Biosystematic Studies in Crepidotus and the Crepidotaceae (Basidiomycetes, Agaricales) M. Catherine Aime Dissertation submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology O.K. Miller Jr., Chair J.O. Falkinham D.M. Porter S.E. Scheckler R.J. Vilgalys May, 2001 Blacksburg, Virginia Key Words: Tubaria, Melanomphalia, Pleurotellus, fungal biology, morphogenesis, species concepts Copyright 2001. Mary Catherine Aime Biosystematic Studies in Crepidotus and the Crepidotaceae (Basidiomycetes, Agaricales) M. Catherine Aime (ABSTRACT) Fungi of the Crepidotaceae are characterized by saprotrophic habit, filamentous cuticle, and brown-pigmented basidiospores that lack either a germ pore or plage. The majority of species belong to Crepidotus, distinguished by their pleurotoid basidiomata. Because of their diverse morphology, the presence of several conflicting classifications, and lack of data regarding the biology, phenotypic plasticity, or phylogeny of these fungi, the present study sought first to determine phylogenetic relationships among the different taxonomic groups as a basis for addressing other aspects of Crepidotus biology and evolution. Sequencing analyses show the Crepidotaceae is not monophyletic, and the family concept is revised. Crepidotus and its sister genus Simocybe are found to be monophyletic. At least nine phylogenetic lineages within Crepidotus were uncovered, although relationships between them could not be resolved. However, none of the previously proposed infrageneric classifications are reflective of phylogeny. Morphological, biological, and phylogenetic species concepts were compared within a single phylogenetic unit, termed the Sphaerula group, showing an unusual amount of phenotypic plasticity exists within species, and a taxonomic revision of these species proposed. Also reported are several unique or unusual aspects of Crepidotus biology, including presence of a prolonged latent period prior to basidiospore germination; spontaneous reversion of differentiated hymenial cells to vegetative growth; and the revelation that structures previously termed pleurocystidia are actually the expression of secondary growth from basidia. Results from mating system, culture, and type studies, reassessment of morphological characters traditionally applied to agaric taxonomy, and a revised life cycle for the Crepidoti are presented. Acknowledgments Many, many, people have been involved in the conduct of this project. Foremost are the members of my doctoral committee, Drs. Orson Miller, Joseph Falkinham, Duncan Porter, Stephen Scheckler, and Rytas Vilgalys. All have, at one time or another, graciously reviewed manuscripts, helped to focus my efforts, and provided much needed critiques and suggestions. Only by benefit of their combined wisdom, guidance, and expertise has this dissertation reached fruition. The culture work could not have been completed during its final year without the assistance of two talented undergraduate researchers, Jessica Ball and Katrina Hochstein. Likewise, I owe an enormous debt to the members of the Mycology Laboratory at Duke University—in particular Drs. Rytas Vilgalys and Jean-Marc Moncalvo, who provided training and unconditional, unlimited access to their facilities during the first three years of the molecular systematic portion of this study. Thanks also to Shian-Ren Liou, Tim James, and Jean-Marc and Jasmine Moncalvo for providing their homes during my sometimes lengthy stays in Durham. Dr. Christoph Neinhuis, formerly of the University of Bonn, Germany, deserves my gratitude for helping to produce most of the scanning electron micrographs of basidiospores. I also wish to thank Doug Slotta and Tracey Bodo Slotta, and Bill Zabaronick whose assistance with technical computer problems during times of duress are greatly appreciated. Basidiomata photographs in Chapter 8, Figs. 8.38-41 and 8.43 are used with the kind permission of Dr. Orson K. Miller. iv I thank Drs. Egon Horak, H. Clémençon, Kenneth Wells, and especially Roy Watling and Tim Baroni, for helpful discussions regarding different aspects of the crepidoti and their biology at various times, from which I have greatly benefited. Dr. Beatrice Senn-Irlet generously provided her notes on the type of C. applanatus and inspired further investigations of these fungi. For their assistance in collecting and/or the donation or loan of Crepidotus material from around the world, without which this study would have been significantly reduced, I thank Dr. Tim Baroni, Dr. Alan Bessette, Dr. Tsutomu Hattori, Terry Henkel, Josh Herr, Yoshinobu Hoashi, Dr. Egon Horak, Sonia Jancovicová, Tim James, Dr. Jim Johnson, Wade Leschyn, Brandon Matheny, Dr. Steve Miller, Dr. Eiji Nagasawa, Dr. Clark Overbo, Dr. Ursula Peintner, Allein Stanley, Dr. Rytas Vilgalys, Dr. Roy Watling, Dr. Kazumasa Yokoyama, the University of Michigan Herbarium, and, of course, Dr. Orson and Hope Miller. Funding for travel and various components of this project was provided by: Mountain Lake Biological Research Station of the University of Virginia, The North American Mycological Association, Sigma Xi, The Mycological Society of America, The U.S. National Science Foundation (#SP-9800003) and Monbusho, The Explorer’s Club Washington Group, The Graduate Student Assembly of Virginia Tech, The Virginia Museum of Natural History, and the Department of Biology of Virginia Tech. I also directly benefited from a grant to Dr. O.K. Miller from the Horton Foundation, and ASPIRES grant (#232095) to Drs. O.K. Miller, E.T. Nilsen, R.H. Jones, D.M. Porter, and H.L. Warren. v Finally, I wish to express my gratitude to Orson K. Miller, Jr., who, as he has done for so many before me and will no doubt continue to do, opened my eyes to the fascinating field of mycology and suggested, as a potentially interesting subject, the study of Crepidotus. vi Contents Introduction 1 Part I. The Crepidotaceae (Imai) Singer 8 Chapter 1. Crepidotus thermophilus comb. nov., a reassessment of Melanomphalia thermophila, a rarely collected tropical agaric. 9 Chapter 2. Reassessment of the Crepidotaceae (Imai) Singer based upon nuclear large subunit rDNA evidence. 24 Part II. Crepidotus (Fr.) Staude 55 Chapter 3. Generic concepts in Crepidotus (Fr.) Staude based upon large subunit rDNA sequences. 56 Chapter 4. Delayed germination of basidiospores in temperate species of Crepidotus (Fr.) Staude. 79 Chapter 5. Crepidotus applanatus s. Josserand: Secondary growth of cheilocystidia and taxonomic and biological implications. 104 Part III. The Sphaerula group 122 Chapter 6. The mating system in two species of Crepidotus. 123 Chapter 7. Intercompatibility tests and phylogenetic analysis in the Crepidotus Sphaerula group complex: Concordance between ICGs and nuclear rDNA sequences highlight phenotypic plasticity within species. 130 Chapter 8. Crepidotus crocophyllus: Phenotypic plasticity and secondary growth of basidia with concomitant spore production. 150 Summary and Conclusions 178 Appendices 1. Phylogenetic analysis of the genera of the Crepidotaceae s.l.—Extended analysis within the euagarics. 185 2. Scanning electron micrographs (SEMs) of basidiospores in the Crepidotaceae s.s. 187 Vita 194 vii List of illustrations Fig. 0.1 Schematic representation of the life cycle of a typical agaric. 7 Fig. 1.1 Crepidotus thermophilus basidiomata TJB 8496. 15 Fig. 1.2 Crepidotus thermophilus inflated tramal elements of the lamellae OKM 27270. 15 Fig. 1.3 Crepidotus thermophilus hyphae of the pileipellis with vacuolar pigments OKM 27270. 15 Fig. 1.4 Crepidotus thermophilus cheilocystidia OKM 27270. 15 Fig. 1.5 Crepidotus thermophilus cheilocystidia OKM 27270. 15 Fig. 1.6 Crepidotus thermophilus scanning electron micrograph of basidiospore TJB 8496. 15 Fig. 1.7 Crepidotus thermophilus basidiospores OKM 27270. 15 Fig. 1.8 Crepidotus thermophilus cheilocystidia TJB 8496. 17 Fig. 1.9 Crepidotus thermophilus basidiospores TJB 8496. 17 Fig. 1.10 Crepidotus thermophilus caulocystidia TJB 8496. 17 Fig. 1.11 Phylogenetic assignment of Crepidotus thermophilus within Crepidotus (Fr.) Staude. 20 Fig. 2.1 Phylogenetic assignment of the genera of the Crepidotaceae s.l. based on a portion from the 5'-end of the nLSU. 34 Fig. 2.2 Topological constraint trees generated with weighted parsimony analysis. 38 Fig. 3.1 Phylogeny of Crepidotus using nLSU rDNA sequences. 66 Fig. 3.2 Phylogeny of Crepidotus using nLSU sequences, showing infrageneric classification of Hesler and Smith (1965). 68 Fig. 3.3 Phylogeny of Crepidotus using nLSU sequences, showing infrageneric classification of Singer (1986). 68 viii Fig. 4.1 Number of weeks between collection of spore deposit and basidiospore germination for all collections of Crepidotus. 93 Fig. 4.2 Spore germination of fall-fruiting collections. 94 Fig. 4.3 Spore germination in Crepidotus malachius following delayed spore plating. 95 Fig. 4.4 Identification of single spore isolates (SSIs) by DNA sequencing. 96 Fig. 5.1 Crepidotus applanatus s. Josserand cheilocystidia MCA 366. 109 Fig. 5.2 Crepidotus applanatus s. Josserand cheilocystidia MCA 838. 109 Fig. 5.3 Crepidotus applanatus s. Josserand cheilocystidia OKM 27606. 109 Fig. 5.4 Crepidotus applanatus s. Josserand cheilocystidia MCA 317. 109 Fig. 5.5 Crepidotus applanatus v. phragmocystidiosus cheilocystidia AHS 57473 (HOLOTYPE). 109 Fig. 5.6 Crepidotus applanatus s. Josserand cheilocystidia OKM 27606. 110 Fig. 5.7 Crepidotus applanatus