, p. 159

FIGURE 54 a, Cyathus olla, Xz;b, C. olla forma anglicus, X2; c, C. earlei, Xz; d, C. pygameus, x$. Figures 54, a, b, reproduced courtesy Mycologia, 1952, 44: 414

152 / The bird's nest fungi

Figure 54, c, courtesy Can. J. Bot., 1962, 40: 1484; Figure 54, d, courtesy Mrs Ellen Trueblood

153 / The genus Cyathus

*intermedius, p. 166 *julietae, p. 161 lentífera (olla), p. 155 lesueurii (stercoreus), p. 168 leveillanus (montagnei), p. 176 *limbatus, p. 172 melanospermus (stercoreus), p. 168 *microsporus, p. 158 *minimus, p. 157 minutosporus, p. 180 *montagnei, p. 176 *nigro-albus, p. 176 *novae~zeelandiae, p. 176 *o//0, p. 155 *olivaceo-brunneus, p. 173 *pallidus, p. 161 pezizoides (Crucib. laeve), p. 146 *pictus, p. 169 plicatus (poeppigii), p. 171 plicatulus (poeppigii), p. 171 *poeppigii, p. 171

puiggarii (stercoreus), p. 168 *pullus, p. 179 *pygmaeus, p. 157 *rudis, p. 177 rufipes (stercoreus), p. 168 rugispermus (stercoreus), p. 168 schweinitzii (v.) (stercoreus), p. 168 scutellaris (Crucib. laeve), p. 148 *setosus, p. 164 similis (olla), p. 155 *sinensis, p. 164 sphaerosporus (pallidus), p. 161 ^stercoreus, p. 168 *striatus, p. 173 subiculosis (stercoreus), p. 168 sulcatus (poeppigii), p. 171 *triplex, p. 163 umbrinus (olla), p. 155 vernicosus (olla), p. 155 wrightii (stercoreus), p. 168

D / SPECIES DESCRIPTIONS, BY GROUPS KEY T O G R O U P S O F C Y A T H U S i

Peridia distinctly plicate or ridged lengthwise externally or internally or both 2

Tunica present, peridia dark or light coloured, hirsute to shaggy Group vu (p. 173)

2

Tunica absent, peridia mostly very dark, pendióles dark Group vi (p. 171)

i

Peridia not distinctly plicate or ridged lengthwise 3

Peridia covered externally by fine matted hairs, no long conspicuous hairs Group i (p. 154)

3

Peridia woolly externally, provided with conspicuous long hairs.

4 4 5

Tunica absent, peridioles shiny black Group v (p. 168) Tunica present, peridioles not shiny black External hairs of the dark-coloured peridium distinctly aggregated into conical tufts or

154 / The bird's nest fungi

mounds, peridia mostly slender, not broad at mouth relative to height Group rv (p. 164) 5

External hairs of peridium not clearly aggregated into tufts, peridia mostly broad relative to height 6

Peridia mostly light-coloured bearing conspicuous long downward-pointing hairs spores mostly ovoid, thin-walled Group ii (p. 161)

6

Peridia mostly dark-coloured, long hairs rare or inconspicuous, spores mostly ellipsoid, thick-walled Group m (p. 163)

G R O U P I, C. O L L A , CtC.

i / Cyathus olla (Batch) ex. Pers., a C. nitidus (Roth) Pers. et j8 C. agrestis Pers., Syn. Meth. Fung., 237. 1801 Cyathus vernicosus DC., Fl. Fr. 2: 270, 1805 Cyathusdasypus Tul. Ann. Sci. Nat. m, i: 85,1844 Cyathia lentífera (L.) White, Bull. Torr. Bot. Club 29: 264, 1902

Crudbulum albosaccum Lloyd, Myc. Notes 7: 1118, 1922 Etym. L. o lia, pot Figures 16, 18, 22, 54a

Like those other members of the Nidulariaceae that are of common and widespread occurrence, this fungus (figure 16), bears a heavy burden of names. The synonomy was recorded at length by Tulasne (1844) and White (1902) and need not be repeated here. As Lloyd tersely remarked, it is time 'mycologists quit discovering it is a new species/ The grey or grey-fawn coloured fruit bodies are usually 10-15 mm hÍ8n anc^/ above all, broad (8-10 mm) for the genus (figure 16) ; they tend to be markedly expanded or flared outwards near the mouth and they are mostly thick-walled. Externally, the peridium is fine-textured; no uneveness or shagginess is apparent to the unaided eye. Internally, the peridium wall is of smooth texture, although it is frequently transversely ridged. The outline of the mouth or lip is commonly wavy and is seldom perfectly circular. The peridioles are conspicuously large (up to 3.5 mm wide), commonly irregular in shape, and provided with a thin tunica and a one-layered cortex. Spores are numerous, ovate, and mostly 10-14 x 6-8 f¿.

The fine-textured peridium wall and the large irregular peridioles (figure 543) make C. olla an easily recognized species ; nevertheless the species is frequently found misnamed in herbaria. It is the most abundant species in Europe (type locality, Ratisbon, Germany) and one of the commonest in North America. It appears not to have been collected farther north than Sweden but ranges far south into South America. It is known also from South Africa, Iran, and Australia. I know of no record of C. olla from the warm moist tropics. C. olla varies considerably in shape and size, but is constant in the nature of the tomentum and in the size and shape of peridioles. The varieties described by Tulasne are, as Lloyd (1906) decided, too variable to make distinction practicable. Cyathus dasypus from South Af155 / The genus Cyathus

rica is surely a form of C. olla with extra-large irregular peridioles. This species is common in North America, especially on the black soils and growing on old wood. In garden plots it fruits upon old boards or upon dead stems of perennial plants. Most of the North American specimens have a welldeveloped emplacement into which a considerable amount of soil is incorporated (figure 54a) ; the emplacement is much less conspicuous in material from other parts of the world. For the most part, C. olla fruits in rather shaded and very moist locations; some races, however, must be tolerant of xeric conditions for it was found fruiting in abundance on the loma in the dry hills around Lima, Peru (Chapter 9, c, 3). In 1922 Lloyd described Crudbulum albosaccum from Argentina on the basis mainly of large peridioles with a white tunica. I have studied the type material from the Lloyd herbarium (No. 32358) and found that the material is in fact Cyathus olla or possibly a closely related undescribed species. The funiculus in the type specimens is that of Cyathus, not of Crudbulum. It is difficult to understand how Lloyd could have made this mistake unless there were present in the packet some peridioles of Crudbulum from another source and which are not there at present. The only unusual feature of the specimens is that the peridioles have a rather thicker, whiter, and looser tunica than is usually seen in Cyathus olla. Otherwise the specimens resemble several South American specimens in the author's herbarium. Lloyd himself noted in his description: Tn general shape and appearance of the cup it (Crudbulum albosaccum) resembles Cyathus vernicosus (= C. olla).' ia / Cyathus olla forma anglicus (Lloyd) Brodie, Mycologia 44: 417, 1952 Cyathus dura White, Bull. Torrey Bot. Club 29: 261, 1902 Figure 54b One of the largest and most striking of all bird's nest fungi. Fruiting bodies are up to 15 mm across the

mouth and 18 mm high (figure 54b). The shape is broadly campanulate, and the mouths are only slightly reflexed but are markedly sulcate or dentate. The exterior is dull grey to grey-brown and covered with a fine-textured tomentum, not at all shaggy. Inside, the cups are smooth, grey-brown, shiny, and irregularly longitudinally grooved. Pendióles are unusually large (3.5-4 mm), flat, and irregular in outline. Basidiospores are ovate, 11.5-12.5 x

7-5-9 M-

This fungus, originally considered to be restricted to England (Lloyd, 1906), has been found in Oregon and Colorado (USA) , Alberta (Canada), and in Argentina. It is a form of C. olla, despite its size and the large pendióles, as is established by the fact that single-spore my celia of typical C. olla were found to be sexually compatible with those of forma anglicus in such a way as to indicate that, of the two pairs of genes governing mycelial interactions, one pair is identical in C. olla and its form anglicus, and one pair is different (Brodie, i95za).

been seen are mostly smaller than C. olla, are browner in colour, and more crucible-shaped, i.e., they have curved sides. Moreover the spores are distinctly more globose than those of C. olla. The species was described from New Zealand and is known only from that country and Australia. It seems to be distinct from those specimens of C. olla that the author has seen from Australia and for that reason it is retained. Cyathus desertorum Muell. & Br., described from Australia, is probably a synonym of C. colensoi. The type specimen (at Kew) resembles a very small pale C. olla except for the form and buff colour, which suggest C. colensoi. The original description of C. desertorum gives spore measurements as 4-5 X 4 ¡JL. However, the author found great variation in the spores in the type : a few were 7-10 x 5-6 [L, roughly globose, but most were 9-12 x 8-9 V" 3 / Cyathus earlei Lloyd, Myc. Writ. 2 , The Nidulariaceae, p. 26, 1906

2 / Cyathus colensoi Berkeley Berk. Fl. N.Z. vol. 2: 192, 1855

Etym. Named for the collector, F.S. Earle Figure 54C

C. desertorum Mueller apud Berkeley, J. Linnean Society, 18: 387, 1881 Etym. Named for W. Colenso For illustration, see Lloyd (1906), Pi. no

In the following notes, a composite picture is given of the species based on the known collections which it has been possible to examine. The fruit bodies are fairly large, 6-7 mm high, 8 mm wide, and have the shape of a wide vase, the mouth often being sulcate and flaring markedly. In colour they are a rich dark brown, ferrugineous in some collections but deep grey-brown in others. The contrast between the deep-coloured exterior and the smooth shiny silvery inner surface (which may be almost white) is a striking feature (figure 540). The tomentum is, as Lloyd described it, made up of short hairs. The outside therefore has a smooth appearance. In fresh specimens, the tomentum is slightly tufted. Lloyd described the fruit bodies as 'rigid/ Actually the wall is thin and brittle, more so than in most species of Cyathus known to me. The basal emplacement is firm and quite conspicuous. The funiculus is very like that of C. olla . Peridioles (up to 2 mm wide) are somewhat irregular in outline, have a thin tunica on

Cups broad, campanulate, 6-7 mm high X 5-6 mm broad, even, smooth, with appressed fine hairs. Even within. Peridioles about 2 mm, black with thin tunica. Cortex thick, a single layer. Spores varying much as to size and shape. Many ellipsoid, 10-12 x 8-1 o fji. Many subglobose, 9-12 ¡JL in diameter.

Lloyd's description of this species (1906) is quoted above as it seems to accord best with the specimens that I have seen. Cunningham (1924) commented as follows: '... somewhat resembles C. olla , but may be distinguished by the smaller differently shaped peridia, smaller peridiola, and more globose spores/ Until more material is studied, I feel uncertain about C. colensoi. Specimens that have 156 / The bird's nest fungi

the upper side, and have a two-layered cortex. Spores are ovate-ellipsoid and vary in size from 12

X 1O fJL tO 22

X 12 ¡JL.

Lloyd expressed the opinion that C. earlei is related to C. olla. In general, C. earlei differs from it in having a browner colour, thinner fruit body wall, which is conspicuously silvery on the inner surface, and in having larger spores. The above is slightly modified from a paper by (Brodie, i96za), in which it was shown that C. earlei exhibits marked similarity to C. olla in the manner of spore germination and the appearance of mycelium. That it is a distinct species, however, was indicated by the failure to obtain hyphal fusions between cultures of the two fungi. C. earlei is a tropical or subtropical species which is now known to occur in Cuba (type), Puerto Rico, Mexico, and Hawaii. It is probably of more frequent occurrence than is realized at present, for I have recently examined a number of specimens from Mexico labelled C. olla that proved to be C. earlei. 4 / Cyathus pygmaeus Lloyd, Myc. Writ. 2, The Nidulariaceae, p. 26, 1906 Etym. L. pygmaeus dwarf Figure 54d Cups small, 4-4.5 mm high x 3.5-4 mm broad, greyish brown, outside smooth, rigid, clothed with appressed hairs; smooth within. Pendióles small, about i mm, with thin tunica. Cortex a single layer, about 30 fji, thicker on the lower side of the peridiole. Spores small, ovate, 12-14 x 8-9 /¿.

Lloyd's description of this beautiful and distinctive species (figure 54d) is paraphrased above. It is one of the smallest Cyathus known. The type consisted of material collected in the state of Washington. Subsequently Lloyd certified two other collections as being this species, one from Los Angeles, California, and one from Santiago, Chile. No other collections were reported until some 157 / The genus Cyathus

were found in the University of Michigan Herbarium, specimens that came from Idaho, Nevada, and Oregon (Brodie, i966b). I have examined Lloyd's type and two other specimens of C. pygmaeus in the Lloyd Mycological Collection. The California plants referred by Lloyd to this species are identical with the type, but the specimen from Chile differs from the type in several respects. Three characteristics of C. pygmaeus which Lloyd did not mention are important in the recognition of the species. The first is the strongly flaring rim of the fruit body (figure 54d) ; the second is the very dark interior of the cup (figure 54d) ; the third is the white, unusually durable epiphragm. C. pygmaeus is one of the smallest species of the genus and is also noteworthy in its xeric habitat. It grows attached to old dead stems of shrubby plants of arid areas and is now known from the following locations: Washington State, Idaho, Nevada, Oregon,1 California. 5 / Cyathus minimus Pat., Journ. de Bot., il : 345, 1897 Etym. L. minimus, smallest For illustration, see Lloyd (1906), p. 26 Peridio albo-rubescente, obconico, 3-4 mm alto, 2-3 mm lato, coriaceo-papyraceo, squamuloso, margine integro angustissimo, intus glabro levique, nunquam striato, pallide rubro; sporangiolis 9-15 in quoque peridio, discoideis, nigris, levibus, vix i mm diámetro, funículo albo minuto munitis; sporis levibus hyalinis, ovoideo-elongatis, 20-23 x 8-10 ¡JL. Hab. in radicibus herbarum in Tonkin.

The above is from Saccardo. Lloyd (1906) apparently based the description, given in his monograph, on examination of a specimen (in i After the foregoing was written, I received an abundant collection of Cyathus pygmaeus which grew on horse manure. The specimens were collected by E. Trueblood, Owyhee Reservoir, Malheur Co., Oregon, i-rv-'09. The unusual substrate is worthy of note as this species and its presumptive relative (C. olla) are not usually coprophilous.

6 / Cyathus canna Lloyd, Myc. Writ. 2, Nidulariaceae, p. 27, 1906 Etym. Canna (L. fromGr.) originally meant a reed, secondarily a reed-pipe or a small boat. The significance of the name is not obvious Figure 55C Cups campanulate, rigid, 7-8 high X 6-8 mm broad, dark brown; externally even, scabrous with short tomentum; internally smooth, white, as if covered with a thin layer of whitewash. Pendióles covered on the upper side with a silvery thin tunica. Cortex double, the outer, thin, composed of small fibrils. Spores small, globose, 9-7 ¡JL.

FIGURE 55 a, Cyathus africanus, X j ; b, C. microsporus, X3; c, C. canna, X}

his own collection) from Patouillard's herbarium. A paraphrase of Lloyd's description follows : Cups very small, 4-5 mm high x 4 mm broad, smooth. Clothed with subappressed hairs. Smooth within. Sporangioles small, about \ mm with thin tunica. Cortex thick, 50 jit but apparently a single layer. Spores elliptical, l8-20 X 10-12 jLL. I have not seen this species. Neither from the original description nor from Lloyd's description and illustration does it seem possible at present to make a positive judgment about it. It appears to possess an unusual combination of characters. The species is retained until more material is found and because of its origin in China. It is strange that C. minimus was not included in a list of the species reported from China, published in 1948 (Tai and Hung, 1948) ; one might infer that it is no better known there than it is elsewhere. 158 / The bird's nest fungi

Adding to his description as given above, Lloyd (1906) later commented: 'they (the cups) resemble those of vernicosus (C. olla) but are even and white within. The globose spores, 8 jn are its feature/ C. canna has not been collected frequently. It resembles C. olla closely in external features, but the tomentum is finer and the colour browner (light ochraceous in all specimens seen by the writer). Microscopically C. canna is distinct from C. olla in two features: C. canna has a double-layered cortex and globose spores; C. olla has a single-layered cortex and ovoid spores. C. canna (figure 55c) is exclusively tropical and is known to occur in Jamaica, Costa Rica, Barbados (type locality), Mexico, and Mauritius. 7 / Cyathus microsporus Tul., Ann. Sci. Nat. m, i: 73, 1844 Etym. Gr. mikros, small, and spora, seed Figure 55b This species is not readily distinguished from other small dark brown species of Cyathus. Cups 5-7 mm high and about 6 mm wide at the mouth. Obconic with a slender base and inconspicuous emplacement ; externally not plicate, sometimes shaggy or covered with appressed hairs; inside, slightly lighter in colour, smooth or faintly ridged (never definitely plicate) . Pendióles black, about 2 mm in diameter,

tunica thin, cortex single-layered as seen in section. Spores 5-6 X 4 ¡JL.

Some specimens, apparently this species, are very small (4 mm high) and fawn or buff, not dark brown. It is possible that more than one species is included within the known variable collections of C. microsporus. Lloyd (1915) referred collections from Florida to this species and concluded that, as they resembled closely C. hookeri Berk., the latter should be considered a synonym of C. microsporus. Berkeley's species occurs in the Eastern Hemisphere, and has larger cups and larger spores than those of C. microsporus. Cunningham (1924) retained C. hookeri as a separate species. The note above is taken from Brodie and Dennis (1945). Little can be added to aid the taxonomist, although I have examined a dozen collections since the above was written. Even the small size of the spores is scarcely diagnostic. The species is supposed to be 'internally, not plicate, even' (Lloyd, 1906) ; however, some specimens are faintly longitudinally ridged even in collections in which most specimens are not ridged. Obviously much more study of this species is needed. Brodie (i968a) noted that cultures of C. microsporus consist of fine-textured colourless mycelium, but this is true not only of species of the C. olla group but also of the C. pallidus group and does not really help to solve the problem of the affinities of C. microsporus. The known distribution of C. microsporus is as follows: San Domingo (type locality), Cuba, Costa Rica, Jamaica, Hawaii, Florida. The writer has in his herbarium a single specimen from Japan (Honshu) which seems to fit descriptions of C. microsporus but has spores 9 x 6 ¡m and is closer to C. hookeri (q.v.) - if indeed that species and C. microsporus are distinct. 8 / Cyathus hookeri Berk., in Hooker's J. Bot., 6: 204, 1854 Etym. L. Named for J. Hooker Peridia campanulate, up to 14 mm high, 10 mm wide 159 / The genus Cyathus

at the mouth, narrowing abruptly into a short stipe 2-3 mm long, 2 mm thick; exterior bay-brown, minutely and densely tomentose, interior even, dark brown, dull; mouth strongly expanded or flaring, margin entire, crenately lobed. Pendióla lenticular, 2-2.5 mm'diam., cortex black; tunica dingy-white, thin. Spores ellipsoid, rounded at both ends, 8-11 x 6-8 ¿¿.

The description is from Cunningham (1924). His material was identified by Lloyd (1906) who had given considerable attention to the possible synonymy of this species with C. microsporus. It appears that fruit bodies and spores of C. hookeri are larger than those of C. microsporus. The dark brown interior of C. hookeri also appears to be a point of marked difference from C. microsporus. Lloyd (1906) remarks: 'It was described as "striate or all even/' I think it should go in Olla/ My own notes, made in 1952 after examining an isotype in Paris, indicate that the specimens are faintly plicate internally and 'pale-buff, shaggy/ The type in Kew is dull ferrugineous and has an 'even, matted tomentum/ This species is known only from India (Khasa), New Zealand, and China (Yunnan), the last recorded by Tai and Hung (1948). I have seen only the type and isotypes of this species. 9 / Cyathus africanas Brodie, Can. J. Bot. 45 : 1653, ^Z Etym. L. African Figure 553 Peridium saccate to irregularly funnel-shaped umber to sepia in colour and arising from a conspicuous basal emplacement which is pale buff; exterior of fruit body not plicate, woolly, hairs mostly of equal length and knotted into tight curls or nodules ; inside moderately smooth with faint and irregular ridges; lip minutely fimbriate, not setose; peridium 4-6 mm wide at the mouth, 6-8 mm high ; epiphragm fragile, whitish. Pendióles thin with peripheral ridge, 2-2.5 mm m diameter, silvery with conspicuous tunica; cortex single, 25-40 ¡JL in

thickness. Spores broadly ovoid, moderately thick-walled, and possessing a distinct apiculus, 6.5 x 8.5 ¿¿wide, 8.5 x 12 jut long.

C. africanus is known only from Mount Kilimanjaro, Tanzania, from the abundant type material. It appears to be a well-marked species, characterized by the saccate peridium (figure 55a), the light-coloured peridium, the peculiar tight curls of the tomentum, and the medium-small basidiospores with their conspicuous apiculi. The species is related to C. earlei and C. badius (see Brodie, 1967). Dikaryon mycelium is Mummy-Brown (Ridgway) in colour, coarse-textured, and the species displays the phenomenon of unilateral dikaryotization (Brodie, 1970^.

15-18 fji x 11-13 ^ iR tne tvPe' are very thick-walled, and possess no apiculi, whereas those of C. africanus are only 8.5-12 x 6.5-8.5 pandare strongly apiculate; C. badius does not possess the peculiar curly tomentum of C. africanus ; the basal emplacement, which is conspicuous in both species, is concolorous with the peridium in C. badius but much lighter coloured than the peridium in C. africanus. C. badius resembles C. earlei, which however has a two-layered cortex and spores considerably longer. From the illustration given by Kobayasi it appears that the cortex of C. badius is a single layer. The species is apparently known only from the Japanese Island of Titizima (type).

10 / Cyathus badius Kobayasi, Bot. Mag., Tokyo,51: 755, 1937

11 / Cyathus confusus Tai and Hung, Sci. Rpts. of National Tsing Hua Univ. 3 : 36,1948

Etym. L. badius, dull brown. For illustration see Kobayasi (1937)

Etym. L. confusus, confused! For illustration, see Tai and Hung

Fructification arising from a circular woolly base 3-4 mm in diameter, broadly obconic, 8-10 mm (rarely 6.5 mm) high, 6-8 mm (rarely 5 mm) broad, outside dull brown (warm Sepia-Bister) tomentose-strigose, with appressed hairs ; margin incurved when young, in age upright fimbriate-denticulate, on the inside lead-coloured, shiny, when dry longitudinally striped, filled with 15-25 pendióles ; epiphragm pallid ochraceous, a powdery membrane. Pendióles lenticular 2.3 mm long, 2 mm wide. 0.6-0.9 mm thick, rugose-reticulate, silvery lead-coloured, shiny, mucilaginous, becoming black; tunica 25-35 JLC thick, pallid grey; exoperidium 25-35 M thick, almost black; endoperidium 160-170 /¿ thick, hyaline. Spores ovoid, hyaline, smooth, 15-18 X 11-13 t*" Funiculus persistent, whitish.

Peridium large, long campanulate or infundibuliform, 11-17 mm hi8n/ 5~9 mm wide at top, exterior light vinaceous cinnamon, shaggy with coarse fasciculate hairs, becoming finally almost smooth or with a few tow-like fibres, not sulcate or rarely faintly so; interior light buff, smooth ; margin fimbriate ; peridiola elliptic or angularly orbicular, Sayal Brown, 2.5-3.5 mm wide, tunica thick', spores elliptic or narrowly obovate, usually tapering toward one end, 7-10 X 5-6.4 /LI, smooth, hyaline, epispore less than i jit thick. Gregarious on rotten wood, Kunming, Nov. i, 1941, W.F. Chiu (7490).

The above paragraph is a translation of the original description. This species resembles C. africanus rather closely. I was at first inclined to believe that the two were identical. After examining the type of C. badius (from Japan) however, it became apparent that the following rather marked differences exist: the spores of C. badius are 160 / The bird's nest fungi

This species is close to C. hookeri, but differs in the larger peridium and peridiolum and smaller spores of different shape. It also differs from C. olla in the darker-coloured peridium with fimbriate margin and smaller spores of different shape (Tai and Hung, 1948), I have not seen the type of this species from Yunnan. The illustration given (a line drawing) suggests a tall form of C. intermedius. It is included close to C. hookeri but only because Tai and Hung indicate this relationship. The

description contains no reference to the cortex and does not clearly suggest any species known to me. GROUP II, C. PALLIDUS, etc.

i / Cyathus pallidus Berk, et Curt., J. Linn. Soc. Bot. 10: 346, 1896 Cyathia pallida (B.&C.) White, Bull Torrey bot. Club 29: 263, 1902 Cyathus sphaerosporus Lloyd, Myc. Writ. 2, Nidulariaceae, 23, 1906 Etym. L. pallidus, pale-coloured Figure 56a, b, c Fruit bodies small, 5-7 mm high and about as wide at the mouth, pale buff-coloured (like bleached straw), distinctly crucible-shaped, i.e., with strongly curved sides and narrow base. The thin and friable nature of the peridium walls is a noteworthy feature. The walls are not at all plicate and the outer surface is covered with long downward-bent hairs. Pendióles are dark grey to black, 2 mm in diameter, with a thin tunica and single-layered cortex. The spores are small, variable but mostly ellipsoid 7.5-15 x 4-8.5 p.

From the above description, C. pallidus is easily recognized (figure 563) in its commonest and typical form. Type material also agrees closely with such a description (figure 56c). Unfortunately the species is variable as to colour and size ; this is known not merely from the fact that colour and form variants often occur side by side in nature, where there is always the possibility of more than one species growing intermixed, but also from the fact that colour and form variants have been observed in laboratory cultures derived from spores from one fruit body of the species. In addition to the commoner small pale form (e.g., in Jamaica) there exists a form with larger fruit bodies of darker colour, in extreme cases as deep as Mummy Brown (Ridgway). Dark forms (figure 56b) appear frequently to have been confused in herbaria with C. microsporus. The latter species, however, has very small spores 161 / The genus Cyathus

and, although woolly, is without the long stiff hairs of C. pallidus. Lloyd (1906) suggested that C. pallidus, C. intermedius, and C. triplex, as he recognized them, constituted a species complex involving intermediate forms. I consider that the two latter species are quite distinct from C. pallidus and have dealt with them in separate groups. Nevertheless, it may well be that more than one species will ultimately be recognized among the collections now labelled C. pallidus. C. pallidus is one of the commoner species in the American tropics and is often found in large numbers growing upon small chips and twigs on moist ground. It grows commonly on compost and mulch in tropical botanic gardens. The species has been found widely distributed in the West Indies (type locality, Cuba). It occurs in Mexico and is known in South America from Brazil and Peru. In the United States it is known to occur in Georgia and Florida. The species is also common in the Hawaiian Islands. It has been reported from China by Tai and Hung (1948), but the Chinese specimens have not been seen by me. It was recorded from Thailand by Dissing (1963) who, however, expressed some doubt as to the identification. Cultures of C. pallidus are snow white (Brodie, 19683) and fruit readily when transferred to sterilized compost. 2 / Cyathus julietae Brodie, Svensk Bot. Tidskr. 61 : 94, 1967 Etym. L. from the name Juliet Figure 56d Peridium large, pale brown to yellowish, broadly obconic with straight sides, thin-walled; 7.5-8 mm wide at mouth, 7-8 mm high; mouth smooth or minutely fimbriate; outside not plicate, covered with very fine hairs and conspicuous long retrorse hairs; inside very smooth, glossy; basal emplacement narrow; epiphragm pale brown to yellowish. Pendióles distinctly elliptical in surface view, black, wrinkled on the upper surface, 1.5-1.75 mm long; tunica thin; cortex a single layer. Spores subglobose to broadly ellipsoid, thin-walled, 5-9 X 5-7 ¡JL.

FIGURE 56 a,Cyathuspallidus, X 2 ;b, C.pallidus, dark wide form, X 2 ;c, C. pa/Wws, type specimen, xi; d, C. julietae, X2 162 / The bird's nest fungi

FIGURE 57 a, Cyathus triplex, spécimen on right smaller and younger than one on left, X3 ; b, C. setosus, X2. 5

C. julietae is distinguished from C. pallidus by its larger size, straighter peridium walls, elliptical pendióles, yellowish peridium walls and epiphragm and its more globose spores. This beautiful species (figure $6d) is known only from Jamaica (type: Hardwar Gap, St Andrew Parish, Brodie, 6641). In culture (Brodie, 19683) the mycelia are snow white and closely resemble those of C. pallidus. GROUP III, C. TRIPLEX CtC.

i / Cyathus triplex Lloyd, Myc. Writ. 2, Nidulariaceae, p. 23, 1906 Etym. L. triplex, threefold Figure 57a Cups 5-6 high x 5 mm broad, smooth within and without, with connivent, spreading, somewhat scabrous hairs. Inner surf ace smooth, silvery white. Peridiole 2 mm, with a very thin adnate tunica. Cortex thick, evidently double, but subhomogene163 / The genus Cyathus

ous and the fibrils slender. Spores ellipsoid, 16-22 x 12-14 M (emended from Lloyd). 'The cups are those of pallidus, but darker and the hairs more scabrous' (Lloyd).

Although Lloyd included C. triplex in the pallidus group, there are several reasons for believing that the two species are not closely affiliated. The fruit bodies of C. triplex are considerably darker than those of C. pallidus, the underlying hairs are coarser, the long downpointing hairs are coarser, the spores are larger, and the cortex is two-layered; all these features seem to the author to set C. triplex rather far apart from C. pallidus. Moreover, the dikaryon mycelium of C. triplex is rosebrown whereas that of C. pallidus is snow white (Brodie, 19683). The dark-buff fruit bodies of this species, taken together with the silvery interior, the thick two-layered cortex and quite large ellipsoid spores, make it readily distinguishable.

The fungus was described from material from the island of Mauritius. It is, however, common throughout the West Indies. It is known also from Florida, Venezuela, Hawaii, the Philippine Islands, and Thailand. 2 / Cyathus setosus Brodie, Can. J. Bot. 45 : i, 1966 Etym. L. setosus, from the conspicuous setose rim of the cup Figure 5/b Peridium large, very dark brown, almost black, broadly conic, symmetrical, thin-walled; 8-10 mm wide at mouth, 7-8 mm high ; mouth beset with s tiff dark setae (almost black) 0.5-1 mm long; outside covered with fine appressed hairs and a few long tangled hairs; inside faintly plicate, conspicuously silvery; basal emplacement distinct but narrow (1.5-2 mm wide); epiphragm white to pale buff, rather thin. Pendióles angular, black, shiny, thick, 2.5 mm or more wide ; tunica absent ; cortex doublelayered and 50 jit thick. Spores ellipsoid, thickwalled, apiculate, 17-24 X 10-14 ¡JL (based on original description).

No other Cyathus has, in addition to such conspicuous setae, so deep a colour, which is almost black yet recognizable purple-brown. There are but few species having cups so large that have so small a basal emplacement. Few species have wider, and no species has plumper, pendióles. C. setosus is easily distinguished from C. triplex by its dark colour, its long setae, and by the lack of tunica. It is probably related to the latter species in its wide form, double cortex, and spore size. This beautiful and unusual Cyathus was collected first in Jamaica in 1966. I have since identified specimens from St Lucia, Trinidad, Guadeloupe, Mexico, and Bolivia. In culture, dikaryon mycelium develops a strong brown pigment suggesting relationship with C. triplex but not with C. pallidus (Brodie, i97ob). 164 / The bird's nest fungi

3 / Cyathus sinensis Imazeki, Bot. Mag. Tokyo, 63 : 96, 1950 Etym. L. sinensis, Chinese (but described from Japan) For illustrations, see Imazeki Peridium obconic, 5-6 mm high, 2.5-5 mm wide, externally cinnamon-brown, densely faciculate woolly, hairs for the most part tufted, grey-brown, composed of yellow-brown hyphae constricted at the septa and 4-1 o ¡JL thick; on the inside not striate, smooth, lead-white ; peridium of three layers, the outer and inner brown, about 70 /¿ thick, plectenchymatous, made up of hyphae 2-3 /¿ thick which are arrayed lengthwise, the middle-layer hyaline, about 40-50 jit thick, pseudoparenchymatous. Pendióles sordid grey, disc-shaped to lenticular, 1.3 mm wide, 0.5 mm thick; tunica membranaceous, very thin; cortex dark-brown, ca. 15-20 ¡JL thick; spores wide ellipsoid, hyaline, smooth, 12.5-18.5 X 8.3-10.3 (usually 13-15 X 9.5) ¡JL.

The above is a translation of the original description. The author has not seen this species, but judging from spore size, thick cortex, colour and white interior it must resemble C. triplex. Imazeki placed it in the section Olla but the tufted hairs and other features seem to make close association with C. olla and its relatives doubtful. The fruit bodies were reported to form on living moss which is a most unusual situation. C. sinensis is known only from Kyushu Island in Japan. G R O U P IV, C. G R A C I L I S etc.

i I Cyathus gracilis Brodie, Can. J. Bot. 51: 1393, 1973 Etym. L. gracilis, slender referring to the narrowly stalked peridium Figure 58a,b Peridium slender, obconic, thin-walled (0.2-0.4 mm), 4-7 mm wide at the mouth, 8-10 mm high, lower third contracted into a slender stalk 2-3 mm in length; outer surface umber to rust colour, covered

FIGURE 58 a, Cyathus gracilis, enlarged to show tufted tomentum, Xio; b, C. gracilis X médius, type specimens, X 4; d, C. crassimurus, X3 ' 165 / The genus Cyathus

3

• c C inter

with distinct conical tufts of hairs (not regularly downward-pointing), not plicate ; basal emplacement distinct, spreading, not massive ; lip minutely fimbriate, not setose; inner surface concolorous with outside or slightly lighter, faintly plicate; epiphragm pale buff, bearing brown hairs. Pendióles 2 mm in diameter, circular; tunica absent or delicate; cortex distinctly double. Spores ellipsoidal, variable, mostly 20 X 10 /x; spore wall thick 2-3 IJL (from original description).

This species from the Philippine Islands is characterized by its slender brown fruit bodies in which the basal portion is long and narrow (figure 58, a,b). External hairs are aggregated into sharp conical tufts, irregularly oriented. The peridiole tunica is absent (or nearly so) and the cortex is distinctly two-layered. C. gracilis appears to possess a combination of characters not present in other species and does not fit easily into any of the recognized groups. It resembles C. intermedius in spore size and shape, and in the tufted tomentum (which, however, is much less conspicuous in C. intermedius), but differs markedly from it in having a two-layered cortex. The species is known only from Luzon in the Philippine Islands. (Type: Lamao River, Luzon, Philippines; R.S. Williams #158, 1903; Brodie #710773) 2 / Cyathus intermedius (Mont.) Tul., Ann. Sci. nat. m, i: 72, 1844 Nidularia intermedia Mont, in Sagra, Hist. Phyt. Pol. Cuba, p. 321, 1838-42 Cyathia intermedia (Mont.) White, Bull. Torrey bot. Club 29: 258, 1902 Etym. L. inter, middle, and médius, middle Figure 58c, type 'C. obverse conicus hispido-tomentosus pallidus, intus brunneus vix striatus ; coronae puis distinctis brevibus ; sporangiis nigris circularibus, pellicula tenuissima involucratis ; substantia media nigrescenti ; sporis ovatis. 'Fungus 8-9 mm altus, ore j-S mm lato; sporangiorum diametrum 2 mm aequat ; crassities i 166 / The bird's nest fungi

mm paulo minor; sporae 15.4 (ju,) longae, 8.8 (jit) basim versus late/

Tulasne's description is quoted above because there exists considerable doubt about the status of this species. Tulasne seemed to emphasize the presence, around the fruit body mouth, of a crown of tufted hairs which are illustrated in his Plate 4, Fig. 5. These are no longer visible in the type specimen in Paris ; they seem merely to have been the uppermost of the tufts of hyphae that cover the outside of the fruit body and not setae as that term is used elsewhere in this book. Moreover, the character that was emphasized by Lloyd (1906) and to some extent by White (1902), namely the tufted tomentum, is difficult to discern in the type material today, although it does appear distinct in Lloyd's photograph (Pi. 109, Fig. 9). Even more puzzling is the problem of the supposed plication of the fruit bodies. Tulasne wrote 'intus vix striatus' (i.e., scarcely striate on the inside), but he placed the species in the section Eucyathus. Lloyd noted 'the striae are absent in most specimens and when present are so faint that I think the plant should go in Olla/ The present writer also noted (in 1952) that plication is very doubtfully a feature of the type. Despite these difficulties, I have taken the view that the description given by Lloyd can be applied to a species which is of fairly common occurrence and which can be identified with reasonable ease. Lloyd's description follows: Peridium broad, campanulate, 5-6 mm high and broad at the mouth, even within and without. (Sometimes faintly striate within.) Pale fawn colour, covered when young with appressed tomentum collected in nodules. This character largely disappears from old specimens. Pendióles thin, about 2 mm in diameter, with a thin tunica. Spores in the type collection elliptical 10 X 16 mic.

From my own study of the type (first in 1952 and again in 1970) it may be noted that there is more variation in spore size than is indicated by

Lloyd's '10 X 16': e.g., 14 X 9, 13 X 10, 15 X 8 IJL. Also the spores in the type are relatively thick-walled (1.5-2 /z). Lloyd gives the colour as 'pale fawn'; actually some specimens are darker, almost ferrugineous. This is also true of some specimens at Kew (no. 168.98) collected by de la Sagra in Cuba (type locality). C. intermedius is a widespread but seldom abundant species in the West Indies. I have also seen what I take to be this species from Florida, Mexico, Venezuela, Colombia, and the Philippines. White (1902) lists C. intermedius from Asia but without reference. Tai and Hung (1948) reported the species from China but I have not seen their material. Dikaryon mycelium of C, intermedius is ivory-coloured or darker (Brodie, 1971^. 3 / Cyathus crassimurus Brodie, Can. J. Bot. 49: 1609, 1971 Etym. L. crassus, thick, and murus, wall, 'thickwalled with reference to thick walls of spores and peridia' Figure 58d Peridium short, wide, broad-obconic, walls 0.3 mm thick ; 6-7 mm wide at the mouth, 5 mm high ; sides incurved when young, flaring in the upper third when old; exterior pale brown, covered with flattened hairs aggregated into tufted downwardpointing nodules ; basal emplacement consisting of a large ball of fungus and soil (5 mm wide) ; mouth beset with stiff brown hairs (o.i to 0.2 mm long); inside of mouth showing remains of white epiphragm; inner surface pale brown, smooth, shiny. Pendióles small (1.25-1.5 mm), angular, pale brown and with shiny tunica ; cortex single ; tunica thick (50 ¡JL), adherent. Spores ellipsoid, 17-20 X 11-12 IJL, very thick-walled (2.5-4 A1)-

This species from Hawaii (figure 58d) could be mistaken for C. pallidus; in both species the colour is pale buff, the fruit bodies have curved sides, and the outside of the fruit body is armed with tufts of down-pointing hairs. However, C. pallidus has a thin-walled peridium whereas that of C. crassimurus is notably thick-walled. 167 / The genus Cyathus

Spores of C. pallidus are thin-walled and small; spores of C. crassimurus are thickwalled and larger. Probably this species is more closely related to C. intermedius because of spore-size, thickness of spore wall, tufted tomentum, and single-layered cortex. In culture (Brodie, i97ib), dikaryon mycelium of C. crassimurus is a medium buff to light brown, suggesting closer relationship to C. intermedius, whose mycelium is ivory in colour, than to C. pallidus with snow white mycelium. This species has been recognized up to now only from the Island of Hawaii (type: near Captain Cook, Hawaii; Brodie, #68124, 1968). Note that it might seem that a characteristic such as the conspicuously tufted tomentum of the species in this group should be emphasized in establishing groups of supposedly related species. However, although it is a feature of the three species described above, they are not grouped solely on this basis, but have other features in common. Moreover, there are other species which have tufted tomentum, but which, in other respects, show no relation to the gracilis group and which are therefore included elsewhere. For example, C. helenae has a tufted tomentum but its taxonomic affiliation is with C. striatus, as has been established by the evidence of partial fertility between the two latter species (Olchowecki and Brodie, 1968). Again, some specimens of C. stercoreus have tufted tomentum, but this species is very far removed from the gracilis group because of its very large spores and other characters. We are here dealing with the old problem that arises from the fact that true relationships are seldom established on the basis of a single character. 4 / Cyathus elmeri Bres., Hedwigia5i : 324, 1912 Etym. Named for the collector A.D.E. Elmer Peridium campanulate, narrow at the base, subsessile, umber coloured, tomentum squamulose, 7-10 mm high, 7-9 mm wide at the top, faintly sulcate,

on the inside almost concolourous, smooth, shiny. Pendióles ash-grey, dull, powdery, 1.3—1.5 mm in diameter; tunica thin, 100-150 ¡JL thick; spores ellipsoidal with a thick wall, 18-22 X 10-12 ju,; hyphae of the tunica fulvous, 2.3^1 thick; hyphae of the funiculus hyaline 2-2.75 ju. Habitat: on stems of palms, Ley te, Palo Philippine Islands (collector, A.D.E. Elmer).

There are several points of difficulty concerning interpretation of the original description as translated above. The peridia are stated to be faintly 'sulcate': In many old descriptions, the term 'sulcate' clearly means 'plicate/ In this instance the Latin diagnosis and the definition in terms of our present-day concept leave doubt as to whether the peridia are smooth or plicate. Also the tunica is described as composed of hyphae which are 'fulvo-fuscis / which leaves doubt as to whether the use of the word tunica is in accordance with the recent concept or whether it refers to the cortex. I examined isotypes of this species at Kew in 1952 and noted the following: colour fawn to buff; form broadly conic with emplacements on some specimens ; outside covered with matted appressed hairs ; mouth fimbriate but not setose; inside broadly but not deeply plicate; peridioles ellipsoid, grey-brown, with distinct pale brown tunica and dense uniform singlelayered cortex; spores 16-20 x 8-10 ¡JL, thick-walled. From this study, I conclude that C. elmeri is more closely related to C. intermedius than to the other species. It differs from C. inter médius in being somewhat larger, in being plicate, in the nature of the tomentum tufts, which are broader and less distinct than those of C. intermedias, and in its spores which tend to be considerably longer than those of that species. The light brown pruinose peridioles also appear to be a unique feature of this species, although judgment on such old material (type collected 1906) may be of doubtful value. It may be a form of C. intermedius, but I consider that it should be maintained until more mater-

168 / The bird's nest fungi

ial is seen. The species is known only from the Philippine Islands. G R O U P V, C. S T E R C O R E U S , CtC.

i / Cyathus stercoreus (Schw.) de Toni in Sacc., Syll. Fung. 7: 40, 1888 Nidularia stercorea Schw., Trans. Am Phil. Soc. 4: 253, 1834 Etym. L. stercorarius, of dung Figure 59a, and many in text (see Index)

If C. stercoreus had no other claim to fame, its lengthy synonomy should evoke attention. This synonomy is recorded elsewhere (Brodie and Dennis, 1954) and it is unnecessary to repeat it. The extreme variability of C. stercoreus as to size and colour of the sporophores has been pointed out by Lloyd (1906) and discussed fully by Brodie (i948b). A description based on many years of experience with this species in its natural habitat and in laboratory culture is given herewith. The fruit bodies are mostly of some shade of golden brown or russet brown but commonly appear almost black in age. The size ranges from 0.5-1.5 cm high and 4-8 mm wide at the mouth. Mostly they have the shape of a rather long, narrow inverted cone (figure 59a), but very great variation in shape may be expected (figure 29). Externally the cups are covered with a shaggy, sometimes tufted rather untidy tomentum, which may be entirely lacking in old worn specimens. The peridia have no plication whatever. The inside of the cups is lead-grey or bluish-black. The emplacement of C. stercoreus is almost always large and conspicuous and of a red-brown colour. The peridiole is black, lacks a tunica, and, in section, shows a two-layered cortex. Spores are large, subglobose, and vary much in size (up to as much as 40 /x) not only in the species but within a single peridiole. In some collections of C. stercoreus no funiculi can be seen, a situation which has frequently resulted in this species being identified as a Nidula !

FIGURE 59a, Cyathus stercoreus, x 3 ; b, C. pictus, x 2.5

From the above, it might seem that C. stercoreus could hardly be missed. However, Brodie (i948b) has illustrated the extreme genetic variation possible in this species as to form and colour, and B. Lu (1965) has shown that the form of the fruit bodies is dependent, in part at least, upon the intensity of the light under which they develop. A sample of the range of fruit body form that exists in this species is illustrated in figures 29, 30. In spite of the variation, C. stercoreus is unique in its brown shaggy non-plicate cups, black pendióles, and large spores. The species is chiefly coprophilous but also fruits on manured soil. It is of world-wide distribution and, as Lloyd notes, 'probably occurs in every country where manure occurs/ One might suspect that this wide distribution is correlated with the distribution of livestock and feed, for the species is not commonly found in undisturbed areas. C. stercoreus has been more extensively studied in laboratory culture than any other of the Nidulariaceae and frequent references to it are made throughout the earlier chapters of this book. 2 / Cyathus pictus Brodie, Can. J. Bot. 49: 1613, 1971 Etym. L. pictus, 'painted/ from the coloured mouthband Figure 59b

169 / The genus Cyathus

Peridium tall, slender with a slender base (3-3.5 mm long, i. 25-1.5 mm wide) ; 8-9 mm high, 5 mm wide at the mouth ; outer surface composed of fine hairs aggregated into low mounds (0.2-0.4 mm wide); coarse hairs absent; pale cinnamon when dry, dark brown when moist; mouth on the outside provided with a distinct red-brown band (0.2-0.3 mm wide) just below rim; edge smooth, devoid of setae, and showing remains of epiphragm as a white line inside; inside smooth, not plicate, lead-grey; emplacement large, firm (7 mm). Pendióles placed deep in cup (one-third or more below lip), black, irregular (1.75-2 mm wide, 2-2.5 mm I°n8)> depressed on upper side; cortex single but subhomogeneous, tunica absent. Spores thick-walled (wall 3-3.5 f¿), globose, 26-32 ¡Ji in diameter (figure 8d).

C. pictus resembles C. stercoreus in the lack of tunica and in spore size but differs from it in the complete lack of long shaggy hairs, the tuberculate fine-textured tomentum, in the nature of the cortex somewhat, and in the coloured mouthband. This species is known only from Mexico. The type material grew on rotted Eucalyptus wood which seems to be a substrate not previously recorded for the Nidulariaceae. 3 / C. fimicola Lloyd, in herb., unpubl. (non C. fimicola Berk., J. Linn. Soc. 18, 387,1881) 'Cups small, about 2-3 x 4-5 mm in diameter, pale,

FIGURE 6o a,Cyathuspoeppigiionsoi\, x2.5 ; b, C. poeppigii, herbarium specimens, X 4 ; c, C. limbatus, X3; d, C. gayanus, X4 170 / The bird's nest fungi

with strigose matted hairs. Pendióles small, black, 1.5 mm. Spores 8 x 16 //,.'

Lloyd's unpublished description was printed by Stevenson and Cash (1936). The name C. fimicola was not validly published as it lacked a Latin diagnosis and it was, in any case, illegitimate as a later homonym of C. fimicola Berk. When the species has been recollected and substantiated it must receive a new name. I consider that this is probably a 'good' species because of its small black pendióles and coprophilous habit. The type (on dung) from Puerto Rico, is paler than most C. stercoreus and the matted hairs quite unlike those of that species. Because of its small spores C. fimicola probably ought not to be included in this group; it is done here largely because so few Nidulariaceae are coprophilous. Two other collections have been identified (by myself) as belonging to this species, although with considerable hesitation. Neither collection is extensive and neither helps to solve the problem of the disposition of C. fimicola. One collection (NYBG) is from Puebla, Mexico, on cow dung; spores of this material average 13 x 10 f¿ and the peridia are similar to those of the type but of a greyer colour. The second collection from Grenada, 'on dung in bamboo pots/ has spores 15 x 8 ¡JL and would suggest a very small C. stercoreus except for the unusually small spores. I believe that more material ascribable to this species must be studied before even a validating diagnosis can be published. G R O U P VI, C. P O E P P I G I I ,

etc.

i / Cyathus poeppigii Tul., Ann. Sci. Nat. m, i: 77, 1844 Cyathus sulcatus Kalchbr., Grevillea, 10:107,1882 Etym. Named for Toeppig/ the collector Figure 6oa, b Fruit bodies reddish brown to dark brown, almost black in age, regular and rather narrowly obconic, felty or shaggy, 6-8 mm high and 6 mm wide at the 171 / The genus Cyathus

mouth. Both outer and inner surfaces deeply fluted or plicate, the plicae about 0.5 mm apart (figure 6oa, b) and fruit bodies in age often splitting along the folds. Peridia black and shiny, tunica absent. Spores mostly elliptical, very large, 30-42 x 20-28 /¿, but variable.

One of the most widely distributed and easily recognized species in the tropics where it often grows in clustered masses on rotting wood and old fibrous mats. Lloyd noted 'it seems to replace C. striatus of temperate regions and to have very much the same habits/ C. plicatulus is an unpublished name applied by Poeppig to his exsiccati from Cuba and usually cited in synonymy. Nidularia plicata Fries is probably a synonym (Palmer, i96ib). C. poeppigii is of almost universal distribution in warm countries : West Indies (type from Cuba), South America, Hawaiian Islands, Asia, Africa, China, etc. In North America it has been reported only from Florida. In culture (Brodie, 19683), dikaryon mycelium of this species is coarse-textured and of an intense purple-brown colour. This species has also been observed (Brodie, 19553) to produce remarkable aberrant fruit bodies from certain cultures. There is far less variability in this species than one tends to expect from a species of Cyathus of such common occurrence. The principal variation is in spore size ; spores of C. poeppigii are always large and variable, but those from certain areas, notably the West Indies, are considerably smaller than those of other areas. When other characteristics are remarkably constant, the author does not feel that spore size ought to be unduly emphasized as a diagnostic feature of C. peoppigii. This species has often been noted to have been labelled C. limbatus in herbaria. The latter, however, has considerably smaller spores and the plications are much coarser (see under C. limbatus). Attempts at various times to obtain union between monospore cultures of C. poeppigii and C. limbatus yielded negative

results (unpubl.), which may be taken as a further indication that they are distinct species. 2/ CyathuslimbatusTul., Ann. Sci, Nat. m, i, 78: 1844 Nidularia striata var. pusilla Berk., Ann. Nat. Hist. 111:397,1839 Etym. L. limbatus, bordered (or fringed) Figure 6oc

Another widely distributed species in warm countries and one which often occurs in great abundance. Cups dark brown, large, 7-10 mm high, 6-7 mm wide at the mouth, hirsute, outer and inner surfaces clearly plicate, the ridges wider apart (0.75-1 mm) than in C. poeppigii. Pendióles 2 mm or more wide, deep brown to black, shiny. Spores 15 X 10 (JL in the type but 16-22 x 10-12 /¿ in other collections.

In general, this species resembles C. poeppigii but the cups of C. limbatus are usually broader for their height, the type (at least) is lighter brown, the cups are not so strongly plicate as those of C. poeppigii and the plications are coarser. C. limbatus often has a conspicuous emplacement. The type came from British Guiana (Tulasne, 1844). Lloyd knew the species only from the West Indies but I have seen material from many parts of the world including China, India, Africa, South America, Hawaiian Islands, Pacific Islands. In culture (Brodie, 1968), dikaryon mycelium is fast-growing and glossy purplebrown, the reverse of cultures becoming almost black. Like C. poeppigii, this species has been observed to produce highly aberrant uni-peridiolar fruit bodies in some cultures (unpubl.).

3 / Cyathus gayanus Tul., Ann. Sci. Nat. m, i : 76-77, 1844 Etym. Named after the collector C. Gay Figure 6od 172 / The bird's nest fungi

The salient features of this species, as paraphrased by Lloyd from Tulasne's description, are as follows: Peridium about 1.5 cm high, 5-6 mm broad, narrow, conic, dark brown, striate within and faintly so without, strigose, hirsute. Pendióles black, large, 3 mm with thick outer wall. Spores subglobose, large, varying from 20 to 32 ¡JL.

This species is readily recognized (figure 6od) by its unusually tall slender peridia which are plicate and dark brown, by its large pendióles, and by its large subglobose spores. For many years it was known only from Chile. Its presence was recorded in Costa Rica (Brodie, 1955) and in Jamaica (Brodie, 1967). I have recently seen one collection from Venezuela (N.Y.B.G. Herb.) which is less elongate than the type, but has the same large spores and pendióles, which are 3 mm or more in diameter. 4 / Cyathus costatus Lloyd ex Stevenson and Cash, Bull. Lloyd Libr. No. 35, Myc. Ser. 8 : 201, 1936 (nom. nud.) Etym. L. costatus, 'ribbed/ for the plicate peridia

This species has not been validly published. If and when more material is collected, a validating diagnosis should be given. Lloyd's manuscript note as published by Stevenson and Cash characterizes C. costatus as follows: 'Cups small, dark, about 2.5-3 mm in diameter, covered with dark, strigose hairs, strongly ribbed, striate. Pendióle small, i mm, black. Spores about 16 x 55 /¿, elliptical - its striate cups and large spores place it close to the common tropical species -C. poeppigii, but the cups are only about one-half as large, the striations much coarser, and its habitat on manure entirely different/ The type locality was Mayaquez, Puerto Rico. I have seen the type, which corresponds well with the description. It may be a form of C. poeppigii, but the small size of the fruit bodies and pendióles, and strongly ellipsoidal spores appear to make C. costatus a distinct entity. No other specimens of this description have apparently been recognized.

5 / C. cheliensis Tai and Hung, Sci. Rpts. National Tsing Hua Univ. 3: 39, 1948 Etym. from type loc., Cheli For illustrations, see Tai and Hung 'Peridium campanulate, 6-8 mm high, 5-7 mm wide at the top; exterior russett, shaggy with coarse hairs, becoming smooth in old specimens; interior brownish, shining, deeply and closely striate near the margin on both surfaces, margin expanding, not fimbriate; peridiola 1.5-2 mm in diameter, elliptic, blackish; spores elliptic or ovate-elliptic, rarely ovoid, rounded at both ends, 16-19 x 8.6-10(12) /¿, smooth, hyaline, epispore 2.9-3.6 ¡JL thick/ 'Gregarious on decaying wood, Cheli, 1939, H.S. Yao (6755 type, 6756).' Tt differs from C. olivaceo-brunneus in the lighter-coloured and more hairy peridium with non-fimbriate margin and thicker-walled and smaller spores' (Tai and Hung, 1948). I have not seen this species from Yunnan. As essential details of cortex and tunica are not given in the diagnosis, one can only note that the description and line-drawing illustration strongly suggest that this is in fact C. limbatus. This is an opinion which can be tested only by an examination of the type which I have been unable to obtain.

6 / C. olivaceo-brunneus Tai and Hung, Sci. Rpts. National Tsing Hua Univ. 3: 39, 1948 Etym. L. oliva, olive, and brunneus, brown, olivebrown with réf. to outside of peridium For illustration, see Tai and Hung Teridium campanulate, 7-8 mm high, 6 mm wide at the top, exterior Olive Brown or Saccardo's Brown, strigose, hairs thin, clustered, scattered, becoming almost smooth in old specimens, distinctly striate about half the length of the peridium ; interior Natal Brown, shining, distinctly striate, margin fimbriate, slightly expanded; peridiola orbicular, Sepia, 1.5-2 mm in diameter; spores elliptic or broadly elliptic 16-20 X 11-15 A6/ smooth, hyaline, epispore 1-1.4 ¡Ji thick/ 173 / The genus Cyathus

'On dead stem of moss, Tali, Aug. 28,1938, H.S. Yao (5518 type)/ 'This species is distinct from C. limbatus in the smaller peridiola and broader spores of different shape. It also differs from C. intermedius in the distinct striae on both surfaces of the peridium which is less hairy' (Tai and Hung). I have not seen this species from Yunnan. No information as to tunica or cortex are given in the diagnosis, and it is difficult from the description to distinguish the species from many other species. Because the spores of C. poeppigii are often within the range given for the above species (see Lloyd, 1906, p. 15) and because there is nothing in the above description to indicate otherwise, I believe that C. olivaceo-brunneus may be a synonym of C. poeppigii, but firm decision must await study of the type. G R O U P V I I , S T R I A T U S , CtC.

i / Cyathus striatus (Huds.) ex Pers., Syn. Meth. Fung., 237, 1801 Cyathus striatus a. Schweinitzii Tul., Ann. Sci. Nat. in, i : 68, 1844 Cyathus hirsutus (Schaeff.) ex Quel., Enchir, Fung. 232, 1886 Cyathus griseus Pers. in Herb. Cyathia hirsuta (Schaeff.) White, Bull. Torrey Bot. Club 29: 259, 1902 Etym. L. stria, furrow, 'with fine ridges or groo ves/ with réf. to plicate peridium Figures i, 2, 41, 6ia, etc. White (1902) lists ten items in synonymy which are not repeated here. Fruit body commonly narrowly obconic but varying to widely obconic, commonly 7-10 mm high, and 6-8 mm wide but variable, usually with a slender base and flaring outwards in the upper third, provided with a distinct emplacement; colour some shade of brown, but varying from grey buff to deep chocolate brown or deep buff; externally covered with an irregular shaggy or woolly tomentum, often with some downward-pointing hairs; mouth, when

FIGURE 6i a,Cyathusstriatus, X 5 ; b , C. helenae, X 4 .5;c, C. berkeleyanus, X 3 ; d, C. bullen, X 3 ;e, C. montagnei, X2.5; f, C. annulatus, x8 174 / The bird's nest fungi

young, provided with short but distinct setae; outside faintly to strongly plicate, inside markedly plicate and smooth, shiny; epiphragm distinct, persistent, usually white; peridioles about 2 mm, frequently roughly triangular and provided with a distinct pale tunica; spores clearly ellipsoid, slightly narrower at one end, 18-20 X 8-10 //,, thick-walled and provided with a notch or apiculus at one end.

study and sketch C. striatus for its beauty and for the fascination provided by its highly developed complex funicular apparatus.

The description given is intended to represent C. striatus as the author has seen it in many variations from many temperate regions. Lloyd (1906) distinguished two forms; the 'type form' from Europe, which he described as dark and having a pronounced tunica; and forma Schweinitzii Tul. from America, pale and with a much thinner tunica. I have seen both forms in Europe and in America and believe no such distinction as Lloyd's is practicable. Many intergrading forms of C. striatus occur in North America alone. In eastern Canada (and the USA) a rather small pale form is common ; in moist conifer forests of Alberta and British Columbia, a very tall dark-coloured form is prevalent (figure 41), similar to the prevalent form of Europe; in the prairie provinces of Canada and the central states of the United States, a large, rather wide, pale form is common. None of these or other forms that exist are really exclusively restricted to any area and cannot be recognized as geographical races. This elegant fungus is very widespread in the temperate world. Lloyd recorded it only from Europe and America but I have in my collection specimens from India, Japan, China, and Mexico. Tai and Hung (1948) recorded it from Yunnan but with hesitation. I have not seen their material. C. striatus fruits almost entirely in an open woodland on small twigs and woody leaf mould, although it is seen occasionally in gardens. In culture, dikaryon mycelium is coarse-textured and of a beautiful shade of golden brown. The species has been recorded to fruit in culture (Leininger, 1915). Every student of plants should be required to

Peridium obconic, thick, flaring outwards sharply in the upper third, 5-6 mm wide at the mouth, 7 mm high ; outer surface pale brown to grey, covered with down-pointing hairs aggregated into nodules ; inner surface grey, shiny, silvery, faintly but distinctly plicate- basal emplacement massive, wider than peridium; lip dark brown towards inside, minutely fimbriate but not setose; epiphragm white, thin; peridioles 2 mm in diameter, angular, with silvery tunica; cortex a single layer; spores ovoid to sphaeroidal, 15-19 X 12-14^1, slightly narrower at one end ; spore wall mostly i. 5 ¡JL thick, occasionally thicker.

175 / The genus Cyathus

2 / Cyathus helenae Brodie, Can. J. Bot. 44 : 1235, 1966 Etym. Named for Helen W. Brodie Figure 6ib

The above description is slightly modified from the original. This beautiful little species is smaller than most forms of C. striatus and, as far as is known at present, is alpine and boreal in habitat except for collections from dry areas of Idaho. The type collection came from Rocky Mountain Park in Alberta, Canada. Despite its resemblance to C. striatus, it is considered to be a distinct species for the following reasons. Morphologically C. helenae is quite distinctive in the presence of tufted tomentum, lack of setae, and faint plication. Ecologically C. helenae invades alpine regions, the far north and desert areas in northwestern America. Biochemically it is unique in the production of a series of diterpenoid substances not previously recognized by chemists (réf. Allbutt et al., 1971; Ayer and Taube, 1972; Ch. XB i). Biochemically it is also distinct from C. striatus in that the indolic substances produced by C. helenae form a chromatogram which is not the same as that produced by C. striatus (Johri and Brodie, 19713 and Ch. x, B 2). Perhaps arguing for conspecificity of the

two entities is the partial fertility among haploid mycelia of C. helenae andC. striatus ; this however is open to question (Olchowecki and Brodie, 1968). Mycelial mating between these two species (without, however, the production of fertile fruit bodies that would allow backcrossing to both parents), cannot be regarded as a sign of conspecificity. Even where some hybrid fertility exists, poor adaptation of hybrids (and their subsequent competitive exclusion) may be a valid reason for maintaining specific rank (see, Chapter 4, €4). 3 / Cyathus montagnei Tul., Ann. Sci. Nat. -L : 70, 1844 Etym. Named for Jean P. Montagne, outstanding French mycologist Figures 443, 6ie Fruit bodies rather short, 8-10 mm high, 8 mm broad at mouth, wide-flaring, dark brown, and hirsute. Tulasne described the colour as ferrugineous ; actually fresh specimens are much darker (mahogany to chocolate brown) but tend to fade. The epiphragm is white and quite conspicuous. Outside, under the hairs, the walls are faintly plicate ; on the inside the walls are clearly and rather widely plicate and lead-coloured or silvery. Pendióles (about 2 mm or more in diameter) are black, shiny, plump, and have a thin tunica. The cortex is apparently onelayered but subhomogeneous, so that it may at times be classed as two-layered. Spores are ellipsoid, 20 x 12 /a.

This species (figure 6ie), which was long known only from Brazil, is apparently quite common in the West Indies and in Central America. Wolf (1949) reported it from Venezuela, Dissing and Lange (1962) from the Congo, and I have seen specimens from the Philippines and Thailand. Dikaryon mycelium of this species (Brodie, 1968) is coarse-textured, pale buff when young, and bright intense brown when old. The mycelium bears a strong resemblance to that of C. striatus.

176 / The bird's nest fungi

4 / Cyathus nigro-albus Lloyd, Myc. Writ. 2, Nid. p. 18, 1906 Etym. L. niger, black, and albus, white For illustrations, see Lloyd, Plate 107, Figures 4, 5 Peridium conic, cup-shaped, 6-7 mm high, 4-5 mm broad at the mouth, externally strigose, hirsute, even, dark brown almost black in colour. Internally silvery white (hence the name), faintly striate. Pendióles 1.5 mm in diameter, with a thin tunica. Spores elliptical, 16-22 x 12 /¿.

Described as above by Lloyd, originally from Samoa, the species was identified a second time by Lloyd (see Stevenson and Cash, 1936) from the Belgian Congo. Regarding the latter specimen, Lloyd wrote, 'If I were revising the genus now, I should probably consider this a dark form of C. limbatus/ It is difficult to understand why Lloyd should have thus compared his C. nigro-albus closely with C. limbatus for the latter species is not silvery internally and it has coarse, obvious plication, whereas C. nigro-albus is but faintly striate. I have seen the type, which seems close to C. montagnei but differs in being smaller, much darker, in having smaller peridioles, and spores more variable in size and shape. I have identified two collections from Fiji as C. nigro-albus. Until more is known of this distinctive and apparently exclusively tropical species, it should be retained. 5 / Cyathus novae-zeelandiae Tul., Ann. Sci. Nat. m, i: 66, 1844 Etym. Named for New Zealand For illustrations, see Lloyd (1906), p. 19 Peridia infundibuliform, 12-14 mm high, 5-7 mm wide at the mouth, tapering gradually to the base and suddenly converging to a short stipe about 2 mm long and i mm thick; exterior dark-brown, covered with appressed tomentum, interior longitudinally striate for about half the depth of the peridium, black, dull; mouth erect or slightly expanded, revolute, striate, margin entire, even. Pendióla lenticu-

lar, 2.3-3 mm diam., black; tunica thin, white. Spores elliptical, somewhat pointed at both ends, 11-13 x 5-6 /¿.

Cunningham's (1924) description is quoted as it seems to agree more closely with my own notes on the type than does Lloyd's somewhat more generalized description. I have seen the type and only one other collection (no. 1117 Herb. HJ. Brodie), obtained by Dr J. M. Dingley, Waitakere Range, Upper Piha Valley, Auckland, New Zealand, July 1948. The latter specimens are very dark brown, very hispid, and have thick-walled spores measuring 11.5-12.5 x 5.5-7.5 M6 / Cyathus chevalieri Hariot and Patouillard, Bull. Mus. Nat. 2, p. 85, 1909 Etym. Named for collector, Chevalier 'Peridio obconico usque ad 2 cm alto, undique hirto leproso, corona brevissima pilosa donato, intus glabro, striato-plicato, nitenti, pallide ferrugineo, sursum obscuriori sub plúmbeo; sporangiolis tenuibus, vix i mm latís, orbicularis, margine tumidulo praeditis; sporis hyalinis, numerosis, ovoideis, 8 x 5 p.'

The diagnosis of this species is quoted from Saccardo, vol. 21, p. 465. I have seen only the specimens in the herbarium of the National Museum in Paris. These (about a dozen plants) are not labelled as type but, as they were collected by A. Chevalier, identified by Patouillard, and came from Oubangui in the Congo, they must be a part of the original collection. From the description, C. chevalieri is unique in the extreme length (2 cm) and narrowness (5-7 mm) of the peridia. The colour is deep brown, the outside roughly hirsute, and the plications distinct internally. The cortex is thick and dark, but one-layered. The specimens do not show the crown of short hairs called for in the description but otherwise correspond closely. In general, this remarkable fungus appears

177 / The genus Cyathus

to resemble C. striatus, but differs in possessing much smaller spores. It is closer to C. novae-zeelandiae, but differs greatly in having very tall slender peridia and smaller pendióles. The original description compares C. chevalieri with C. microsporus, C. berkeleyanus, and C. lesueurii (C. stercoreus), but, from what I have seen, C. chevalieri is utterly unlike any of these species. 7 / Cyathus rudis Pat., Champ, de Madagascar, Mém. Acad. Malagache, fase, vi: 35,1927 Etym. L. rudis, rough, raw, unculti va ted etc., application not clear, refers to rough exterior Peridium conic, cup-shaped, 5-10 mm high, 5-8 mm wide at the mouth, striate on the inner surface, covered on the outside with broad red-ferrugineous squamules hiding the external striae which are rather faint. Interior silvery white. Pendióles black-brown with thin tunica, i mm wide. Spores abundant, elliptical, 9-12 x 5 ¡JL. Related to C. novae-zeelandiae which has the same spores but peridioles double the size. On cow dung under shaded woods. Ankaramy, December (transí, of original descr.).

Examining an isotype in Paris, I noted that the species does resemble C. novae-zeelandiae, but seems to differ from it in the following points: spores considerably smaller (8 X 5 /¿), peridioles distinctly smaller (i mm), coarse external hairs tufted and less evenly distributed than in C. novae-zeelandiae, fruit bodies somewhat broader and not so high. In the type, no structures which could be called broad squamules are evident. As noted above, the hairs are irregularly tufted. I have seen, in addition to the type, only two other specimens, which are in the herbarium of the National Museum in Paris. One from New Caledonia is close to the type but has slightly taller cups; another, from Amboina, agrees fairly well with the type but is rather worn. No other specimens appear to be known. Whether or not C. rudis is truly coprophil-

ous cannot be stated at present. Only for the type material is the substrate noted as dung. This fungus seems to be fairly well defined and is retained as a valid species.

rather broadly obconic when fresh, deep buff or sepia brown (dark reddish brown in the type) but usually fade markedly upon drying and frequently specimens in the same collection fade to different degrees. The outside is hirsute or shaggy when fresh but the tomentum is fragile, for almost all the loose 8 / Cyathus berkeleyanus (Tul.) Lloyd, hairs are easily rubbed off and old specimens appear Myc. Writ. 2, Nidulariaceae, p. 19, 1906 smooth! The outside is strongly plicate, but in very Cyathus microsporus var. berkeleyanus Tul. Ann. woolly specimens the plication may not be evident. Sci. Nat. m, i: 74, 1844 The inside is also plicate, but the degree of plication Cyathia berkeleyana (Tul.) White, Bull. Torrey varies greatly from specimen to specimen and the Bot. Club 29: 258, 1902 inexperienced collector is very likely to place some Etym. Named for M.J. Berkeley 1803-1889, famous specimens in the 'smooth' section of a key. In colour British mycologist the inside is usually the same as the outside, but may Figures 44b, 6ic be darker or lighter. Pendióles are dark brown in Tulasne (1844) considered this species to be a variety colour, 1.5-3 mm in diameter, commonly but not invariably elliptical and provided with a thin tunica. of Cyathus microsporus. White (1902) and later The cortex of the pendióle is composed of only one Lloyd (1906) pointed out that C. berkeleyanus bears layer (cf. C. poeppigiif C. limbatus, and other plilittle resemblance to C. microsporus and should be cate species). Spores are small, mostly subglobose, held as a distinct species. The type collection was and 6-9 X 4-7 fji in size. from Rio de Janiero, Brazil (Chas. Darwin) fide Lloyd.

No one writing about this species seems to have been aware of its great variability in size, colour, plication, and (to a lesser extent) spore size, or at least to have mentioned it. I found C. berkeleyanus repeatedly on bamboo pots in Jamaica, and, almost always, there seemed to be about four variants among the specimens of a single collection. This was so perplexing that I frequently resorted to sorting them out into different piles under a stereoscopic microscope, only to find that each pile, despite marked differences, could not be identified as any species other than C. berkeleyanus. That such variation in a collection from nature is not the result of several species growing in proximity, is established by the fact that when ten soil-pot cultures - all subcultures of one dikaryon mycelium - fruited in the greenhouse, much the same degree of variation was observed. In place of Tulasne's description, the following is offered to cover the variation exhibited by this species and yet emphasize its salient characters. The fruit bodies (6-8 mm high, 4-6 mm wide) are 178 / The bird's nest fungi

Because the range of spore size of C. berkeleyanus overlaps that of C. pallidus, pale weakly plicate specimens of the former species are easily mistaken for dark forms of the latter, especially since both species have a one-layered cortex. C. berkeleyanus is of widespread distribution in the tropics and often occurs in great abundance. It is also a very difficult species to recognize except by a process of elimination. I suspect that my own very extensive collections of this species are not all correctly identified. Intensive study of this species, using new techniques such as scanning electron microscopy, may well reveal some reliable and constant character by which it should be characterized rather than those in use. A specimen carefully compared with the type and taken as its equivalent is illustrated in figure 6ic, right, while in figure 6ic, left is shown a representative of a very common form of the species prevalent in the West Indies. Specimens labelled C. berkeleyanus are known from almost all parts of the West Indies and from Florida, Cuba, Mexico, Bolivia,

Brazil, and the Hawaiian Islands. It was reported from China by Teng (1935), but I have seen no material of this species from the eastern hemisphere. Dikaryon mycelium of C. berkeleyanus is white when freshly transferred and becomes only slightly off-white in age. It is fluffy, fine-textured, and slowgrowing at room temperature. If the mycelium is of any value in species differentiation, then C. berkeleyanus is not closely related to C. striatus and C. poeppigii with which Lloyd associated it (Brodie, 1968), for both the latter species produce coarse, deeply pigmented mycelium. 9 / Cyathus bulled Brodie, Bull. Torrey bot. Club 94: 68-71, 1967 Etym. Honouring Prof. A.H.R. Buller Figures 19, 6id Teridium obconic with curved sides and abruptly narrowed at base, pale grey to linen colour; 5-9 mm high, 5-8 mm at mouth; externally covered with a fine tomentum and long connivent down-pointing hairs ; externally strongly plicate in the upper third ; internally plicate, silvery; lip smooth or minutely fimbriate. Emplacement small or lacking. Epiphragm snow-white, beset with fawn-coloured vertical tufts of hairs. Pendióles 2-2.5 mm *n diameter with thick tunica, silvery when fresh, shiny and dark brown when old; cortex single. Spores variable, 5-8.5 IUL in diameter, spherical to subglobose, thick-walled' (Brodie, 1967).

Because of its very pale colour and long external hairs, this species (figures 19, 6id) may in the past have been confused with C. pallidus. C. bulleri, however, is strongly plicate whereas C. pallidus is smooth or at least not regularly plicate. Moreover C. bulleri has an epiphragm beset with vertical tufts of brown hyphae whereas that of C. pallidus is snow white. C. bulleri is the only very pale, strongly plicate species known to occur in the tropics. Detailed comparison with other species may be found in Brodie (1967^). The species has been identified from the West Indies (the type is 179 / The genus Cyathus

from Guadeloupe, Brodie, No. 668oa), the Hawaiian Islands, and Mexico. In culture, dikaryon mycelium is finetextured, remains almost snow-white, and recalls that of C. berkeleyanus (Brodie 19/ob). C. bulleri has been shown to fruit readily in pure culture (Brodie, -Ly/ob). 10 / Cyathus annulatus Brodie, Can. J. Bot. 48, 749, 1970 Etym. L. annulatus, ringed, with reference to lip of peridium Figure 6if Peridium broadly and regularly obconic, flaring only slightly in the upper quarter, 7-10 mm wide, 7-12 mm high, pale brown, covered with fine tufted tomentum; basal emplacement small, inconspicuous ; inner surface of peridium pale buff, shiny, very evenly but delicately striate ; lip of peridium marked by a distinct deep brown ring 0.5 mm wide; pendióles small (1.5-1. 75 mm), subtriangular, with shiny tunica ; spores variable in shape from evenly ellipsoid to ovate, to subspherical, thick-walled (2-3 /x), 15.5-17 /i> 15-19 M (Brodie, 19703).

This readily recognized species (figure 61 f) is taxonomically related to C. striatus and C. helenae, the only other plicate species known to occur in temperate North America. It is recognized at present only from the Cypress Hills of Alberta (type collection) and is characterized by the distinctive deep brown ring which colours the inner surface of the lip of the fruit body and by the shape of the basidiospores. 11 / C. pullus Tai and Hung, Sci. Rpts. National Tsing Hua Univ. 3 : 38, 1948 Etym. L. pullus, very dark For illustration, see Tai and Hung Teridium campanulate, 8-10 mm high, 7 mm wide at the top, exterior appressed tomentose, Verona Brown, lighter at the upper part (Tawny Olive), closely striate on both surfaces; interior Bister Brown, somewhat shining, margin non-fimbriate,

pendióla Drab, orbicular, 2-2.5 mm in diameter; spores ovoid to subglobose, hyaline, smooth, bluntly pointed at one end or rounded at both ends, 8.6-11 X 6.4-7.9 ^' epispore i ¡JL thick/ 'On soil, Kunming, Aug. 28, 1938, F.L. Tai (5510) type/ 'It is close to C. Hookeri, but differs in the darker-coloured and less hairy peridium which is also striate on both surfaces with fimbriate margin and thinner walled spores' (Tai and Hung, 1948).

This species from Yunnan has not been seen by me. As neither tunica nor cortex is mentioned in the description, it is difficult to visualize this species, and the line drawing accompanying the description indicates only a clearly striate peridium, the character which causes it to be herewith included in the striatus group. If, as described, the peridia are striate on both surfaces, then the species would not appear to be at all closely related to C. hookeri with which Tai and Hung associate it. E / D O U B T F U L SPECIES

1 / Cyathus ambiguus Tul., Ann. Sci. Nat.3 : 75,1844 This name can probably be considered as a synonym of C. poeppigii (or possibly of C. limbatus) for the following reasons: (i) Tulasne himself, who described it from Colombia, wrote: 'this species resembles very closely those above described under the names of C. poeppigii andC. limbatus' ; (2) writing of the type, Lloyd commented, 'The cups are of the same general nature as those of Cyathus limbatus ... ' ; (3) I examined the type (in Paris) and noted close resemblance to the species named above. Specimens cited by Wright (1949) from Argentina as C. ambiguus are, in my opinion, probably a large form of C. poeppigii with spores 30-45 x 20—25 I*" 2 / Cyathus elegans Speg., Sacc. 16: 230, cited as Fg. Arg. novi v. crit., p. 185,1899 Isotype specimens of this species (No. 16639 180 / The bird's nest fungi

Herb. Inst. Spegazzini) do not conform to the original description in the following principal respects: (i) the outside of the peridium is not 'subglabrato / but bears rather sparse long hairs ; (2) the inside of the peridium is shiny but brown, not 'argénteo' ; (3) no tunica is evident on the black shiny peridioles. The author found no basidiospores in the isotype, as noted in the description 'Sporae non visae.' Sectioned peridioles revealed the presence of masses of yeast cells with which the Cyathus had evidently become contaminated. C. elegans is apparently a slender longstemmed form of C. stercoreus as Spegazzini himself noted, close to forma Lesueurii (Ann. del Museo Nacional de Buenos Aires, 1898). 3 / Cyathus minutosporus Lloyd, Myc. Writ. 7,1325,1924 'The material is very scanty and there are no good cups for illustration. The spores are the character, however. They are in great abundance and truly minute, measuring 2 X 4 . They are only about one third the size of those of Cyathus microsporus which has the smallest spores heretofore known/

Lloyd's description (above) could be dismissed on the grounds that the species is not described except in the one sense of spore measurements. The type was collected by A. V. Duthie, Heidelberg, South Africa (no date given). I have examined the type material (Cat. No. 24889) from the Lloyd Herbarium and can report the following. The type consists of only one reasonably complete specimen (about 5 mm in height), the rest of the packet containing only small fragments of fruit bodies and a few loose peridioles. Fragments are dark reddish-brown in colour, externally shaggy, plicate externally and (probably) smooth internally. The emplacement was evidently rather wide and of loose organization. Peridioles are 1.5-2 mm wide, rather plump and possibly bore a weak tunica. Spores are about 4 x 2-3 JLC. These 'remains' suggest small dark speci-

mens of C. berkeleyanus because of the plication, and not C. microsporus. Since neither of the two latter species is known to occur in Africa and because of the very small spores of C. minutosporus, I do not feel that the name can be considered as a synonym of either of the other two. It cannot legally be recognized as a valid species, but if found again it should be easily recognized by collectors of African material because of the minute spores.

fungus occupies. Through the courtesy of Dr Svercek of the National Museum of Prague, I have been able to examine Velenovsky's type material. There can be no question about its identity as one of the many common forms of Crucibulum laeve. The type specimens are moderately dark in colour, but not 'atrofuscus/ and the presence of a typical Crucibulum funiculus leaves no doubt as to generic identity.

4 / Cyathusboninensis S. Ito et Imai, Trans. Sapporo Nat. Hist. Soc. 15: 8, 1937

TWO

It has not been possible to examine the type specimen of the Japanese fungus but there is little reason to doubt that it is a slender, tall 'form' of Cyathus stercoreus. The describers state that it 'is allied to C. stercoreus, from which it is distinguished by the darker coloured and less shaggy peridia and the presence of tunica, as well as by the smaller spores/ Neither of the first two characters would be distinguishing in such a comparison. As for the matter of tunica, if a true tunica were present, the species could not be the same as C. stercoreus. The diagnosis as published does not indicate that a tunica is present, but states instead 'pendióles blackish or black ... smooth, shiny ....' It is apparent that the sense of the term tunica here is not that of modern usage (see Chapter 11, c, i). If a true tunica were present the peridioles could not be black and shiny. Moreover, since many forms of C. stercoreus have spores well within the range of size given for C. boninensis, this feature likewise does not justify the validity of the latter name. 5 / Cyathus atrofuscus VeL, No vit. Mycol. Nov., p. 94, 1947 Velenovsky's description seemed to pose some problems as it suggested that the fungus occupies a position intermediate between Crucibulum and Cyathus, which no known

181 / The genus Cyathus

RECENTLY PUBLISHED SPECIES OF

CYATHUS

Two additional species, Cyathus ellipsoideus Brodie and C. crispus Brodie, have been published too late for inclusion in the main body of the book. i / Cyathus crispus Brodie: to be added to Group vi of the present monograph Brodie, HJ. 1974. Cyathus crispus, a new species from Ghana. Can. J. Bot. 52: 1661-3. Cyathus crispus sp. nov. from Ghana has goldencoloured, strongly plicate peridia covered externally by curls of hyphal hairs. The small dark brown peridioles are radiately wrinkled ; they are provided with a two-layered cortex and long narrow basidiospores. The new species is most closely related to C. limbatus Tul.

2 / Cyathus ellipsoideus Brodie: to be added to Group vu Brodie, HJ. 1974. A new plicate Cyathus from India. Can. J. Bot. 52: 247-9. A pale-coloured, strongly plicate species of Cyathus (Nidulariaceae), at present known only from Mysore (India), is described as C. ellipsoideus sp. nov. It is the only species known to possess peridioles and basidiospores both of which are ellipsoidal in outline. Cyathus ellipsoideus bears close relationship to C. berkeleyanus and C. bulleri but has much larger spores than either.

Nidulariana

Nidulariana i

Nidulariana 2

'The Nidulariaceae have previously been the subject of numerous dissertations: there is scarcely a treatise on the origin and reproduction of fungi in which they are not mentioned extensively, either to furnish arguments for writers who favour the existence of seeds in the lower plants or, on the other hand, as objects of controversy as to whether or not they possess internally the true reproductive bodies. ... it appeared to us, however, that there still remained some points to be cleared up, the manner of fructification to be discovered, a great number of details of organization to be elucidated, etc. Therefore we took up the work anew, and, even if we have not found a solution to all the problems that the story of the Nidulariaceae presents, at least we shall have made a contribution towards making it more complete.... they should not be reproached for an analytical anatomical work who present, above all, descriptions which are at the same time concise and significant, searching with the greatest care the essential features that form the critérium of each species.... the truth still, perhaps remains to be discovered/ Translation from the monograph on the Nidulariaceae by L.R. and Ch. Tulasne, Ann. Sci. Nat. (3)1: 41 et seq., 1844.

(Notes made by the late Dr A.H.R. Buller, 1941, but never published. At the time, Buller was working at the University of Manitoba, Winnipeg, Canada.)

182 / The bird's nest fungi

1 / University of Manitoba, Sept. 3, 1941 'Dr. H.B. came to my office today and I revealed to him the secret of organization in Cyathus stercoreus. Got out dried fruit bodies brought from Baton Rouge (La.), pulled out dry peridioles and attached parts and put them in water on a slide. Saw no explosive swelling in these dry specimens, but with a needle I pulled out the attachment cords from their purses. Put peridioles with their cords on writing paper and found, when they had dried, only part that sticks fast is the swollen end of the cord; the rest of the cord and the peridiole are easily shaken free when one inverts paper. ... The reason why cord is so strong is that it is composed of solid hyphae, clamps form solid knots and lumen of hyphae seems to be entirely suppressed/ 2 / University of Manitoba, Sept. 24,1941 'This morning Dr B. came in and gave me his sketches showing, in Cyathus vernicosus (C.

olla) and C. striatus, that middle piece joins sheath at about its middle and enters wall not as a thin string but as a flared-out mass of hyphae. This afternoon, I verified this with C. striatus. Pulled off peridiole and saw that end of purse had been pulled off and left at free end of anchor. Under microscope could see a depression at one end, also saw fibers of anchor as he described. Pulling at peridiole seems to lengthen and then alter shape of sheath. VerifyV Nidulariana 3: Fairy Goblets In many of the oldest writings about bird's nest fungi, the elegant vase-like fruiting bodies are referred to as Elfin Cups, Fairy Goblets, Corn Bells, and using other fanciful names derived, no doubt, from folklore: from such sources, the following lines of unknown origin are derived: 'It is called the Elfin Wood, they said and pointed far far up to where the mist filled a broad bowl of the valley and spewed downward between dark mountain crags. The name they gave urged the Wanderer on as he left the grassy plain below and plodded ever upward, weary yet unable to rest. The way grew darker and what little of the failing light remained was dimmed by long garlands of ghostly lichen plants that burdened the arms of old trees, grey-green and smelling of smoke, although there was no smoke. The moss was deeper over the mouldering fallen trees; it hid their forms, and the Wanderer stumbled wearily while all about him the grey green was a veil over his eyes. There was naught to clear the mist and give reality to form save the bright tawny dome of a lone toadstool, too large for a toad, perhaps too small for an Elf, if there were Elves. Only the Wanderer's steamy breath moved and there was no sound at all. 'Anon he saw an opening in the wood where more light was and where the mist lurked not so low. Here there was sound, a low chattering

183 / Nidulariana

sound, not loud but gruff and in a tongue he thought he knew. Carefully, lest he should betray himself, he drew nearer to the opening until he could descry six small figures, each seated on a small red toadstool which is called a russule. Each figure was a small man; some men had long beards and some had none and though the faces were of old men, yet they were young, or else ageless. The oldest man, as it seemed to the Wanderer, smoked a long pipe and was nodding wisely while another spoke. The Wanderer found that the tongue was unknown to him, yet it had a familiar ring - as though it should be within his ken. Then the oldest man reached for and held aloft a brown goblet of wondrous beauty, slim-stemmed and graceful and shining within as though of silver. At this, the others of the band raised their goblets and one pronounced a toast and the Wanderer saw that they all drank from Fairy Goblets ../ Nidulariana 4 (The following is a translation of a page from the diary of the late Dr René Vandendries, the renowned Belgian mycologist who died in March 1952.) Rixensart, Belgium; July 24, 1950 After a separation of eighteen years, I had the great pleasure of seeing again my friend and former collaborator, Dr B. He had just come from the International Botanical Congress in Stockholm and, before leaving Europe for his home, he spent several days with me in my villa 'La Chanterelle/ Our first greetings over, my friend proceeded to show me photographs, drawings and specimens of the Nidulariaceae concerning which he had spoken before the Congress. I was astonished by his story. He then put this question to me: 'During the eighteen years that have passed since I last visited you, have you ever found any of these Bird's Nest Fungi?'

FIGURE 62 The late Dr René Vandendries and thé author finding bird's nest fungi in Belgium, 1950

'I can remember/ I replied, 'only once finding a single species in the Forest of Fontainebleu, near Paris/ My questioner looked at me with a mischievous smile and added, 'What about your own garden? Shall we look there?' 'Gladly, but it would be little short of miraculous to find your beautiful little Bird's Nests full of eggs in my garden/ We walked down the steps of the stone stairway leading to the terrace. Right and left were embankments on which roses had formerly been planted and on which there now flourished cherry-laurels and flowering shrubs. A solitary rose bush had escaped the disaster of a ten-degree-below-zero frost two years before. The surviving rose had received an application of manure in the spring. It was about to flower. My friend pushed aside the dense foliage, pulled away some dead leaves and showed me a group of the little brown cups of Cyathus striatus. Many of the cups still contained pendióles. I uttered a cry of astonishment. Our trembling hands probed elsewhere. The ground was 184 / The bird's nest fungi

covered with a veritable culture of the beautiful peridiole-filled vases. The keen eye of my specialist friend soon discovered some ejected peridioles attached by their funiculi to foliage about 50 cm from the nearest fungus cup (a perfect confirmation of the story he had told me a few minutes before!). 'Now let us look farther/ he said. Along the hedge which bounded my garden, not far from the spot we had already explored and under some shrubs, my friend showed me, in an area as large as the palm of the hand, seven white buttons ... seven future vases of still another species (Cyathus olla). These we left to mature and to open in due course. To my friend, delighted as I with our discovery, I remarked, 'I had eyes but I did not see. There is a kindly god of mycologists who strews in their paths prodigious numbers of treasures which crowd about them and which they often fail to see'/ (But see figure 62 !) Nidulariana 5 Several botanists have suggested to the author that birds could possibly distribute peridioles by mistaking them for seeds. If peridioles were mistakenly eaten by birds and were capable of passing undamaged through the birds' digestive apparatus a very important means of long-range distribution would exist, for birds fly far and might transfer fungi across large water and mountain barriers. Said H.B. 'The sight I like best Of all sights that my vision have blest, Is the view ever-glorious Of Cyathus stercoreus With its eggs overflowing its nest/ Dr Thorvaldur Johnson Nidulariana 6 Not all technical assistants are as highly paid, ignorant, or unwilling as many of those of the 'more developed' countries. In the photograph

shown are seen a few of the author's assistants in Jamaica. They were careful and assiduous collectors of fungi, they appeared to be eager to learn, and (best of all) they contributed vastly to the fun of hunting for treasure by their limitless good-will and their infectious smiles. After these boys were shown two specimens of what turned out later to be an undescribed species (Cyathus setosus) and being asked to search for more, they returned in less than an hour with handsful, and only one other species was intermingled with their collections ! (figure 63). Nidulariana 7 Some form of representation of mushrooms and other fungi has been found in many of the most ancient efforts of man in the graphic and ceramic arts. Frequently the fungus was generalized or symbolized, but at times it was represented in enough detail to allow specific identification of the subject. Few fungi however have achieved the notoriety of having been the subject of that important (if sometimes reprehensible) medium of communication, the so-called 'comic strip' of our time. It is quite possible that the facts of the irresistibly interesting mode of spore dispersal of the Bird's Nest Fungi became known to more persons through Mr Ed Dodd's little sketches, reproduced herewith, than through the medium of over a hundred scientific works listed in this book. In any case, Mr Dodd's artistic effort (figure 64) is worth preserving. 185 / Nidulariana

FIGURE 63 A few good companions and able assistants in Jamaica, 1966

Nidulariana 8: Advice to a Student The late Professor Buller was not, as far as I am aware, often inclined to express himself philosophically: it appeared to me that he believed that his time and energy could be more profitably expended in observation and experiment. On rare occasions, some of his credo which was implied in what he called his motto, 'carpe fungu m ' - was passed on to his students. The following is the only written example of which I have any knowledge. 'If the fungi are a "treacherous tribe," as has been said, it may well be largely because we have placed too much reliance on some of the means of studying them. Certainly, some of the cytological pictures of the past have, more recently, been discredited; in taxonomic work, too many mycologists have based their dicta too frequently on limited herbarium material and have not been familiar with a wide range of living specimens; on the physiological side, there has too often been a tendency to forget that a fungus growing under natural conditions is rarely, if ever, a pure culture closely comparable to what we prize in our laboratories. Use your powers of direct observation of the fungus as it grows in nature. Use them to the fullest extent of your ability, remembering - of

FIGURE 64 'Comic' strip by Mr Ed Dodd presenting the story of the bird's nest fungi for the newspapers. Reproduced by permission of Publishers-Hall Syndicate

course - that the conclusions you draw from observation alone may frequently be erroneous. But do not worry unduly about teleology ; I would much prefer to arrive at a new possible concept through a teleological approach than to

186 / The bird's nest fungi

miss seeing a new road. For, after all, we cannot find the truth unless we look along all possible roads!' A.H.R. Buller - from a letter to H.J.B., Jan. 10, 1931

There are no more appropriate words with which to close than those of the great Linnaeus as quoted by Tulasne in the second volume of the Carpología: 4

... so that new things are always presented for the delight of his curiosity, lest the thread-bare common objects should make him weary/ Linnaeus, Cui bono?, 1752, § 14

Selected Bibliography

Alexopoulos, C.J. 1962. Introductory Mycology, 2nd ed. New York: John Wiley, pp. 613 Allbutt, A.D. et al. 1971. Cyathin, a new antibiotic complex produced by Cyathus helenae. Can. J. Microbiol. 17: 1401-7 Andrews, P.M. 1900. Notes on a species of Cyathus common in lawns at Middlebury, Vermont. Rhodora, 2: 99-101 Ayer, W.A. and H. Taube. 1972. Metabolites of Cyathus helenae. Cyathin A3 and Allocyathin B3, members of a new group of diterpenoids. Tetrahedron Letters, No. 19: 1917-20 Bowerman, Constance A. and J. Walton Groves. 1962. Notes on Fungi from Northern Canada, v Gasteromycetes. Can. J. Bot. 40: 239-54 Brams, Richard F. 1950. The effect of pepsin, trypsin and Upase on the germination of certain fungus spores. Master's Thesis, Indiana University (unpubl.) Bref eld, O. 1877. Botanische Untersuchungen iiber Schimmelpilze. in Heft. Basidiomyceten i. 226 pp., Leipzig: Felix Brodie, Harold J. i948a. Tetrapolarity and unilateral diploidization in the bird's nest fungus Cyathus stercoreus. Am. J. Bot. 35: 312-20 - i948b. Variation in the fruit bodies of Cyathus stercoreus produced in culture. Mycologia, 40: 614-26 - 1949. Cyathus vernicosus, another tetrapolar Bird's Nest Fungus. Mycologia, 41: 652-9 - 1950. Notes on two little-known Bird's Nest Fungi from southern United States. Mycologia, 42: 186-90 - i95ia. The splash-cup dispersal mechanism in plants. Can. J. Bot. 29: 224-34 188 / Selected bibliography

- i95*h. The splash-cups of Polyporus conchifer. Can. J. Bot. 29: 593-6 - 19510. Two heterothallic species of the genus Nidula. Mycologia, 43: 329-37 - i952a. Inter fertility between two distinct forms of Cyathus olla. Mycologia, 44: 413-23 - i952b. Nature's splash guns. Natural History (New York), 61: 403-7 - 1953. Sexuality, inheritance studies and nuclear migration in the Nidulariaceae. Proc. vii Int. Bot. Congr. Stockholm, 1950, p. 431. Stockholm: Almqvist and Wiksell - 19553. Morphology and culture characteristics of a highly aberrant Cyathus. Am. ]. Bot. 42: 168-76 - i955b. Springboard plant dispersal mechanisms operated by rain. Can. ]. Bot. 33: 156-67 - 19550. Cyathus gayanus from Costa Rica. Mycologia 47: 266-8 - 1956. The structure and function of the funiculus of the Nidulariaceae. Svensk Bot. Tidskr. 50 : 142-62 - 1957. Raindrops as plant dispersal agents. Proc. Indiana Acad. Sci. 66: 65-73 - 1958. Renewal of growth and occurrence of twin fruit bodies in the Nidulariaceae. Svensk Bot. Tidskr. 52: 373-8 - 19623. Culture and taxonomy of Cyathus earlei. Can. J. Bot. 40: 1483-5 - i962b. Twenty years of Nidulariology. Mycologia, 54: 713-26 - 19663. A new species of Cyathus from the Canadian Rockies. Can. J. Bot. 44: 1235-7 - i966b. Cyathus pygmaeus from Northwestern United States. Mycologia, 58: 973-6

- 196/a. Cyathus setosus, a new member of the Nidulariaceae from Jamaica. Can. J. Bot. 45: 1-3 - 1967!). Cyathus bullen, a hitherto undescribed fungus of the Nidulariaceae from the West Indies. Bull. Torrey bot. Club. 94 : 68-71 - 1967^ A new species of Cyathus in the pallidus group. Svensk Bot. Tidskr. 61: 93-6 - i907d. New record of a large Cyathus from Western Canada. Mycologia, 59: 532-3 - 19676. Cyathus africanus, a previously undescribed Bird's Nest Fungus. Can. J. Bot. 45: 1653-5 - 1967^ New records of Nidulariaceae from the West Indies. Trans. Brit. Mycol. Soc. 50: 473-8 - 1968a. Tetrapolarity and comparison in culture of some tropical species of Cyathus. Svensk Bot. Tidskr. 62: 201-16 - i968b. The Nidulariaceae of Canada. Canadian Field Naturalist. 82 : 2-14 - i97oa. A previously unnamed species of Cyathus from the Cypress Hills, Alberta. Can. J. Bot. 48 : 749-50 - i97ob. Sexuality patterns in some new and little-known species of Cyathus. Svensk Bot. Tidskr. 64: 44-50 - 197OC. Crucibulum parvulum, a very small new bird's nest fungus from Northwestern North America. Can. J. Bot. 48: 847-9 - i97ia. Cyathus pictus, a large-spored bird's nest fungus from Mexico. Can. J. Bot. 49: 1613-14 - i97ib. Cyathus crassimurus sp. nov. from Hawaii. Can. J. Bot. 49: 1609-11 - i97ic. Crucibulum cyathiforme, a new bird's nest fungus from Colombia. Can. J. Bot. 49: 2009-10 - 1973. A new species of Cyathus from the Philippines. Can. J. Bot. 51: 1393-4 Brodie, Harold J. and R. W.G. Dennis, 1954. The Nidulariaceae of the West Indies. Trans. Brit. Mycol. Soc. 37: 151-60 Buller, A.H.R. 1942. The splash cups of the bird's nest fungi, liverworts and mosses (Abstract). Proc. Roy. Soc. Can. m, 36: 159 Burnett, V.H. and M.E. Boulter. 1963. The mating systems of the Gasteromycetes Mycocalia denudata and M. duriaeana. New Phytologist, 62 : 217-36 Cejp, K. 1958. Nidulariales (Hnizdovkotvare) in Flora CSR, B, i : 633-82, Praha Cejp, K. et J.T. Palmer. 1963. Rody Nidularia Fr. a Mycocalia J.T. Palmer v Ceskoslovensku a 189 / Selected bibliography

Mycocalia sphagneti J. T. Palmer sp. nov. z Anglic. Ceská Myk. (Praha), 17: 113-26 Christiansen, M.P. 1941. Studies in the larger fungi of Iceland. The Botany of Iceland. Vol. m, partn, No. 11: 191-225 Clusius, C. 1601. Rariorum Plantarum Historia. Antverpiae. (cited from Cat. Lib. Arnold Arb) Coker, W.C. and J.N. Couch. 1928. The Gasteromycetes of the Eastern United States and Canada, pp. 201. Chapel Hill: University of North Carolina Press Cooke, W. and C.G. Shaw. 1952. Notes on Alaskan fungi. Research Studies State Coll. Wash. 20: 15-20 Cunningham, G.H. 1924. A revision of the New Zealand Nidulariales, or 'Bird's-nest Fungi.' Trans. N.Z. Inst. 55: 59-66 Demoulin, V. 1969. Les Gasteromycetes. Introduction à l'étude des Gasteromycetes de Belgique. Les Naturalistes Belges. 50: 225-70 Diehl, W.W. 1941. The taxonomy of Zenker's Leptostroma Camelliae. Mycologia, 33 : 215-19 Dissing, H. 1963. Studies in the Flora of Thailand 25. Discomycetes and Gasteromycetes. Dansk Botanisk Arkiv. 23(1): 115-30 Dissing, H. and M. Lange. 1962. Gasteromycetes of Congo. Bull. Jard. Bot de l'état (Brussels) 32 : 325-416 Dodge, B.O. 1941. Discharge of the sporangioles of bird's nest fungi. Mycologia, 33 : 650-4 Duss, R.P. 1903. Enumeration méthodique des Champignons recueillis à la Guadeloupe et à la Martinique. Lons-le-Saunier (Impr. etlithogr.) Lucien Declume Eckblad, F.E. 1955. The Gasteromycetes of Norway: The epigaean genera. Nytt Mag. Bot. 4 : 19-86 - 1962. Gasteromycetes from the Canary Islands. Nytt Mag. Bot. 9: 135-8 - 1970. Gasteromycetes from Iraq, Iran and Afghanistan. Nytt Mag. Bot. 17: 129-38 - 19713. Gasteromycetes of Finnmark (Northernmost Norway). Astarte, 4(1): 7-21 - i97ib. Spores of Gasteromycetes studied in the scanning electron microscope (SEM), i. Norw. J. Bot. 18: 145-51 Eidam, B. 1877. Die Keimung der Sporen und die Entstehung der Fruchtkôrper bei den Nidulariaceen. Beitr. z. Biol. d. Pflanzen v. Cohn. m (2): 221-48 Fischer, E. 1933. Gasteromycetes. Natürlichen

Pflanzenfamilien 2nd. éd. /a: 1-122 Fries, Nils. 1936. Crudbulum vulgare Tul. and Cyathus striatus Pers., zwei Gasteromyceten mit tetrapolar Geschlechtsverteilung. Bot. Notiser (Lund), 19361567-74 - 1940. Researches into the multipolar sexuality of Cyathus striatus Pers. SymbolaeBot. Upsaliensis 4(i) : 1-39 Fries, Nils. 1943. Über das Vorkommen von geographischen Rassen bei Crudbulum vulgare Tul. Archiv. fur Mikrobiologie 13 : 182-90 - 1948. Heterothallism in some Gasteromycetes and Hymenomycetes. Svensk Bot. Tidskr. 42 : 158-68 Fries, R.E. 1910. Om utvecklingen af fruktkroppen och peridiolerna hos Nidularia. Svensk Bot. Tidskr. 4: 126-38 - 1911. Über die cytologischenVerhàltnisse bei der Sporenbildung von Nidularia. Zeit. f. Bot. 3 : 145-65 Fries, TH.C.E. 1914. Zur Kenntnis der Gasteromyceten-Flora in Torne Lappmark. Svensk Bot. Tidskr. 8: 235-43 Fulton, I.W. 19500. Unilateral nuclear migration and the interactions of haploid mycelia in the fungus Cyathus stercoreus. Proc. Nat. Acad. Sci. 36: 306-12 - i95ob. Nuclear migration and the interaction of haploid mycelia in Cyathus stercoreus. Thesis, Indiana University (unpubl.) Garnett, E. 1958. Studies of factors affecting fruiting body formation in Cyathus stercoreus. (Schw.) de Toni. PHD Thesis, Indiana University (unpubl.) Guzman, G. andT. Herrera. 1969. Macromicetos de las zonas áridas de México, ii Gasteromicetos. An Inst. Biol. Univ. Nal. Auton. México. 40: 1-92 Hesse, R. 1876. Keimung der Sporen von Cyathus striatus Willd., einer Gastromycetenspecies. Jahrb. Wiss. Bot. 10: 199-203 Hoffman, H. 1859. Über Pilzkeimungen. Bot. Zeit. 17: 209-17 Hollos, L. 1904. Die Gasteromyceten Ungarns. Leipzig: O. Weigel Humphries, W.J. 1940. Physics of the Air. 3rd éd. New York and London: McGraw Hill Imazeki, R. 1950. Fungi collected at Mt. Wu TaiShan. Bot. Mag. Tokyo. 63: 93-6 I to, S. and S. Imai. 1937. Fungió í the Bonin Islands, i. Trans. Sapporo Nat. Hist. Soc. 15: 1-12 Johri, B.N. 1969. Cyathin, a previously unknown

190 / Selected bibliography

antibiotic complex. Thesis, University of Alberta (unpubl.) Johri, B.N. and H.J. Brodie, i97ia. Extracellular production of indolics by the fungus Cyathus. Mycologia, 63: 736-44 - i97ib. The physiology of production of the antibiotic cyathin by Cyathus helenae. Can. J. Microbiol. 17: 1243-5. - 1972. Nutritional Study of Cyathus helenae and related species. Mycol. 64: 298-303. Johnson, M.M. 1929. The Gasteromycetae of Ohio. Ohio Biological Survey, 4(7): 273-352 Kambly, P.E. and R.E. Lee. 1936. The Gasteromycetes of Iowa. Univ. Iowa Studies, 17(4) : 121-85 Kennedy, M.E. 1956. The breeding systems of the Nidularia denudata complex in relation to those of other fungi. PHD Thesis, University of Liverpool Killermann, S. 1931. Die Nidularia Fr. Gruppe. Krypt. Forsch. bayer, bot. Ges. 2: 194-8 Kobayasi, Y. 1937. Fungi Austro-Japoniae et Micronesiae, i. Botan. Mag. 51: 755-57 Lange, M. 1948. The Gasteromycetes of Greenland. Medd. Gr0nland. 147(4): 1-32 Larsen, P. 1932. Fungi of Iceland. The Botany of Iceland. 2(3): 453-607 Leininger, H. 1915. Physiologische Untersuchungen iiber Cyathus striatus Willd. Ber. d. deutsch. bot. Ges. 33: 288-300 Lloyd, C.G. 1906. Myc. Writ. 2: The Nidulariaceae, 1-30 Lloyd, C.G. 1910. Myc. Writ. 3: 455 - 1915. Myc. Writ. 4: Letter 58, July 1915 - 1917. Myc. Writ. 5: 731 - 1922. Myc. Notes. 7: 1118 - 1923. Myc. Notes. 7: 1176 - 1925. Myc. Notes. 7: 1363 Lockwood, L.B. 1929. Peridia of Crudbulum vulgare. Proc. Indiana Acad. Sci. 38: 105-7 Lohman, M.L. 1938. Notes on Indiana Fungi 1937. Proc. Indiana Acad. Sci. 4: 88-92 Lu, Benjamin C. 1962. A new fixative and improved propiono-carmine squash technique for staining fungus nuclei. Can. J. Bot. 40: 843-7 - 19643. Chromosome cycles of the Basidiomycete Cyathus stercoreus (Schw.) de Toni. Chromosoma. 15: 170-84 - 1964^ Polyploidy in the Basidiomycete Cyathus stercoreus. Amer. J. Bot. 51: 343-7 - 1965. The role of light in fructification of the

basidiomycete Cyathus stercoreus. Amer. J. Bot. 52 : 432-7 Lu, Benjamin and H. J. Brodie, 1962. Chromosomes of the fungus Cyathus. Nature (London) 194: 606 - 1964. Preliminary observations of meiosis in the fungus Cyathus. Can. J. Bot. 42: 307-10 Lu, Shih-Hsiung. 1973. Effect of calcium on fruiting of Cyathus sterocoreus. Mycologia, 65: 329-34 Maire, R. 1902. Recherches cytologiques et taxonomiques sur les Basidiomycètes, pp. 209. Thesis, Paris. (AnhangzuBull., Soc. Myc. France 18, 1902) (cited in L. and Syd.) Martin, G.W. 1927. Basidia and spores of the Nidulariaceae. Mycologia, 19: 239-47 - 1939. New or noteworthy fungi from Panama and Colombia, iv. Mycologia, 31: 507-18 Martinez, A. 1956. Las Nidulariales Argentinas. Rev. Invest. Agrie., Buenos Aires, 10: 281-311 Miles, P.G. 1953. Studies of Cyathus stercoreus with special reference to nuclear behavior as revealed by the phase microscope. PHD Thesis, Indiana University, Bloomington, Indiana Molliard, M. 1909. Le cycle de développement du Crucibulum vulgare Tul. et de quelques Champignons supérieurs obtenus en cultures pures. Bull. Soc. Bot. France, 56: 91-6 Olchowecki, A. 1967. Culture studies and mating reactions in Cyathus helenae Brodie and related species. Master's Thesis, University of Alberta, PP- 45 Olchowecki, A. and HJ. Brodie. 1968. Sexuality and mycelial characteristics of Cyathus helenae and the related fungus Cyathus striatus. Can. J. Bot. 46: 1423-9 Olive, L.S. 1946. Some taxonomic notes on the higher fungi. Mycologia, 38: 534-47 Overholts, L.O. 1926. In FJ. Seaver and C.E. Chandon, Scientific Survey of Puerto Rico and the Virgin Islands. £(i): 1-208, N.Y. Acad. Sci. Overstreet, R.A. 1955. Morphology, Development and Taxonomy of the genus Nidula. Master's Thesis, Indiana University Palmer, J.T. 1957. Two species of Nidularia Fr. Section Sorosia Tul. in Yorkshire. Naturalist, Lond. Jan-Mar: 1-4 - 19583. Revise Nidularia arundinacea Vel. a jeji srovnání s príbuznymi druhy. Ceska Mykol. (Praha) 12: 132-6 - i958b. Observations on Gasteromycetes. vi. Three British species of Nidularia Fr. Section Sorosia Tul.: Ecology and distribution. Trans. 191 / Selected bibliography

Brit. Mycol. Soc. 41: 55-63 - i96oa. Nidularia pulvinata (Schw.) Fr. In Europa? Ein Exsikkat aus Killermanns Sammlung Z. Pilzk. 26: 37-44 -i96ob. Zur Ôkologie und Systematik von Nidularia farota (Roth ex Pers.) Fr. Druhy sjezd evropskych mykologû, Ceskoslovensko, pp. 19-21, Praha - i96ib. Observations on Gasteromycetes. x. The Nidulariaceae of Persoon's herbarium. Persoonia iU): 433-51 - 19613. Observations on Gasteromycetes. ix. The conservation of Nidularia Fr. and the separation oiMycocalia. J.T. Palmer, gen. nov. Taxon. 10: 54-60. Utrecht Palmer, J.T. 1963. Deutsche and andere Arten der Gattung My co cali a. - 1968. A chronological catalogue of the literature to the British Gasteromycetes. Nova Hedwigia, XV: 65-178 Ray, }. 1686. Historia Plantarum, i. London. Saccardo, P.A. 1888. Nidulariaceae, Sylloge Fungorum, vol. 7 et seq. Patavii Sachs, J. 1855. Morphologie des Crucibulum vulgare Tulasne. Bot. Zeit. 13: 833-845, 849-61 Schmitz, J. 1842. Morphologische Beobachtungen aïs Beitràge zur Leben und Entwicklungsgeschichte einiger Schwàmme aus der Klasse der Gastromyceten und Hymenomyceten. Linnea. 16: 141-215 Smith, A.H. 1951. Puffballs and their allies in Michigan. Ann Arbor: University of Michigan Press Sosin, P.E. 1960. De speciebus novis et curiosis Gasteromycetorum ex Oriente extremo. Not. Syst. e Sect. Crypt. Inst. Bot. Acad. Nauk SSSR, 13 : 207-14 Stevenson, A. and E.K. Cash, 1936. The new fungus names proposed by C.G. Lloyd. Bull. Lloyd Lib. 35, Myc. Ser. 8: 1-209 Tai, F.L. and C.H. Hung. 1948. Nidulariales of Yunnan. Sci. Rep. Nat. Tsing Hua Univ. 3 : 34-41 Teng, S.C. 1935. Notes on Gasteromycetes from China. Sinensia6: 701-24 Tulasne, L.R. and C. Tulasne. 1844. Recherches sur l'organisation et le mode de fructification des champignons de la tribu des Nidulariées, suivies d'un essai monographique. Ann. Sci. Nat. m(i): 41-107 - 1861-65. Selecta fungorum carpología, 3 vols. Paris.

Velenovsky, J. 1947. Novitates mycologicae novissimae, pp. 1-167, Praga Walker, L.B. 1920. Development of Cyathus fascicularis, C. striatus, and Crucibulum vulgar-e. Bot. Gaz. 70: 1-24 White, V.S. 1902. The Nidulariaceae of North America. Bull. Torr. Bot. Club. 29: 251-80 Wilkins, W.H. 1954. Investigations into the productionof bacterios tatic substances by fungi. Brit. J. Exp. Pathol. 35: 28-31

192 / Selected bibliography

Wolf, F.A. 1949. Notes on Venezuelan fungi. Lloydia 12 : 208-19 Woodward, Carol H. 1943. Egg-throwers of the Mushroom World. New York Bot. Gard. 44: 2 74~8 Wright, Jorge E. 1949. Contribución al catalogo de Gasteromycetes argentinos I. Lilloa2i: 191-224 Zenker, J.C. 1834. Ueber einen neuen Pilz auf Camellia japónica. Flora 17: 211-13

Glossary

Basidiomycetes A l l the higher fungi in which nu­ clear fusion and meiosis occur in a basidium, on which there generally form basidiospores borne on sterigmata Clamp Connection A n arching bridge-like connec­ tion between two adjacent cells characteristic of the dikaryotic mycelium of many basidiomycetes (figure 14, b, d) Cortex A firm bounding tissue, specifically in this book, the main wall of the peridiole (Ch. 11, c, 2). Component hyphae commonly dark-coloured and with thickened walls (figures 7c, 48) Dikaryon (adj. dikaryotic) Refers to the mycelium with cells containing two sexually compatible nuclei (figures 3, 33c) Diploid Refers, in most basidiomycetes, only to the fusion nucleus in the basidium in which the sporophytic (in) chromosome number occurs (figure 3) Dentate Toothed, with the teeth pointed and di­ rected outward Emplacement The solid rounded mass of hyphae at the lower and narrow end of the fruit body; soil is sometimes incorporated into the emplacement (Chapter 7, F) Epiphragm The membrane of hyphae covering the mouth of the fruit body when the latter is ap­ proaching maturity and, by rupturing, exposing the peridioles (Chapter 5, E) Funiculus The complex cord by means of which peridioles of some Nidulariaceae are attached to the fruit body (Chapter 7, B) Funicular Cord That part of the funiculus which is specifically a cord or cable composed of strong spirally twisted hyphae (Chapter 7, B, 4)

193 / Glossary

Gasteromycetes Those basidiomycetes whose spores are passively released from their basidia within closed fruit bodies, rather than forcibly discharged in the open Gleba The inner, fertile portion of the fruit body of the Gasteromycetes Glycerine Jelly A mixture of glycerine and gelatine in water, often including a preservative, used for making a semi-permanent mounting for a micro­ scopic object Hair In the Nidulariaceae, aggregated hyphae on the outside of the peridium, often clustered to form downward-pointing tufts Haploid The gametophytic (in) chromosome number of all nuclei in the Nidulariaceae except the fusion nucleus in the basidium Hapteron The adhesive free end of the funicular cord in Cyathus (Chapter 7, B, 4) Heterokaryon (adj. heterokaryotic) A mycelium whose cells contain genetically different nuclei. In almost all Nidulariaceae, the fruiting mycelium is a heterokaryotic dikaryon. In rare instances of self-fertility the fruiting mycelium is normally a homokaryotic dikaryon; but such species may in­ corporate nuclei with genetic differences through hyphal fusions and exchanged nuclei. Heterozygous The situation in which the two members of a pair of genes located on homologous chromosomes and influencing a given trait are not identical (cf. homozygous) Homokaryon A mycelium whose nuclei are all genetically alike. In most self-sterile basidiomy­ cetes, the mycelium derived from a single basidiospore is a homokaryotic monokaryon; but in some Nidulariaceae the cells of such mycelia

contain one to several identical nuclei ; it is preferable to refer to such mycelia simply as homokaryons or homokaryotic mycelia. Homozygous The situation in which the two members of a pair of genes located on homologous chromosomes and influencing a given trait are identical Eypha (pi. hyphae) The slender filaments from which the body of a fungus is built up Infundibuliform Resembling a funnel; obconical Lip Extreme rim of peridium Middle Piece That part of the funiculus (q. v.) which attaches the sheath, or basal piece, to the purse (q. v.). A cord about 0.4 mm wide and i mm long (Chapter 7, B, 2) Monokaryon A mycelium with one nucleus per cell. In most self-sterile basidiomycetes, used in reference to the mycelium derived from a single spore. Because of the inconstant number of nuclei per cell in some Nidulariaceae, it is preferable to avoid this term and to refer simply to the homokaryotic mycelium (cf. homokaryon). Mycelium The aggregate of hyphae that make up a fungus colony Pendióle The lens-shaped dissemination unit of the Nidulariaceae, externally bounded by a wall of greater or less complexity. Each peridiole contains within it, the sexually produced basidiospores. Peridium With reference to the Nidulariaceae, the cup-shaped structure that holds the pendióles; strictly, the outer bounding wall of the fruit body (Chapter i, A; Ch. vu, C-E) pH (potens Hydrogenis) A measure of acidity or alkalinity, using a numerical scale that represents the log reciprocal of the hydrogen ion concentration in gram molecules/litre; pH/ is neutral, lower figures increasingly acid and higher ones increasingly alkaline Plicate Folded, as of the peridium of various Cyathus spp. (zigzag in cross-section) Prim ordium The first formed part or rudiment of an organ

194 / Glossary

Pseudoparenchyma A tissue composed of mycelium whose cells are so compacted that superficially they resemble the nearly isodiametric cells of parenchyma in higher plants Purse The uppermost part of the funiculus ; a sac or envelope composed of loosely woven hyphae (Chapter 7, B, 3) Sclerotic Having thickened and/or hardened walls Seta (pi. setae] A rigid slender bristle; in the Nidulariaceae, specifically the bristles composed of compacted hyphae at the mouth of the peridium in some species (Chapter 11, B, 6) Sheath (or basal piece) That part of the funiculus of Cyathus that is attached to the peridium (Chapter 7/ B' ï) Splash-Cup A cup-shaped structure with a flaring rim, from which disseminating units are thrown by the energy of falling water drops, e.g. the peridium of a bird's nest fungus Sporocarp A general term for a fungus fruit body of definite form. The term basidiocarp is sometimes used for that of a basidiomycete. Striâte Ridged, scored, or with lines. Sometimes used (inexactly) to describe a plicate peridium Sulcate Grooved, often broadly and irregularly. In older descriptions, sometimes used to describe a plicate peridium. Should be avoided because the inference is that grooves are cut in one face of the peridium Tomentose Thickly and evenly covered with somewhat appressed or matted hairs or hyphae Trama The fungal tissue composing the pileus or bearing the hymenium or fertile layer (Chapter 10) Tunica The outermost covering layer of the peridiole. Conspicuous in Crucibulum and many species of Cyathus and consisting usually of a layer of interwoven hyphae whose walls are scarcely or not at all thickened or coloured (cf. cortex)

Index

(For individual species, the principal taxonomic reference of several is in bold face. An asterisk * indicates reference to an illustration.)

Agrobacter tumifaciens : sensitivity to cyathin 120 Allbutt, A.D. et al. : cyathin 120 antimicrobial nature of cyathin 120 aphrodisiac: possible action 119 basal piece 6, 80, 81* basidia: form 23, 25"; metamorphosed 25 basidiospores: 24*, 25*, 27*; nuclei in 10, number 25 ; separation from basidia 25 ; wall 23 ; 'notch' in spores 23; longevity 114; - germination: early studies 16; difficulty 26; development and maturation 25 ; technique 27 bird's nest fungi: English names 3, 183 ; ingestion by humans 119 bolas 88 Brodie Medium 36 Buller, A.H.R. v; early study of splash dispersal 18 ; quotation from notes by 18, 19, 182 calcium: effect on fruiting 47, 116 chromosomes: first counts 21 Clusius, C. : earliest reference to bird's nest fungi 14, 22 colour: of fruit bodies 3, 6; as a taxonomic character 127 coprophilous species 101,102 cords, mycelial 10, 52, 53* cortex 130*, 131 Crucibulum : funiculus 85*, 86,147; splash dispersal 89, 96; genus 147; key 148 195 / Index

-

(albosaccum) 149,155 cyathiforme 146*, 149* (emodense) 144 laeve : 146*, 148; basidiospore, curved 23 ; basidiospore failure 25 ; mycelial cords 52 ; twinning 58 ; regeneration 58; peridium layers 69 ; splash experiments 96 ; on corn stems 105 ; on driftwood 107 - parvulum 146*, 149 - (vulgare) 148 Cunningham, G.H.: New Zealand Nidulariaceae 16 (Cyathia = Cyathus} 150 cyathin 120 Cyathus \ genus 150; groups 150;key 154; species and synonyms 151 - (affinis) =stercoreus (q.v. 168) - africanus 158*, 159; apiculus 23;indolics 121 — (ambiguus) — poeppigii 171 - anglicus : olla forma anglicus 152*, 155 - annulatus 174*, 179; isolation in Cypress Hills "7 - (atrofuscus) = Crucibulum laeve 148,181 - badius 160 - (baileyi) =stercoreus (q.v. 168) berkeleyanus 174*, 178; fruiting 48; on bamboo pots 106*, 107 ; on saline substrate 112 - (boninensis) = stercoreus 168 - (brazlaviensis) = stercoreus (q.v. 168) - bulleri 174*, 179; fruiting 48, 5o* - (byssisedus) — montagnei (q.v. 176) - canna 158* - cheliensis 173 - chevalieri 177 - colensoi 156

-

(complanatus) = olla (q.v. 154) confusus 160 costatus 172 crassimurus 165*, 167 crispus 181 (dasypus) = olla (q.v. 154) (deformis) = Crucibulum laeve 148 (desertorum) = colensoi 156 (dimorphus) =stercoreus (q.v. 168) (dura) = olla (q.v. 154) earlei 153*, 156 (elegans) =stercoreus (q.v. 168) ellipsoideus 181 (emodensis) = Nidula(q.v. 144) elmeri 167 (fascicularis) — olla (q.v. 154) fimicola 169 gayanus 170*,172 gracilis 164, 165* (grisews) =striatus (q.v. 173) helenae 174* ,175 ¿nutrition 36; antibiotics 37, 120 ; mating with Cyathus striatus 43 ; alpine habitat 107 - (hirsuta) = striatus (q.v. 173) - hookeri 159 - intermedius 165*, 166; type specimen 165 - julietae 161,162* - (laevis) = Crucibulum laeve (q.v. 148) - [lentífera] = olla (q.v. 154) - (lesueurii) = stercoreus (q.v. 168) - (leveillanus) = montagnei (q.v. 176) - limbatus 170*, 172; fruiting 48; aberrant 61; eaten by man 119 - (melanospermus) = stercoreus (q.v. 168) - microsporus 158* ; small spores 23 ; eaten by man 119 - minimus 157 - (minutosporus) 180 - montagnei 174*, 176; on jute sacks 106*, 107 - nigro-albus 176 - (niveo-tomentosa) =Nidula(q.v. 142) - novae-zeelandiae 176 -olla 152*, 154; variation 42, 63 ; development and structure 65 ; swelling of basidiospores 29 ; germlings 29 ; hybridization with forma anglicus 42*; fruiting 48*, 49*,54*; morphogenesis 65 ; nuclei 76 ; funiculus 86 ; splash experiments 95 ; xeric habitat 109 ; taxonomy 154 ; forma anglicus : hybridization with C. olla 42 ; distribution 111 ; taxonomy 155

196 / Index

- olivaceo-brunneus 173 - pallidus 161,162* ; fruiting 48, 49* ; pendióles on Camellia 18,111 ; type specimen 162* - (pezizoides) — Crucibulum laeve (q.v. 148) - pictus 169* - (plicatus) = poeppigii (q.v. 171) - (plicatulus) =poeppigii (q.v. 171) -poeppigii 170*, 171; large spores 23, fruiting 48 ; aberrant fruit bodies 60*, 61 ; funiculus 86; plication 89,170* - (puiggarii) = stercoreus (q.v. 168) - pullus 179 - pygmaeus 153*, 157; size 128 - rwc//s 177 - (rufipes) = stercoreus (q.v. 168) - (rugispermus) — stercoreus (q.v. 168) - (schweinitzii) = striatus (q.v. 173) - (scutellaris) = Crucibulum laeve (q.v. 148) - setosus 164; setae 89; 163* - (similis) = olla (q.v. 154) - sinensis 164 - (sphaerosporus) = pallidus (q.v. 161) - stercoreus 168,169*; genetic variation 9,12, 21, 62, 63 ; sexuality 20, 39; paucity of spores 25 ; germlings 24*, 29 ; germination temperature 28; nutrition 36, 37; fruiting 46, 47,48, 51*,55*; twinning 58;nursehyphae 26; nuclei 72-9 ; entangled cord 85 *; splash experiments 96 ; on manured soil 102 ; on coco mat 107,108* ; and Fusarium 113 ; abortion of funiculus 168; taxonomy 168 - striatus 4 ",5 *,173,174*; as a splash cup 80-92; development and structure 63 ; spore notch 23 ; matings with Cyathus helenae 43; morphogenesis 63 ; hollow stipe 66 ; emplacement 68; funiculus 67, 80, 81*, 85*; plication 4*, 89 ; splash experiments 97 ; habitat 101,107; parasitized by Hypocrea 113*; antibiotics i2o;indolics 121; taxonomy 173 - (subiculosus) = stercoreus (q.v. 168) - (sulcatus) = poeppigii (q.v. 171) - triplex 163*; spore notch 23 - (umbrinus) = olla (q.v. 154) - (vernicosus) = olla (q.v. 154) - (wrightii) — stercoreus (q.v. 168) Diehl, Wm. : proof that Leptostroma is a Cyathus 18 dikaryotization: regular 38; unilateral 41,42* diploidization: see dikaryotization; see also mycelium (dikaryon)

dispersal: by rain 7; early concepts 15; early work 17; funiculus and peridiole as a bolas 88; experimental proof of rain dispersal 93; without rain 114 - see also distribution and splash dispersal dissection (of fruit body) 83 * distribution, long-range 109; by herbivores no; by agricultural practices in; as seed contaminants 111; by rafting on wood in; by small animals 112; geographical 116 diterpenoids 121 Dodge, B.O., on peridiole dispersal 19 ecology 101-18; endemic species 117 emplacement 90*; development of 68 endemism 117 epiphragm 4*, 6, 54, 55*; pressure beneath 56*; development and rupture 54 fibres (plant), Nidulariaceae on 105 Fries, N., sexuality in Nidulariaceae 20 fruit body: structure (general) 4; colour 6; as splash mechanism 7; early studies of 14; variability 9,12, 61*, 62*, 63 ; developmental stages 63, 64*, 65*; tropisms 57, 58*; twinning 59*, regeneration 59*; uniperidiolar fruit bodies 60*, 61; growth regions 69; morphological differences among genera 69; form related to function 80; angle of sides 88; position related to function 90; phylogenetic series by function 92; abundance 107; longevity 114; intermittent growth 115; cupulate, other than bird's nest fungi 123,124*; size as published 127 fruiting: time required 48; efforts to achieve 46—52; effect of light 47; effect of temperature 48; species fruited 48; on soil 48*, 49*, 50*; effect of substrate surface 51; mycelial cords as precursors 52, 53*; mycelial knots 52; effect of calcium 46*, 47 funicular cord: 80, 81 * ; origin of 67; structure and function 80, 81*; technique for dissection 82, 83*; explosive swelling 83 ; length and tensile strength 84; modified clamps in 81*, 83 ; time and place of extension 87 - see also funiculus funiculus: parts of 80-6, 81*; in different genera 86; formation of 67; function and structure 86; action as bolas 88 Fusarium, on Cyathus 113 gelatinization:ofbasidia 25*, 26; of peridiole tissue

67

197 / Index

germlings (sporelings): types of 29; early death 31 glossary 193 (Granularia) 134,137; castanea 138; denudata 138; duriaeana 138 habitat: sites for finding Nidulariaceae 101-9; dung 101; wood 102; dead herbaceous stems 103; leaves 105; fibres 105 ; fruits 104*, 105 ; soils 105; xeric 109; abundance of fruit bodies 105; saline substrate 112; association with puffballs 112; unusual sites 114 hapteron 5*, 9, 81*, 83 ; operation of 87; structure 83; adhesiveness 84 herbivores: and dispersal no hybridization 42^,43 hymenium 26*,65*,67 hyphal elements (of peridium) 135* Hypocrea latizonata, parasite on Cyathus 113* indolics 121 insects: Brefeld's incorrect view 15; and oidia 35 Johri, B.N., on cyathin 121 Johri's Medium 36 lichens, cup structures of 123 life cycle, diagram of 8* light, effect on fruiting 47, 48, 57, 58* Lloyd, C.G., monograph 7,16 Lu, B.C., on chromosomes 74*, 75*, 76, 77* lysis (of young mycelia) 31 Martin, G. W., rain splash dispersal 17; nurse hyphae 17, 26 mating systems 37-41; mating type alleles 39; distribution (numerical) of mating types 41 metabolites: cyathin 120; indolics 121 middle piece 5*, 81*, 82 Miles, P.: on nuclei 72, 73* mycelial knots 10 mycelium - monokaryon (= homokaryotic): sporelings 24*, 29; septation 31; external appearance 32, 33*, 34*; pigments 32; blotchy 32; microscopic aspects 35; nuclear distribution 35; oidia 30*, 35 ; growth rate 35; growth requirements 36; interactions among mycelia 37-41; self sterility 40 - dikaryon (= heterokaryotic): formation of 44-6; unilateral dikaryotization 41, 42*; external appearance 34*, 44; colour 44; microscopic

aspect 45*, 46; survival of variant cultures 63 ; longevity no Mycocalia : fruiting 51; sexuality 39; relation with soil acidity 114; compared with Nidularia 133, 135*;genus 137;key 137;denudata 138 - (arundinacea), as Nidularia 138 - duriaeana 138 - minutissima 138 - (fusispora) as Nidularia 138 - reticulata 139 - sphagneti 140 New Zealand, Nidulariaceae of 16 Nidula 30*, 142; germination of spores 30*, 31; sexuality 39; twinning 58, 59*; regeneration 58, 59*; morphogenesis 70; splash experiments 98; isolated occurrence 109; and bracken 112; genus 142;key 142 - Candida 144,145* - emodensis 144,145* - macrocarpa 145* - (microcarpa) — niveo-tomentosa (q.v. 143) - (microcarpa varrugispora) ---niveo-tomentosa (q.v. 143) - niveo-tomentosa 142,143*, 145* Nidularia: peridiole wall 69; splash dispersal 88; compared with Mycocalia 133,135*, genus 133 - (australis) = farcta (q.v. 136) - bonariensis 141 - campor 141 - (candida) = Nidula 144 - (castanea) = Mycocalia duriaeana (q.v. 138) - (confluens) = farcta (q.v. 136) - (duriaeana) = Mycocalia duriaeana (q.v. 138) - farcta 136* - (globosa) = farcta (q.v. 136) - (heribaudii) = Cyathus olla (q.v. 154) - (intermedia) — Cyathus intermedius (q.v. 166) - (minutissima) — My cocalia minutissima (q.v.

i38)

- (microspora) = farcta (q.v. 136) - (pisiformis) = farcta (q.v. 136) - (plicata) = Cyathus poeppigii (q.v. 171) - pulvinata 134,136*; fruiting 51 - (reticulata) = Mycocalia reticulata (q.v. 139) - (striata) — Cyathus limbatus (q.v. 172) Nidulariaceae: family 3; genera of 3; key to genera 6; number of species in various genera 7; monographs 7,16; lists by country 21 Nidulariales, order 3 nuclei: early studies 71; in Nidularia farcta 71,

198 / Index

72; phase microscopy 72, 73*; dikaryon behaviour 72, 73; mitochondria, 73*, 75; staining with propionocarmine 76; chromosome numbers 76; chromosome morphology 76,77; meiosis in basidia 76; centrioles and spindles 75 *, 76; mitosis in mycelium 78; nucleoli 73 *, 77*-78 nursehyphae 24*, 26 nutrition (see mycelium) nuts, as substrate 104* oidia 30*, 35 Olchowecki, A.: on cyathin 120 palisade 67 Palmer, J.T. 21, 25, 51,102,114,132,133 parasites (of Nidulariaceae) 112,113* peridiole: erroneous concepts about 14-16; section 26*; numbers of spores in 25; numbers of 63 ; development 66, 67; weight 84; arrangement 91; adhesive coating 91; germination as a whole 85*, no peridium 4, 5* ; development 66, 67; layers of 69; elastiticy of wall 88; outer covering 128 phototropism (of fruit bodies) 57, 58* phylogeny 92, 123 plication 4*, 89, 90* purse 5*,81*, 82, 84 raindrops: size 94; velocity of fall 94 regeneration 58, 59* sacks, as substrate 106* setae 89; in Cyathus setosus 89,163*; function 90 sexuality: patterns, general 8*, 37-43 ; early studies 20; modern studies 37-43; mating systems 39; unequal distribution of sex types 40, 4* sheath (see basal piece) Sphaerobolus 3,15 splash cups: of bird's nest fungi, compared 92; in other plants 122; splash corona (photo) 89* splash dispersal 11*; in Nidularia and Crucibulum 88; angle of splash cup sides 88, 89; experimental proof 94, 99*; observation in nature 93, 94; raindrops, diameter and velocity 94; observation during experiments 95-100; maximum horizontal distance 98; in other plants 122 sporelings (see germlings) sporocarp (see fruit body)

sterigmata 25*; inNidularia and Crucibulum 25; sessile spores in Cyathus 25 taxonomy: characters used, review 126; macroscopic characters 127; microscopic characters ^ , , s T, temperature (and growth) 35-6 , ( . i T x tropisms (see fruit body) 0 1 1 , * ss tomentum i28;nodular 151,159,165 , 166, 175 _t / ji T _, Tulasne, L.R. 7,13,14,16; concepts re dispersal 15; quotation from monograph 182 tunica 65*, 67, 81*, 129; difficulty of determining 129,130*, 131; use of term 129

199 / Index

twinning 58,59* unilateral diploidization (dikaryotization) 41, 42* Vandendries, R. 184* Walker, L.B. Structure and development of , ,„ . , , , , * ,* n Cyathus and Crucibulum 63-70,64 , 65 7 J x ^7 „ , ^ J TATl r White, V.R. 16,22,142 , XT.j , . ^ wood: Nidulanaceae on 102 xeric habitat 109 zinc: possible nutritional role 37

This book was designed by ANTJE LINGNER

under the direction of ALLAN FLEMING

and was printed by University of Toronto Press