Phylogenetic Relationships and Species Richness of Coprophilous Ascomycetes Åsa Nyberg Kruys Department of Ecology and Environmental Science Umeå University Umeå 2005 AKADEMISK AVHANDLING som med vederbörligt tillstånd av rektorsämbetet vid Umeå universitet för avläggande av filosofie doktorsexamen framläggs till offentligt försvar i Stora hörsalen, KBC, fredagen den 25 november 2005, kl. 9.00. Avhandlingen kommer att försvaras på engelska. Examinator: Dr. Mats Wedin, Umeå Universitet Opponent: Ass. Prof. Thomas Laessøe, Department of Microbiology, University of Copenhagen, Denmark. ISBN 91-7305-949-8 © Åsa Nyberg Kruys Printed by Solfjädern Offset AB Cover: Ascus of Sporormiella antarctica with eight 13-celled ascospores. Design by Åsa and Nic Kruys. Organization Document name UMEÅ UNIVERSITY DOCTORAL DISSERTATION Department of Ecology and Environmental Science Date of issue SE-901 87 Umeå, Sweden November 2005 Author Åsa Nyberg Kruys Title Phylogenetic relationships and species richness of coprophilous ascomycetes. Abstract Coprophilous ascomycetes are a diverse group of saprobes, of which many belong to three families, Delitschiaceae, Phaeotrichaceae and Sporormiaceae, within the large order Pleosporales. The natural relationships and circumscription of these families are unclear, especially within the family Sporormiaceae, where the generic delimitation have been questioned. There is also a need to understand how different ecological processes affect species richness and occurrence of coprophilous ascomycetes in general. The aim of this thesis was therefore to test earlier classifications of coprophilous taxa within Pleosporales, using phylogenetic analyses of DNA sequences; and to study how the habitat, dung type and herbivores´ food choice may affect the species richness and species composition of coprophilous ascomycetes. A phylogenetic study shows that coprophilous taxa have arisen several times within Pleosporales. Sporormiaceae and Delitschiaceae are separate monophyletic groups and should continue to be recognized as two distinct families within Pleosporales. Phaeotrichaceae forms a monophyletic group, and is, unexpectedly, a strongly supported sister-group to Venturiaceae, but if they belong to Pleosporales or not, remains unresolved. Testudinaceae and Zopfiaceae, which previously had an unclear position in Ascomycota, are shown to be members of Pleosporales and should be treated as two separate families. The genus Eremodothis is, however, not related to Testudinaceae, but is nested within Sporormiaceae and should be transferred to Westerdykella. The natural relationships within Sporormiaceae are still not fully resolved and consequently, I suggest a rather conservative generic classification, accepting Preussia, Sporormia, Westerdykella, as well as Sporormiella, despite that the latter is not conclusively well supported as monophyletic. Characters previously used in the taxonomy and classification of Sporormiaceae, as choice of substrate, presence or absence of an ostiole, presence or absence of germ slits, and spore ornamentation, were all homoplastic and not very useful for circumscribing monophyletic groups. Field-studies of moose (Alces alces), mountain hare (Lepus timidus) and roe deer (Capreolus capreolus) dung resulted in several new species records, which suggests that coprophilous ascomycetes in boreal Sweden are poorly known. Fungal species richness and occurrence on moose dung varied significantly between habitats. Species diversity was negatively associated with amount of insect attack, and insects feeding either on the dung and/or the fungi may be an important factor explaining the observed pattern. Species richness of coprophilous fungi varied also significantly between different dung types. A study of moose, mountain hare, and roe deer dung did not show any consistent patterns in respect to the animals´ digestive system. There was, however, a general strong positive relationship between the total number of ascomycete species and the number of plant species foraged by the three herbivores. Fungal species with large spores (≥ 50 µm) were over-represented on roe deer dung, and under-represented on moose dung, while the reverse was found for species with small spores (<10µm). This suggests that the foraging level of the herbivore, which in turn mirrors species-specific differences in spore dispersal of the fungi, may be an important factor in explaining species richness and diversity of the coprophilous community. Key words: ß-tubulin, Bayesian analysis, dung, forage, fungi, moose, parsimony, phylogeny, Pleosporales, spore dispersal, Sporormiaceae Language: English ISBN: 91-7305-949-8 Number of pages: 28 + 4 papers Signature: Date: 25 Oktober 2005 List of papers This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I. Kruys Å, Eriksson OE, Wedin M. Phylogenetic relationships of coprophilous Pleosporales (Dothideomycetes, Ascomycota), and the classification of some bitunicate taxa of unknown position. Manuscript. II. Kruys Å, Wedin M. A study of the natural relationships and traditionally used taxonomic characters in Sporormiaceae (Pleosporales, Ascomycota), utilizing multi-gene phylogenies. Manuscript. III. Nyberg Å, Persson I-L. 2002. Habitat differences of coprophilous fungi on moose dung. Mycological Research 106: 1360-1366. IV. Kruys Å, Ericson L. Species richness of coprophilous ascomycetes in relation to variable food intake by herbivores. Submitted manuscript. Paper III is published with the kind permission of the publisher. TABLE OF CONTENTS 1. INTRODUCTION .............................................................. 5 1.1 Natural relationships of the coprophilus Pleosporales.................. 6 1.2 Focus on Sporormiaceae................................................. 7 1.3 Species richness of coprophilous ascomycetes ......................... 8 2. OBJECTIVES.................................................................. 9 3. MATERIAL AND METHODS .................................................10 3.1 Molecular methods......................................................10 3.2 Field studies.............................................................10 4. MAJOR RESULTS AND DISCUSSION........................................11 4.1 Phylogenetic relationships of coprophilous families in Pleosporales..11 4.2 Phylogenetic relationships within Sporormiaceae.....................13 4.3 The effect of habitat on coprophilous fungi on moose dung ..........16 4.4 Species richness in relation to variable food intake by herbivores ..17 4.5 New records of coprophilous ascomycetes in Sweden ................19 5. FINAL CONCLUSIONS.......................................................19 6. ACKNOWLEDGEMENTS.....................................................21 7. REFERENCES ................................................................21 8. POSTSCRIPT .................................................................27 APPENDICES: PAPER I-IV 1. INTRODUCTION A dung pile is a tremendously rich substrate sparkling with life! Fungi, bacteria, arthropods, and mosses compete for space and nutrients, and interact in the degradation of the heterogeneous substrate. Coprophilous (dung-loving!) fungi are a diverse group of saprobes including taxa from most major fungal groups. This thesis will, however, focus on Ascomycota, which is the largest group of fungi with more than 32 000 species described (Kirk et al. 2001). The number of coprophilous ascomycetes has not been estimated, though. Coprophilous ascomycetes can be found on various dung types from all over the world, but are more frequent on dung of herbivores than carnivores (Lundqvist 1972, Richardson 2001). In addition, they have seldom been reported on reptile or amphibian dung, indicating that coprophily of fungi developed among the warm-blooded animals (Webster 1970). Some species are strictly coprophilous, others occur on different ephemeral substrates. The strictly coprophilous ascomycetes have a remarkable life-cycle, dung Æ plant Æ animal gut Æ dung (Fig. 1). Thus, they do not disperse between habitat patches (Wicklow 1992). Instead they have to pass the digestive system of an animal, and spore germination may even be triggered by gastric juices (Sussman & Halvorson 1966, Furuya & Naito 1980). The spores of coprophilous ascomycetes are often darkly pigmented, with thick walls, and are probably well protected against both gastric juices and the harmful UV-light of the sun (Ingold & Hudson 1993). Fig. 1. The life-cycle of a strictly coprophilous pyrenomycete. A fruit-body that is produced on a dung pile, will discharge its spores when mature. The spores are often surrounded by mucilage or have gelatinous appendages, and attach easily to the plant parts on which they land. When a plant is foraged by a herbivore, the spores will follow and be transported through the digestive gut. Finally, when ending up in a new dung pile, the spores will germinate and produce new fruit- bodies. Drawing inspired by Bell (1983). 5 Coprophilous ascomycetes are advantageous organisms to study; a great range of various morphologically and systematically different taxa can appear on a fresh dung pile within few weeks, there is also no shortage of dung in nature, and a majority of the species are possible to grow in vitro. Consequently, coprophilous fungi have been subject to early and extensive studies (Massee & Salmon 1901, Lundqvist 1972, Richardson 2001). The dark, thick-walled spores can be found in ancient