A World-Monograph of the genus Pseudombrophila 3930167107, 9783930167104

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LIBRI BOTANICI Vo!. 14

A World-monograph of the Genus PSEUDOMBROPHILA

(Pezizales, Ascomycotina)

by

J. van Brummelen

With 33 text-figures 8 coloured plates and 17 monochrome plates

IHW-VERLAG 1995

L1BRI BOTANICI Vcl. 14

A World-monograph of the Genus Pseudombrophila (Pezizales, Ascomycotina)

LIBRI BOTANICI Vol. 14

A World-monograph of the Genus PSEUDOMBROPHILA

(Pezizales, Ascomycotina)

by

J. van Brummelen

With 33 text-figures 8 coloured plates and 17 monochrome plates

IHW-VERLAG 1995

Die Deutsche Bibliothek - CIP-Einheitsaufnahme Brummelen, Joop van: A World-monograph of the Genus Pseudombrophila (Pezizales, Ascomycotina) / by J. van Brummelen. - Eching bei Miinchen : IHW-Verl., 1995 (Libri Botanici ; Vo!. 14) ISBN 3-930167-10-7 NE:GT

Impressum:

ISBN 3-930167-10-7 Author:

Dr. J. van Brummelen Rijksherbarium - Hortus Botanicus P.O. Box 9514 NL - 2300 RA Leiden

Production:

Berchtesgadener Anzeiger Griesstatter Str. 1 D - 83471 Berchtesgaden

Publication:

IHW-Verlag Postfach 1119 D - 85378 Eching bei Miinchen Telefax:

nat. 089-3192257 internat. +49-89-3192257

© 1995

All rights reserved. No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publisher.

CONTENTS SUMMARY

5

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 GENERAL PART History

6 6

Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Structure of the fungus TAXONOMIC PART

10 18

The genus Pseudombrophila . . . . . . . . . . . . . . . . . . . . . . . . 18 Key to the sections of Pseudombrophila

19

Section Pseudombrophila

20

Key to the species of section Pseudombrophila

20

Section Nannfeldtiella

65

Key to the species of Nannfeldtiella

65

APPENDIX. Insufficiently known and excluded species

83

REFERENCES

89

INDEX

93

COLOURED PLATES

97

MONOCHROME PLATES

101

3

SUMMARY The genus Pseudombrophila (Pyronemataceae, Pezizales) is described and discussed. The genus is divided into two sections: Pseudombrophila and Nannfeldtiella. Nineteen species are distinguished in section Pseudombrophila and nine in section Nannfeldtiella, on a world basis. Four species have been newly described and seventeen new combinations were necessary under Pseudombrophila. The names of excluded and insufficiently known taxa are discussed in an appendix. Svrcekomyces pallidus is redescribed and referred to the genus Leucoscypha.

INTRODUCTION The genus Pseudombrophila Boud. (Pyronemataceae, Pezizales) is the subject of a worldwide revision. Many species previously described in other genera were recognized as belonging to Pseudombrophila, while a number of new species have been described through the years. Others were described several times because of the absence of a modern treatment and insufficient knowledge of specific variability. Several species were newly described in the genus Fimaria Velen. since attention was paid to this forgotten name (VAN BRUMMELEN, 1962). In the present study Fimaria is included in Pseudombrophila. Since most published descriptions proved to be insufficient to recognize the species, a study of authentic material was necessary to make full descriptions and illustrations of the species. As far as possible this study is based on living material. But, as many species are extremely rare or restricted to unusual habitats, the major part of the material studied consisted of dried specimens. Studies of type-specimens have been undertaken and included whenever possible. In cases where type specimens were obviously absent, published and unpublished illustrations and descriptions have sometimes helped in recognizing species. Several specific names are only known from short, insufficient descriptions. These names are included in the list of insufficiently known species. However, a few of the oldest of such names have consistently been interpreted in the same way as a result of the existence of exsiccatae distributed under this name by early mycologists, like K. W. G. L. Fuckel, G. L. Rabenhorst, P. A. Karsten, and M. C. Cooke. In such cases illustrative specimens or epitypes (GREUTER, 1994: Art. 9.7) have been indicated to stabilize the use of these names. The purpose of this investigation is to evaluate the morphological variation among the species of Pseudombrophila and produce a taxonomic treatment. To accomplish these goals a number of different techniques have been used, including the study of macro-morphology, pigmentation, differentiation of hairs, ascoma ontogeny, ascus and ascospore ultrastructure (VAN BRUMMELEN, 1986a, 1994), and development and structure of ascospore ornamentation.

ACKNOWLEDGEMENTS The author wishes to thank the directors and curators of the institutes mentioned on p. 7-8 for the loan of material or for permission to examine specimens in their care. Valuable material has also been gratefully received from Dr. O. Aas, Mr. J. Astier, Mr. A. Ayel, Mr. B. Hannf, Mr. J. Breitenbach, Prof. F. D. Calonge, Dr. H. Dissing, the late Dr. J.-c. Donadini, Mr. H. Engel, Mr. G. Fortoul, the late Mr. W. D. Graddon, Prof. G. S. de Hoog, Mr. R. Kristiansen, Mr. W. D. 1. Kuijs, Prof. N. Lundqvist, Mr. W. Matheis, Dr. E.

5

Ohenoja, Mr. J. T. Palmer, Mr. H. & Mrs G. Piepenbroek-Groters, Dr. A. Raitviir, Mr. L. Riousset, Dr. S. Sivertsen, Mr. G. J. M. Verkley, and Prof. J. Webster. Warmest thanks are also extended to Ms Dr. S. M. Francis for reading the manuscript and improving the English text; to Dr. R. A. Maas Geesteranus for correction of the Latin diagnoses of the new taxa; and to Prof. T. Schumacher for permission to reproduce his colour-slide of Pseudombrophila guldeniae. The 'Netherlands Organization for the Advancement of Research (N.W.O.)' is gratefully acknowledged for a grant which financed a visit to the herbaria at Uppsala and Stockholm. The author is also most grateful to the 'French National Scientific Research Center (C.N.R.S.)' for a grant to visit French herbaria. GENERAL PART HISTORY

The genus Pseudombrophila was first proposed by BOUDIER (1885) for three species: Helotium pedrottii Bres., Peziza chartarum Quel., and Peziza pluvialis Cooke. He placed it in his new family 'Ciliaries' with the following diagnosis: 'Poils courts et obtus; spores sans sporidioles; couleur pale; especes subfimicoles, stipitees ou obconique'. ROLlAND (1888) included Pseudombrophila theioleuca from France. From BOUDIER's (1907) presentation of his new classification of the discomycetes it became clear that he had a rather narrow conception of the genus Pseudombrophila. In his description he stresses the presence of receptacles with an outer decoration of fascicles or isolated bunches of coloured, septate and blunt hairs of the type found in Melastiza Boud. and Anthracobia Boud. In this enumeration only six species of Pseudombrophila were recognized, while most species now belonging to Pseudombrophila, with less conspicuous bunches of submarginal hairs, were included in his large heterogenous genus Ascophanus Boud. Before the establishment of Pseudombrophila its species were mostly arranged under Peziza Dill. ex L.: Fr. in the works of Batsch, Fries, Sauter, Berkeley, Nylander, Karsten, Cooke, Schroter, E. C. Hansen, and Phillips. Later, after the publication of new classifications by FUCKEL (1870) and SACCARDO (1884, 1889, 1895), these species were also classified under Humaria Fuckel (Fuckel, Saccardo, Quelet, Rehm, and Velenovsky), Pyronema Carus (Saccardo, Spegazzini, Rehm, and Bisby), Lachnea (Fr.) Gill. (Saccardo and Velenovsky), and Neottiella (Cooke) Sacc. (Saccardo and Boudier); while species with spherical ascospores were listed under Cubonia Sacc. (Saccardo, Boudier, Grelet, Faurel & Schotter, and Durand). New species of Pseudombrophila were described by LE GAL (1937), SVRCEK (1966, 1988), and HARMAJA (1979, 1986). VELENOVSKY (1934) described the new genus Fimaria Velen. with four species and a variety. He segregated it from the large genus Humaria (Fr.) Boud. sensu Rehm. He described the genus Fimaria as: 'Genus, omnino generi Humaria affine, sed apoth. margine late membra-naceo-limbata, pierumque in cilias acutas fimbriata, paraph. simpl.-filif. vel ramosae. In fimis, raro in limis.'

Fimaria was emended by VAN BRUMMELEN (1962) by lectotypification. The genus was redescribed and four species were distinguished. Later, several other species of Fimaria were added by SVRCEK & KUBICKA (1965), ECKBLAD (1968), SVRCEK & MORAVEC (1969), JENG & KRUG (1977), TORRE & CALONGE (1978), GRADDON (1980), and CALONGE (1984). In the present treatment Fimaria is recognized as belonging to the genus Pseudombrophila. ECKBLAD (1968) introduced the genus Nannfeldtiella Eckbl. for a species, N. aggregata Eckbl., associated with the subiculum of Byssonectria at places where elk is living. Because

6

the ascospore ornamentation was of a non callose-pectic nature Nannfeldtiella was incorporated in the family Sarcoscyphaceae Le Gal ex Eckbl. Further study of this fungus by KORF (1972) revealed that its ascospore markings are of a callose-pectic nature (,cyanophilie') and not typical for the Sarcoscyphaceae. HARMAJA (1979) transferred it to Pseudombrophila and described some other closely related species from the same habitat. MORAVEC (1976) introduced the new generic name Svrcekomyces for Pseudombrophila guldeniae Svrcek, which he considered 'rather similar' or possibly 'even identical' with Nannfeldtiella aggregata. PFISTER (1984) compiled a number of coprophilous species with spherical ascospores from the synonymy of Ascophanus dentatus Boud. and provided a key to species of Fimaria. VAN BRUMMELEN (1986a) studied the structure and development of the ascus top and the ascospore wall in two species of Pseudombrophila and two species of Fimaria with light and electron microscopy. Ascus tops were found with roughly delimited ascostomes and opercula. The general structure and the dehiscence mechanism of the ascus are very similar and the same as those found in genera of the Pyronemataceae. The ascospore ornamentation, although sometimes very different at maturity under the light microscope, develops initially in the same way from a smooth electron dense, blue staining ('cyanophilous') secondary wall. In the smooth-spored species the initial layer is permanent. In the rough-spored ones it usually breaks up to form an irregular granular pattern. In an additional study of Nannfeldtiella aggregata, which shows mature ascospores with a rather conspicuous, incomplete net-work of thick and fine lines, VAN BRUMMELEN (1994) found a similar initial development of the secondary ascospore wall. But on further ripening the investing membrane is lifted, creating large spaces only filled with a few widely spaced fibrillar elements. At maturity the secondary wal1 becomes densely filled with fibrillar elements constituting the final pattern of ornamentation. MATERIALS AND METHODS

Whenever possible observations were made on living material, but the major part of the material consisted of dried specimens. LIVING MATERIAL. - - Since most species of Pseudombrophila are coprophilous, a great number of samples of dung of different origin was col1ected and incubated to isolate fruit bodies of fungi growing on them. The methods for isolation and cultivation were essential1y the same as used before (VAN BRUMMELEN, 1967). In general fruit bodies of Pseudombrophila appear rather late in the sequence of species fructifying on dung. The optimal temperature for developing fruit bodies of Pseudombrophila is between 10 and 15°C. This may explain the fact that they are less frequent in cultures at room temperature. Although fruit bodies of Pseudombrophila can be found in nature throughout the year, they are more frequent in spring and in mild winters. HERBARIUM MATERlAL. ~ Most of the material studied in this work consisted of dried specimens. Herbaria from which specimens, including type material, were examined, are indicated in the text by the fol1owing abbreviations. Institutes visited personal1y have been marked with an asterisk (*). B BG BPI BR C

Botanischer Garten und Museum Berlin-Dahlem, Berlin, Germany *. Universitets Botaniske Museum, Bergen, Norway. The National Fungus Col1ection, U.S. Department of Agriculture, Beltsville, Maryland, U.S.A. *. Jardin Botanique National de Belgique, Meisse, Belgium *. Botanical Museum and Herbarium, Copenhagen, Denmark *.

7

CONC DAOM E FRS G H HBG K KUO L

LPS M MA MPU NY NYS

o

OULU PAD PC PRM S TRH TRTC TU

TUR UPS W WU

Laboratoire de Biologie Marine du College de France, Concarneau, France *. Mycological Herbarium, Plant Research Institute, Department of Agriculture, Ottawa, Canada. Royal Botanic Garden, Herbarium, Edinburgh, Great Britain *. Falconers' Museum, Forres, Great Britain *. Conservatoire et Jardin Botanique, Geneve, Switzerland *. Botanical Museum, University of Helsinki, Helsinki, Finland. Staatsinstitut fUr Allgemeine Botanik, Hamburg, Germany. Royal Botanic Gardens, The Herbarium, Kew, Great Britain *. Department of Natural History, Kuopio Museum, Kuopio, Finland. Rijksherbarium - Hortus Botanicus, Rijksuniversiteit, Leiden, The Netherlands*. Inclusive of the mycological collections formerly placed in the herbaria of Groningen (GRO) and Utrecht (U). Instituto de Botanica C. Spegazzini, La Plata, Argentina. Botanische Staatssammlung, Miinchen, Germany. Instituto 'Antonio Jose Cavanilles', Madrid, Spain. Insitut de Botanique, Universite de Montpellier, Montpellier, France *. Herbarium of the New York Botanical Garden, Bronx, New York, U.S.A. * Herbarium of the New York State Museum, Albany, New York, U.S.A. Botanisk Museum, Oslo, Norway *. Department of Botany, University of Oulu, Oulu, Finland. Instituto Orto Botanico dell'Universita, Padova, Italy *. Museum National d'Histoire Naturelle, Laboratoire de Cryptogamie, Paris, France *. Mycological Department of the National Museum, Praha, Czechia. Naturhistoriska Riksmuseet, Section for Botany, Stockholm, Sweden * Botanical Department, Museum of the Royal Norwegian Society for Science and Letters, Trondheim, Norway. Cryptogamic Herbarium, Department of Botany, University of Toronto, Canada. Herbarium of the Department of Taxonomy and Geobotany of the Tartu State University, Tartu, Estonia. Botanical Institute of the University, Turku, Finland. Institute of Systematic Botany, University of Uppsala, Uppsala, Sweden *. Naturhistorisches Museum, Wien, Austria *. Botanisches Institut und Botanischer Garten der Universitat Wien, Austria *.

For a more accurate indication of herbarium specimens, especially where labelling is not wholly adequate, the customary abbreviation of the herbarium name is followed by the author's revision number. STUDY OF HERBARIUM MATERIAL. Reasonable swelling of this dried material was usually obtained by leaving a fragment for two or more hours in a small tube of water. But rehydration and swelling were better when a few drops of a wetting agent, like Photo-Flo (Kodak) or Invadine (Geigy) were added to 100 ml of water. Repeated warming (to about 70 QC) hastens the rehydration. In difficult cases, with thin-walled tissues or old material, the addition of a small amount of ammonia (up to 1 %) gave the best results. Moreover the alkalinity, produced by the addition of ammonia, made the material far less brittle during later handling. Too fast or insufficient rehydration, which may easily occur in mounting media with a low water content, like lactic acid, lacto-phenol, or Melzer's reagent, leads to poor preparations with all kinds of artefacts, such as deformed asci, collapsed paraphyses, and spores with airbubbles. MICROSCOPIC EXAMINATION. - - For microscopic examination small fragments of living fruit bodies were placed in a drop of tap water on a slide and the elements then spread

8

under gentle pressure. Vital observations could only be made during a few minutes, because of the quick disintegration of cell components. As vital stains, brilliant cresyl blue (Colour Index 51010; Rowe, 1956-58; Lillie, 1969), neutral red (Cl. No. 50040), and trypan blue (Cl. No. 23850) were used in very weak solutions (under 0.01 %) in tap water. Suitable objects were also studied with interferencecontrast and phase-contrast optics. Whenever possible spore measurements were made in water on ascospores that had just been forcibly discharged from mature living asci. To obtain a higher accuracy and better reproducibility of visual size measurements of ascospores the sheared-image measurement, as described by DYSON (1960), was constantly used. This method avoids the considerable measuring deviations due to the use of oilimmersion objectives with high apertures, the so-called aperture error (LOCQUlN, 1956, 1984; CHARMAN, 1963) or the hypercentric perspective (MICHEL, 1964). The latter shows the equatorial plane of spores smaller than more distant planes. Fully rehydrated herbarium material was used as fresh material for further microscopic study and the preparation of microtome sections. Microtome sections, IQ-15 ,um thick, were made with a freezing microtome from material embedded in polyvinyl lacto-phenol (Gurr) as previously described (VAN BRUMMELEN, 1967). Ascospore ornamentations and hyphal walls in sections and squash mounts were stained in a 0.1---0.5 % solution of methyl blue (Cl. No. 42780) or trypan blue (Cl. No. 23850) in lactic acid or lacto-phenoI. Methyl blue was used as a dye for staining the ornamentation of ascospores in discomycetes by the late Dr. M. Le Gal after the production of [the synonymous] Poirrier blue C 4B ('bleu C 4B de Poirrier'; GUEGUEN, 1905) was discontinued (VAN BRUMMELEN, 1967). It is still the best microscopic stain available for ascospore ornamentation. The observation with visual microscopy of fine details of spore ornamentations stained with methyl blue or trypan blue can be improved by the use of a complementary monochromatic light filter.

In the literature there exists some confusion about the dyes for staining spores. For a long time the terms cotton blue and aniline blue have both been differently applied to mixtures of two dyes, now called methyl blue (Cl. No. 42780) and water blue I (Cl. No. 42755) (LILLIE, 1969; MATHEIS, 1975). Although it appears that both components are interchangeable with each other in most stains, it would be better to relegate the terms cotton blue and aniline blue to the history of staining, and use the proper name of the single dye substituted for the mixture in accordance with the standardized names of the Colour Index (ROWE, 1956--58; LILLIE, 1969). Thus confusion can be avoided of current with older procedures, and the nomenclature of dyes will correspond to actual practice.

In the staining of sections trypan blue gave sharper results than methyl blue, since it showed a greater affinity with certain parts of the hyphal walls and less with the cytoplasmatic contents (cf. BOEDIJN, 1956; DADE, 1960; DADE in GURR, 1965). As an iodine-containing reagent on the possible blue staining of ascus walls Lugol's solution (0.3 g 12 , 0.6 g KI, 100 ml H2 0) was used. This has the advantage, because of the relatively Iow iodine content as compared with Melzer's reagent, of a clear test without too much background staining of cellular contents by the excess of iodine (cf. BARAL, 1987). Since iodine by itself is also very reactive, its addition immediately terminates all vital observations. The most successful method of applying the iodine solution is by placing a small drop at one edge of the cover-glass of a preparation in water, while the water is sucked away at the opposite edge with a piece of blotting paper. During continuous microscopic observation all eventual changes caused by the reagent can be followed.

9

ILLUSTRATIONS. - In the text-figures the following abbreviations have been used: CORT. for detail of cortical excipulum seen from outside; DS for diagrammatic section of fruit body; MS for median section through fruit body. In the diagrammatic sections the density and extend of pigment are indicated as dotted areas, mainly in the hymenium and the cortical excipulum. All ascospores are drawn from preparations stained with methyl blue and reproduced at 1600 times. Most spores are depicted as seen in side view, a few in optical section. Under-cast letters in the line-figures refer to the origin of the material, when more than one specimen is depicted. STRUCTURE OF THE FUNGUS

Despite a considerable amount of similarity throughout the genus Pseudombrophila, many structural differences of tissues and elements can be observed between the taxa. MYCELIUM. - - In several species of Pseudombrophila mycelium is often rather abundant and clearly present at the base of the fruit bodies or as a subiculum on the surrounding substratum. The mycelium consists of hyaline, septate, cylindrical or curved, branching hyphae, about 3-5 Jim wide. The mycelium of Pseudombrophila must not be confused with the subiculum of Byssonectria terrestris on which fruit bodies of Pseudombrophila sect. Nannfeldtiella are frequently found. The subiculum of Byssonectria consists of rather stiff, thick-walled, cylindrical, hyaline hyphae of varying thickness (1(}---20 Jim wide). SCLEROTIA. - Pseudombrophila bulbifera is well known for the production in culture of bulbils or microsclerotia of 75-100 Jim diam. (HOTSON, 1912). Of a different nature are the conspicuous, large, dark brown, hairy sclerotia (5-15 mm diam.) formed on old cow dung by P. ripensis. From these sclerotia, after a period of rest, apothecia develop by breaking open of the cortex of the sclerotium. In later stages the remnants of the sclerotia can be found immersed in the substratum near the base of the fruit bodies. Such immersed sclerotia are also found in P. disciformis, but here no cortical outer layer is differentiated. ANAMORPHS. - - Very little is known about the production of anamorphs in Pseudombrophila. It is known (pers. comm. Prof. S. de Hoog, Baarn), that P. cervaria produces anamorphs which belong to Basifimbria Subram. & Lohda. APOTHECIA. - - The development of ascomata varies in some respects in different species of Pseudombrophila. The stage at which the hymenium becomes exposed to the exterior especially may differ and can explain some of the later structural variation. All species produce subglobular initial stages. In e.g. P. hepatica, P. leporum, P. cervaria, and P. theioleuca these are fully closed at first and the excipular roof over the hymenium opens in the mid-mesohymenial phase, when the hymenium is in process of ripening and ascospores are being formed in the most advanced asci (VAN BRUMMELEN, 1967: 25, p1.17). In other species, like P. laciniata and most species of Pseudombrophila sect. Nannfeldtiella (e.g. P. guldeniae and P. microtetraspora), the hymenium becomes exposed at an earlier stage, during the pro hymenial or early-mesohymenial phase. The results of the forcible disruption of the excipular roof can be observed in most species of Pseudombrophila sect. Pseudombrophila, in the later stages, by the shape of the rim of the excipular margin. When the margin is not damaged during the opening process it is visible as a complete membranaceous, raised rim or collarette, as known in species of Cheilymenia (VAN BRUMMELEN, 1986b). But in other cases during further expansion the margin of the receptacle becomes torn to form a dentate or fimbriate rim.

10

In most species growth is fairly equally distributed over the whole fruit body and restricted to intercalation of new elements and increase in size of such elements. In a few species, however, the activity of a submarginal growing zone can be observed. From the margin of the hypothecium new elements may be formed in the direction of both the hymenium and the excipulum by the activity of such a growing zone (CORNER, 1929a, 1929b). By the activity of the annular growing zone new paraphyses are formed towards the proximal side and cortical hyphae towards the distal side. Both types of elements are inserted between the outside of the hymenium and the inner side of the already existing cortex. Here they constitute a zone of radiating, relatively undisturbed hyphae, usually rather thin-walled with inflated ends. This may lead to an excipular margin which is regular and smooth towards the hymenium and rough or with disrupted cortical tissues towards the outside. Morphologically the apothecium consists of two main parts: the disc and the receptacle. The disc represents the spore-producing part of the fruit body, the hymenium, consisting of asci surrounded by paraphyses. The receptacle is the hymenium supporting structure, which consists of the excipulum and the hypothecium, a thin layer beneath the hymenium in which the ascogenous hyphae branch and form croziers. The excipulum in Pseudombrophila is usually clearly differentiated into an outer cortical excipulum, or cortex, and an inner medullary excipulum, or medulla. For the study of the different tissues median sections of fruit bodies are essential (Fig. 1). RECEPTACLE. - The shape of the receptacle is subglobular, subcylindrical, or turbinate at first becoming cupulate or scutellate later. A short stipe or a narrow base are present in several species. The colour is typically reddish or purplish brown, even in the palest species (P. virginea). In a few species the pigment is more yellowish brown or brown. The outer surface is typically tomentose or covered with appressed red-brown hairs. Some species may show small isolated tufts of dark hairs close to the margin, while P. bulbi/era and P. ripensis possess both hairs and excipular warts. The margin of the receptacle usually ends in a raised membranaceous rim that soon becomes dentate, crenulate, or fimbriate. Only P. coprina, P. disci/ormis, and P. earina show entire and smooth margins at all stages. In mature fruit bodies the margin expands and the delicate structure of the rim may stretch out and become less pronounced. DISC. - . The surface of the disc varies from concave to flat. In a few species the usually smooth surface may become roughened by the protruding tips of ripe asci. The colour of the disc is usually similar to that of the receptacle, but more intense or darker. Only in P. minuta, P. theioleuca, and P. virginea is the disc is colourless or white. HYPOTHECIUM. In most species the hypothecium is a clearly differentiated layer. But sometimes in species with very small fruit bodies it is discontinuous. It is composed of tightly entangled hyphae consisting of small plasma-rich, isodiametric or slightly oblong cells. MEDULLARY EXCIPULUM OR MEDULLA. - This is the layer that fills the interior part of the fruit body and may vary considerably in thickness. In most species it is composed of rather loosely interwoven hyphae of textura intricata, but often also subglobular elements of textura globulosa may be present. The terminology used to describe arrangements of cells in fungal tissues is the same as used before (VAN BRUMMELEN, 1967; Fig. 2). In median sections of young fruit bodies the remains of the ascogonium can sometimes be observed in the central part of this layer.

11

CORTICAL EXCIPULUM OR CORTEX. In Pseudombrophila this layer is always clearly differentiated from the underlying medulla. It usually consists of rather large thick-walled elements of textura globulosa or angularis, which constitute an outer protection of varying thickness. Only in P. fuscolilacina is the cortex extremely thin and consists of intermingled hyphae of textura intricata. The thickness of the cortex halfway between margin and base is a most constant character and useful for comparison. Through the activity of the submarginal growing zone a secondary thickening of the margin occurs and the width of the cortex may increase considerably. PIGMENTS. - The pigmentation of the fruit body forms one of the most typical characters of the genus Pseudombrophila. Pigment is always present in varying amounts in the exterior region of the cortical excipulum and in most species between the tips of paraphyses and asci in the hymenium. Only in P. minuta, P. theioleuca, and P. virginea is pigment absent from the hymenium. The typical colour of the pigment is reddish to purplish brown. Only rarely is it more yellowish brown (P. cervaria, P. bulbifera, and P. ripensis) or brown to dark brown (P. coprina, P. laciniata, P. microtetraspora, and P. minor). Microscopically the pigment is intercellular and amorphous, without crystals. It does not easily dissolve in water or any of the usual mounting media for observation. HAIRS. The occurrence of excipular hairs is one of the main characters of the genus Pseudombrophila. Hairs in discomycetes were defined by CORNER (1929a) as 'cortical hyphae with a prolonged period of rapid growth'. They need not always be strongly modified by having thickened or coloured walls. All hairs in Pseudombrophila fall in the category 'hyphoid hairs' of ECKBLAD (1968). They originate from the outermost layer of cortical cells and might by others possibly be interpreted as modified hyphal outgrowths. At least three types of hairs occur in species of Pseudombrophila. The most common type is present in all species as appressed, cylindrical, hyphoid hairs of greatly varying length, with reddish or purplish brown pigment on their walls. In most species they form a more or less dense tomentose covering of the upper part of the receptacle (MH in Fig. 1). In a few species, which includes P. merdaria, the type species of Pseudombrophila, these hairs form rather dense fascicles that may break up into isolated patches or tufts of hairs. Especially at the margin of the receptacle their length is restricted and the obtuse ends become obvious (TH in Fig. 1). This was the type of hair BOUDIER (1885, 1907) had in mind when he defined Pseudombrophila. They resemble very much the tufted hairs found in Melastiza and Anthracobia. In other species, especially those formerly arranged under Fimaria (VAN BRUMMELEN, 1962; SVRCEK & KUBICKA, 1965; ECKBLAD, 1968; SVRCEK & MORAVEC, 1969; TORRE & CALONGE, 1978; GRADDON, 1980; CALONGE, 1984), such isolated patches and tufts of submarginal hairs are usually absent, while the appressed hairs may become rather scarce or inconspicuous (MH in Fig. 1). Another type of hair is present in all representatives of Pseudombrophila sect. Nannfeldtiella and in P. petrakii. These hairs are flexuous or undulate, rather tough, branched, septate, hyaline without adhering pigment, and standing away from the cortical surface. They usually form a more or less woolly, white covering of the lower part of the receptacle (BH in Fig. 1; Pis 22b, 22d, 23c). A third type of hair is present in some species with an active submarginal growing zone, like e.g. P. laciniata, P. merdaria, P. microtetraspora, and P. minor. Here the cortical hyphae bordering the hymenium may elongate beyond the margin, while their ends inflate. They are rather thin, cylindrical, septate, hyaline, with clavate tips. Because of their delicacy they cannot be easily found in dried material (ST in Fig. 1; Fig. 3b, 3c). HYMENIUM. - The hymenium in Pseudombrophila varies considerably in thickness, from less than 100 fim to over 250 fim. Although asci increase greatly in length during ripening,

12

1

ST

/RlM

SUBI

I

2

textura prismatica

textura globulosa

textura angularis

textura intricata

textura

epidermoidea

textura oblita

textura porrecta

Fig. 1. Diagram of the construction and terminology of a sessile apothecium. HYM, hymenium; HYP, hypothecium; MED, medulla or medullary excipulum; COR, cortex or cortical excipulum; MAR, margin of receptacle; RIM, rim or extreme margin; COL, collarette; SUBS, substratum; SUBI, subiculum; ASC, ascogonium; BH, basal hairs; TH, tufted hairs; ST. secondary thickening of the cortex, with apically inflated elements; MER (as large black dot), meristematic submarginal growing zone. Fig. 2. -

Terminology for arrangement of cells in fungal tissues (from van Brummelen, 1967).

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their tips only rarely protrude for 10-20 ,urn above the level of the paraphyses. The thickness of the hymenium is measured from the bases of the asci to the ends of the paraphyses in mature hymenia. The characteristic hymenial pigment is restricted to the upper part of the hymenium. It forms an intercellular, amorphous, reddish brown or brown substance between and over the paraphyses. When seen from above, the mature hymenium may look like a dark layer perforated by the hyaline tips of ripe asci. PARAPHYSES. - The paraphyses start to develop before the asci and during development the tips become exposed. They are filiform or cylindrical, branched, septate, and hyaline. The tips of the paraphyses are not, or scarcely, thickened in most species of Pseudombrophila. But in P. misturae, P. bulbi/era, P. fuegiana, and P. hepatica the considerable enlargement of the tips forms a distinguishing character. When the tips of paraphyses are clearly swollen, often the subterminal elements may also show some swelling. Sometimes the branching of the paraphyses is very frequent in the upper part. The repeated bifurcation and the intercalation of pigment may give the paraphyses the aspect of a very regular and rigid palisade. Amorphous, reddish brown or brown pigment is present between the paraphyses in all species of Pseudombrophila, with the exception of P. minuta, P. theioleuca, and P. virginea, where the pigmentation is restricted to the outer zone of the cortical excipulum. AscI. - The asci are cylindrical with a rounded apex and are rather uniform throughout the genus. In only a few species do the mature apices extend somewhat (10-20 ,urn) above the level of the paraphyses. The length of the asci varies considerably from less than 100,um in P. equina, P. maekinenii, and P. microtetraspora to over 200,um in P. laciniata, and even up to 230-300,um in P. ripensis, forming a useful distinguishing character within the genus. No part of the ascus stains blue with iodine. The fine structure of the ascus in Pseudombrophila has been the subject of a special investigation (VAN BRUMMELEN, 1986a). This study with electron microscopy revealed the double nature of the lateral ascus wall and the presence of an opening mechanism by a roughly delimited operculum, very similar to the 'Octospora type' as defined by VAN BRUMMELEN (1978) or the 'Otidea-Aleuria type' of SAMUELSON (1978). This opening mechanism is characteristic of all genera of the Pyronemataceae studied so far and forms one of the main reasons to place Pseudombrophila in this family. ASCOSPORES. The ascospores provide some of the most valuable characters for the recognition of species of Pseudombrophila. As the result of three nuclear divisions of the fusion nucleus in the ascus eight nuclei are formed. In most cases eight normal ascospores will develop, but sometimes in a small part of the asci one or a few spores may abort and a smaller number of spores ripens. Since some of these spores are larger than normal they are not included for measuring purposes. In three species four ascospores are regularly formed in an ascus. In P. guldeniae the typical spore number is four, but in certain collections a varying number of asci, often in the same fruit body may also develop eight normal ascospores of a smaller size. In P. microtetraspora of the eight nuclei, initially present, four usually abort and remain for a long time visible as 'ghosts', while the other four develop normally into ornamented spores. But occasionally some of the asci develop eight normal spores of a smaller size. In P. minor the normal ascospore number is eight, but in certain collections part of the asci develop only four normal spores. Apparently there exists a certain amount of genetic instabi-

14

lity in the sporogenesis in part of the populations of these three species and perhaps also in a few other species of Pseudombrophila sect. Nannfeldtiella. The shape of the spores is very characteristic of most species of Pseudombrophila. Species with globose and shortly to oblong ellipsoid spores are found. There is no reason to place species with globose spores in a separate section, or even a genus, as was done by e.g. SACCARDO (1889), BOUDIER (1907), DURAND in HOTSON (1912), GRELET (1944), and FAUREL & SCHOTTER (1965). Although the length/width ratio varies for each species within certain values, the average proved to be a very valuable character for delimiting species. The length of the spores, varying from 8 to 38 .um, provides another such character. As is usual in spore measurements, the sometimes strongly variable height of spore ornaments is not included. The contents of the ascospores appear rather homogeneous and are typically pale yellowishbrownish in all species of Pseudombrophila. Oil drops were sometimes observed in ripening spores, but these always disappeared before maturity. Air or gas inclusions are sometimes reported for ascospores of Pseudombrophila as 'de Bary bubbles'. They are produced by abrupt dehydration of living spores or insufficient and too fast rehydration of dried material. Mounting media especially apt to produce them are: lactic acid, lacto-phenol, chloralhydrate, and Melzer's reagent (cf. also BARAL, 1992). Thick-walled spores are more susceptible than thin-walled ones. With more natural media and careful, gradual rehydration these artefacts can be fully avoided (see 'Materials and methods'). The wall of the ascospore consists of a primary and a secondary wall. The primary wall again is composed of two layers, an inner endospore and an outer epispore (Fig. 4). The latter is very dense and can be considered as the outer delimitation of the ascospore proper, and can only with great difficulty be penetrated by gasses and liquids, which may explain the formation of the ' de Bary bubbles'. The secondary wall or perispore is responsible for the ascospore ornamentation, which offers, in Pseudombrophila, valuable characters for the delimitation of species and sections. In Pseudombrophila ascospores occur with smooth, irregularly warted, striate, or incompletely reticulate surfaces. The perispore can easily be stained with solutions of methyl blue or trypan blue. Usually little attention was paid to the presence of a smooth uniform perispore, but HARMAJA (1974) recorded such a layer for a number of genera of the Pezizales. The ultrastructure of the development of the ascospore wall in Pseudombrophila was the subject of a separate study (VAN BRUMMELEN, 1986a, 1994). This study showed that the different patterns of ascospore ornamentation form a continuous series (Fig. 4). In all species of Pseudombrophila a smooth rather uniform secondary wall develops initially (state IV in Fig. 4). For most species in the section Pseudombrophila this is also the final state and is known as 'smooth ornamentation' (a in Fig. 4). In P. merdaria (P. deerrata) and P. petrakii (P. obliquerimosa) the smooth secondary wall develops by a process of local breaking down either into a pattern of irregular granules or warts or into a pattern of oblique ridges (b in Fig. 4). In P. guldeniae (syn. Nannfeldtiella aggregata) the smooth secondary wall is lifted locally to form an intermediate pattern of large irregular vesicles of which only the thin superficial layer stains with methyl blue (state V in Fig. 4c). This state was described by ECKBLAD (1968) as being typical for Nannfeldtiella aggregata Eckbl. But on further ripening the spaces of the vesicles are gradually filled with fibrillar elements to form an incomplete reticulum of broad and narrow lines, now easily staining with methyl blue (state VI in Fig. 4c).

15

3

midmesohymenial phase

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Fig. 3. - Scheme of the development of ascomata in Pseudombrophila. - a. Pseudombrophila cervaria, P. hepatica, P. leporum, and P. theioleuca; opening in the mesohymenial phase without active submarginal growing zone. - b. Pseudombrophila merdaria and P. petrakii; as in b, but with active submarginal growing zone. - c. Pseudombrophila guldeniae, P. microtetraspora, and P. minor; opening in the prohymenial phase, with active submarginal growing zone. Fig. 4. - Diagrammatic schemes of secondary spore wall development in Pseudombrophila. - a. Pseudombrophila cervaria and P. theioleuca. - b. Pseudombrophila merdaria and P. petrakii. - c. Pseudombrophila guldeniae. - I -VI. Successive stages of development. Note that at the intermediate stage IV a 'smooth ornamentation' is present in all cases.

16

The reticulate pattern is characteristic of all species of Pseudombrophila sect. Nannfeldtiella. It varies in density and structure, while sometimes conspicuous apical elongations or apiculi

are produced. Apiculi develop somewhat independently and are visible during ripening as terminal amorphous bodies staining at first only pale blue with methyl blue. At maturity their shape may be conical, cylindrical, or hemispherical. HABITAT. - Species of Pseudombrophila occur on a wide variety of substrata. Some are strictly coprophilous and grow on many kinds of dung. Others are found growing on soil or vegetable debris contaminated by dung, urine, or urea. Rarely, species are reported from bare soil without obvious contamination by dung or urine. A single species, P. disciformis, is known only from burnt ground. Some species, like P. earina, P. merdaria, and P. ramosa, are found on decaying stems and leaves of plants or on rotting materials (paper, millboard, cloth, compost, or animal cadavers). Most representatives of Pseudombrophila sect. Nannfeldtiella and P. petrakii are known from the sleeping places of elk and deer, where they grow in close association with the subiculum of a species of Byssonectria. The white, rather tough subiculum is covered with fruit bodies of the Byssonectria and also with those of one or even several species of Pseudombrophila. BENKERT (1988) has suggested that there might be a parasitic relationship. This is not very probable, since some species usually found growing associated with Byssonectria can also be found in other habitats. The shape and extent of the subiculum are subject to wide variation. Often the subiculum is rather extensive and may cover fallen branches and leaves, but sometimes it may be restricted to small patches of white mycelium largely covered by the substratum. In such cases it is difficult or even impossible to ascertain the association with Byssonectria without the presence of fruit bodies. Only a single species of Byssonectria is concerned. This is known under a great number of names, most of which can be found in the treatments of this genus by RIFAI (1968), ECKBLAD (1968), BENKERT (1988), and PFISTER (1993). Although the species concept differs among these authors, the correct name for the species under any of these concepts is Byssonectria terrestris (Alb. & Schw.: Fr.) Pfister, of which the isotype could be studied from the Persoon herbarium in L. DISTRIBUTION. - Since species of Pseudombrophila are only rarely collected and studied and several species are known only from a single locality, relatively little can be said about their distribution. Most species were collected from Europe and northern America. Other parts of the world are still unexplored. Only P. bulbifera is known from tropical regions. Overall the genus shows a more or less boreaI-antarctic type of distribution. Pseudombrophila cervaria, P. coprina, and P. theioleuca clearly show such a distribution.

Pseudombrophila argentinensis, P. fuegiana, and P. minuta are known only from the extreme south of Argentina. A larger group of species shows a boreal type of distribution, either restricted to the boreal part of Europe or represented also in the northern part of North America. Some species, like P. equina, P. hepatica, P. misturae, P. petrakii, and P. virginea are restricted to the temperate regions of Europe.

17

TAXONOMIC PART THE GENUS PSEUDOMBROPHlLA BaUD. Pseudombrophila Boud.• Bull. Soc. mycol. Fr. 1: 106. 1885; Hist. Class. Discom. d'Eur. 65. 1907. - Lectotype (Seaver, 1927; Clements & Shear. 1931; Eckblad, 1968): Helotium pedrottii Bres. [= Pseudombrophila merdaria (Fr.) Brumm.]. Fimaria Velen.• Monogr. Discom. Boh. 1: 331. 1934. - Lectotype (van Brummelen, 1962): Fimaria murina Velen. [= Pseudombrophila hepatica (Batsch) Brumm.]. Ramulina Velen., ap. bot. tech. 4: 147. 1947 [not definitely accepted]. ustulata Velen. [= Pseudombrophila merdaria (Fr.) Brumm.].

Monotype: Humaria

Anthracobia subg. Pseudoanthracobia Svrcek, Sb. n