ARIZONA MEMBERS OF THE GEASTRACEAE AND LYCOPERDACEAE (BASIDIOMYCOTA, FUNGI) Scott T. Bates

Transcription

1 ARIZONA MEMBERS OF THE GEASTRACEAE AND LYCOPERDACEAE (BASIDIOMYCOTA, FUNGI) by Scott T. Bates A Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science ARIZONA STATE UNIVERSITY December 2004

2 2004 Scott Thomas Bates All Rights Reserved

3

4

5 ABSTRACT Within Arizona there is a diverse assemblage of biotic communities that support an equally diverse fungal biota. In an effort to record this fungal biodiversity, a monograph was produced documenting 22 taxa in the Geastraceae and 28 taxa in the Lycoperdaceae that are present in the state. Members of these families are more commonly called earthstars and puffballs, respectfully. Of the 50 taxa presented, 29 species are reported from Arizona for the first time and one species, Calvatia bicolor, is a new record for the United States. In addition to various widely distributed species, rare or understudied species, such as Calvatia cf. leiospora and Holocotylon brandegeeanum, are treated and thorough descriptions are given. As part of a larger study, internal transcribed spacer (ITS 1 and 2, including 5.8S) regions of the nuclear ribosomal RNA gene were sequenced. Sequence data were used in phylogenic analyses to assist in clarifying the taxonomic position of 39 species within the Lycoperdaceae. Basidiospore morphology was also examined ultrastructurally using field-emission scanning electron microscopy. Spore ultrastructure and additional morphology characters contribute to the understanding of natural relationships within the Geastraceae and Lycoperdaceae. In addition to contributing taxonomic knowledge, the monograph will aid in establishing baseline data for fungal diversity in Arizona. iii

6 iv

7 ACKNOWLEDGEMENTS I thank Dr. R.W. Roberson - mycologist, artist, mentor and ultrastructure explorer - for affording me the privilege of working in his lab over the course of the past two and a half years. I have learned a great deal about fungi, microscopy and mycology there. Dr. T.H. Nash III, another fine mentor, has been tremendously generous in providing me with a space to work, support and first hand knowledge of the ins-and-outs of field biology and herbarium work. He has also opened my eyes to the fascinating field of lichenology. Dr. D.E. Desjardin, expert advisor and mycologist extraordinaire, has been a key component in making this study possible. He and his field course in the Sierra Nevada provided my first introduction to how one might live and breath mycology. Dr. M.F. Wojciechowski provided expertise in molecular phylogenetics and was most generous in offering lab space to carry out that work. Bill Sharp of the School of Life Sciences Electron Microscopy facility and John Wheatly the Center for High Resolution Electron Microscopy have offered tutelage in the operation of the scanning electron microscope and other related techniques. Samantha Standley and Jonathan Noyes-Elfstrom assisted in genomic DNA isolation and preparation of materials for PCR and sequencing. Dr. S. Bingham at the ASU DNA laboratory oversaw nrrna gene sequencing. Dr. K.J. Hoober and Dr. J.M. Briggs played a crucial role in my entering into the Plant Biology graduate program at ASU. Dr. R.L. Gilbertson and was extremely generous in offering material for loan from the Gilbertson herbarium. Dr. A.Y. Rossmann and Dr. L.A. Castlebury helped in facilitating my visit to the National Fungus collection, and Erin B. McCray prepared loan materials from that institution. Dr. R. Fogel provided material for loan from the Fungus v

8 Collection at the University of Michigan herbarium. Dr. D.Q. Lewis researched specimens in Morgan s type collection and provided material for loan from ISC. Darvin DeShazer has been a steady guiding force, mycological companion and continuous source of support. He has also been most gracious in allowing me access to his extensive collection of mycological literature. Fred Stevens was the first to introduce me to puffball taxonomy, and Mike G. Wood kindly provided specimens and literature. The enthusiasm these men have shown for mycology has been an inspiration. Dr. V. Demoulin sent reprints and was generous in providing a copy of his Ph.D. dissertation. Many members of the Arizona Mushroom Club assisted the project in one way or another. In particular, Lynn Theriot and Janie Thom graciously provided specimens. Matthew R. Keirle, Andy Wilson, Peter Werner and Denise Gregory all inspired me early on to take my mycological studies to a new level. Dr. F. Bungartz and Frauke Ziemmeck, Rob Bills, Frank Farruggia, Karen and Bill Iselin, James Lendemer, Ken G. Sweat, Maho Uchida and Dr. A.M.L. van de Meene have all assisted in many ways. A special thank-you is in order for those institutions and organizations that provided financial support. They are ASU s School of Life Sciences and the Michael A. Cichan Memorial Award, the ASU Graduate and Professional Student Association, Sonoma County Mycological Society and Sigma XI. I am also indebted to mycologists past and present whose collections have greatly assisted in making this project possible; mainly Dr. R.L. Gilbertson, Dr. W.H. Long, C.G. Lloyd and Dr. J.S. States. Finally, I thank Dr. C.R. Leathers for initially introducing me to the science of mycology and the Arizona mycota. vi

9 dedicated to my father, W.F. Bates, without his initial support this thesis would not have been possible and to my wife Tonya R. Boschmann for her patience, love and support throughout this project vii

10 viii

11 TABLE OF CONTENTS Page LIST OF TABLES... xii LIST OF FIGURES...xiii INTRODUCTION... 3 MATERIALS AND METHODS Methods Monography...10 Basidiospore ultrastructure...11 DNA extraction...11 PCR amplification...13 Phylogenetic analyses...14 Specimens examined and herbaria ECOLOGY MORPHOLOGY AND TAXONOMIC CHARACTERS Macromorphology Gasterocarp...25 Peridia...26 Ostiole...30 Peristome...32 Gleba...32 Subgleba...33 Columella...34 ix

12 Page Mycelial layer...34 Rhizomorphs...35 Micromorphology Basidia and Sterigmata...36 Spores...36 Capillitia...38 Peridia...41 TAXONOMIC REVIEW AND SYNOPSIS OF INFRAGENERIC TAXA Polyphyly of the Gasteromycetes Geastraceae Geastrum...45 Myriostoma...46 Lycoperdaceae Bovista...48 Calvatia...50 Handkea...52 Holocotylon...53 Lycoperdon...54 Morganella...56 Mycenastrum...57 Vascellum...58 PHYLOGENY OF THE LYCOPERDACEAE x

13 Page Molecular phylogeny Spore ultrastructure and phylogeny in the Lycoperdaceae TAXONOMY Artificial Key to Arizona Earthstars (Geastraceae and Astraeus) The Geastraceae Artificial Key to Arizona Puffballs (Lycoperdaceae) The Lycoperdaceae REFERENCES APPENDIX I: REPORTED SYNONYMY FOR NORTH AMERICAN EARTHSTARS APPENDIX II: REPORTED SYNONYMY FOR NORTH AMERICAN PUFFBALLS xi

14 LIST OF TABLES Table Page 1. Origin of records for Geastraceae species (with Astraeus hygrometricus) included in this study Origin of records for Lycoperdaceae species included in this study The Geastraceae (with Astraeus hygrometricus) and their occurrence within various biotic communities The Lycoperdaceae and their occurrence within various biotic communities Species used in analysis of ITS and 5.8S nuclear rrna gene sequences xii

15 LIST OF FIGURES Figure Page 1. Biotic communities present in Arizona Strict consensus tree produced using ITS sequence data Maximum likelihood tree produced using ITS sequence data Bootstrap tree produced using ITS sequence data Distribution of Geastrum arenarium Distribution of Geastrum campestre Distribution of Geastrum corollinum Distribution of Geastrum coronatum Distribution of Geastrum elegans Distribution of Geastrum fimbriatum Distribution of Geastrum floriforme Distribution of Geastrum fornicatum Distribution of Geastrum hieronymi Distribution of Geastrum kotlabae Distribution of Geastrum lageniforme Distribution of Geastrum minimum Distribution of Geastrum quadrifidum Distribution of Geastrum rufescens Distribution of Geastrum saccatum Distribution of Geastrum schmidelii xiii

16 Figure Page 21. Distribution of Geastrum smardae Distribution of Geastrum smithii Distribution of Geastrum striatum Distribution of Geastrum triplex Distribution of Geastrum xerophilum Distribution of Myriostoma coliforme Distribution of Astraeus hygrometricus Distribution of Bovista aestivalis Distribution of Bovista dermoxantha Distribution of Bovista plumbea Distribution of Calvatia bicolor Distribution of Calvatia booniana Distribution of Calvatia craniiformis Distribution of Calvatia cyathiformis Distribution of Calvatia fragilis Distribution of Calvatia cf. leiospora Distribution of Calvatia pachyderma Distribution of Calvatia rugosa Distribution of Disciseda arida Distribution of Disciseda candida Distribution of Disciseda cervina Distribution of Disciseda pedicellata xiv

17 Figure Page 43. Distribution of Holocotylon brandegeeanum Distribution of Lycoperdon lividum Distribution of Lycoperdon marginatum Distribution of Lycoperdon molle Distribution of Lycoperdon perlatum Distribution of Lycoperdon pulcherrimum Distribution of Lycoperdon rimulatum Distribution of Lycoperdon umbrinum Distribution of Morganella pyriformis Distribution of Mycenastrum corium Distribution of Vascellum intermedium Distribution of Vascellum lloydianum Distribution of Vascellum texense Capillitia, endoperidial hyphae and spores: (a) Geastrum arenarium; (b) Geastrum campestre; (c) Geastrum corollinum; (d) Geastrum coronatum Capillitia, endoperidial hyphae and spores: (a) Geastrum elegans; (b) Geastrum fimbriatum; (c) Geastrum floriforme; (d) Geastrum fornicatum Capillitia, endoperidial hyphae and spores: (a) Geastrum hieronymi; (b) Geastrum kotlabae; (c) Geastrum lageniforme; (d) Geastrum minimum Capillitia, endoperidial hyphae and spores: (a) Geastrum quadrifidum; (b) Geastrum rufescens; (c) Geastrum saccatum; (d) Geastrum schmidelii xv

18 Figure Page 60. Capillitia, endoperidial hyphae and spores: (a) Geastrum smardae; (b) Geastrum smithii; (c) Geastrum striatum; (d) Geastrum triplex Capillitia, endoperidial hyphae, exoperidial elements and spores: (a) Geastrum xerophilum; (b) Myriostoma coliforme; (c) Astraeus hygrometricus; (d) Bovista aestivalis Capillitia, exoperidial elements and spores: (a) Bovista dermoxantha; (b) Bovista plumbea; (c) Calvatia bicolor; (d) Calvatia booniana Capillitia, exoperidial elements and spores: (a) Calvatia craniiformis; (b) Calvatia cyathiformis; (c) Calvatia fragilis; (d) Calvatia cf. leiospora Capillitia, exoperidial elements, endoperidial hyphae and spores: (a) Calvatia pachyderma; (b) Calvatia rugosa; (c) Disciseda arida; (d) Disciseda candida Capillitia, endoperidial hyphae, exoperidial elements and spores: (a) Disciseda cervina; (b) Disciseda pedicellata; (c) Holocotylon brandegeeanum; (d) Lycoperdon lividum Capillitia, exoperidial elements and spores: (a) Lycoperdon marginatum; (b) Lycoperdon molle; (c) Lycoperdon perlatum; (d) Lycoperdon pulcherrimum Capillitia, exoperidial elements, endoperidial hyphae and spores: (a) Lycoperdon rimulatum; (b) Lycoperdon umbrinum; (c) Morganella pyriformis; (d) Mycenastrum corium Capillitia, exoperidial elements and spores: (a) Vascellum intermedium; (b) Vascellum lloydianum; (c) Vascellum texense; (d) capillitia types; intermediate type, Lycoperdon type, Bovista type, Calvatia type xvi

19 Figure Page 69. Gasterocarps: (a) Geastrum arenarium; (b) Geastrum campestre Gasterocarps: (a) Geastrum corollinum; (b) Geastrum coronatum Gasterocarps: (a) Geastrum elegans; (b) Geastrum fimbriatum Gasterocarps: (a) Geastrum floriforme; (b) Geastrum fornicatum Gasterocarps: (a) Geastrum hieronymi; (b) Geastrum kotlabae Gasterocarps: (a) Geastrum lageniforme; (b) Geastrum minimum Gasterocarps: (a) Geastrum quadrifidum; (b) Geastrum rufescens Gasterocarps: (a) Geastrum saccatum; (b) Geastrum schmidelii Gasterocarps: (a) Geastrum smardae; (b) Geastrum smithii Gasterocarps: (a) Geastrum striatum; (b) Geastrum triplex Gasterocarps: (a) Geastrum xerophilum; (b) Myriostoma coliforme Gasterocarps: (a) Astraeus hygrometricus; (b) Bovista aestivalis Gasterocarps: (a) Bovista dermoxantha; (b) Bovista plumbea Gasterocarps: (a) Calvatia bicolor; (b) Calvatia booniana Gasterocarps: (a) Calvatia craniiformis; (b) Calvatia cyathiformis Gasterocarps: (a) Calvatia fragilis; (b) Calvatia cf. leiospora Gasterocarps: (a) Calvatia pachyderma; (b) Calvatia rugosa Gasterocarps: (a) Disciseda arida; (b) Disciseda candida Gasterocarps: (a) Disciseda cervina; (b) Disciseda pedicellata Gasterocarps: (a) Holocotylon brandegeeanum; (b) Lycoperdon lividum Gasterocarps: (a) Lycoperdon marginatum; (b) Lycoperdon molle Gasterocarps: (a) Lycoperdon perlatum; (b) Lycoperdon pulcherrimum xvii

20 Figure Page 91. Gasterocarps: (a) Lycoperdon rimulatum; (b) Lycoperdon umbrinum Gasterocarps: (a) Morganella pyriformis; (b) Mycenastrum corium Gasterocarps: (a) Vascellum intermedium; (b) Vascellum lloydianum Gasterocarps and morphological characters: (a) Vascellum texense; (b) eucapillitia; (c) paracapillitium; (d) gleba of Arachnion album; (e) gleba of Holocotylon brandegeeanum Basidiospore ultrastructure under SEM: (a) Geastrum arenarium; (b) Geastrum campestre; (c) Geastrum corollinum; (d) Geastrum coronatum; (e) Geastrum elegans; (f) Geastrum fimbriatum Basidiospore ultrastructure under SEM: (a) Geastrum floriforme; (b) Geastrum fornicatum; (c) Geastrum hieronymi; (d) Geastrum kotlabae; (e) Geastrum lageniforme; (f) Geastrum minimum Basidiospore ultrastructure under SEM: (a) Geastrum quadrifidum; (b) Geastrum rufescens; (c) Geastrum saccatum; (d) Geastrum schmidelii; (e) Geastrum smardae; (f) Geastrum smithii Basidiospores and Eucapillitia under SEM: (a) Geastrum striatum; (b) Geastrum triplex; (c) Geastrum xerophilum; (d) Myriostoma coliforme; (e) Geastrum type eucapillitia of Geastrum kotlabae; (f) heavily encrusted eucapillitia of Geastrum corollinum Basidiospore ultrastructure under SEM: (a) Bovista aestivalis; (b) Bovista dermoxantha; (c) Bovista plumbea; (d) Bovista plumbea; (e) Calvatia bicolor; (f) Calvatia booniana xviii

21 Figure Page 100. Basidiospore ultrastructure under SEM: (a) Calvatia craniiformis; (b) Calvatia cyathiformis; (c) Calvatia fragilis; (d) Calvatia cf. leiospora; (e) Calvatia pachyderma; (f) Calvatia rugosa Basidiospore ultrastructure under SEM: (a) Disciseda arida; (b) Disciseda candida; (c) Disciseda cervina; (d) Disciseda pedicellata; (e) Holocotylon brandegeeanum; (f) Holocotylon brandegeeanum Basidiospore ultrastructure under SEM: (a) Lycoperdon lividum; (b) Lycoperdon marginatum; (c) Lycoperdon molle; (d) Lycoperdon perlatum; (e) Lycoperdon pulcherrimum; (f) Lycoperdon rimulatum Basidiospore ultrastructure under SEM: (a) Lycoperdon umbrinum; (b) Morganella pyriformis; (c) Mycenastrum corium; (d) Mycenastrum corium; (e) Vascellum intermedium; (f) Vascellum lloydianum Basidiospores and Eucapillitia under SEM: (a) Vascellum texense; (b) Chlorophyllum molybdites; (c) elastic Lycoperdon type eucapillitia of Bovista aestivalis; (d) fragile Calvatia type eucapillitia of Calvatia pachyderma, disarticulating at the septum; (e) inflated septum of Calvatia craniiformis; (f) elongated pores in the eucapillitium of Calvatia cf. leiospora Eucapillitial ultrastructure under SEM: (a) pores in the eucapillitium of Calvatia booniana; (b) fragile Calvatia type eucapillitia of Disciseda candida; (c) medium sized pores in the eucapillitium of Disciseda cervina; (d) slit-like pores in the eucapillitium of Handkea utriformis; (e) spine on eucapillitium of Mycenastrum corium; (f) spiny Mycenastrum type eucapillitia of Mycenastrum corium xix

22 Figure Page 106. Arizona Field Mycologists: (a) William H. Long; (b) Jack S. States; (c) Robert L. Gilbertson xx

23 The study of mycology is not a matter of exact measurement. It is rather a study of variation, a study of change. All things that live change. C.G. Lloyd 1909, referring to variation in the exoperidium of Lycoperdon species

24 2

25 INTRODUCTION Arizona possesses a wide range of biotic communities from arid Sonoran desertscrub to lush Petran subalpine conifer forest and the barren alpine tundra of the San Francisco Peaks. The variety of habitats within these communities provides for a rich heritage of biological diversity. This diversity is also reflected in the mycota found in the state. Unfortunately, much of this potential well-spring of species richness remains unrecorded. Two families of basidiomycetaceous fungi are presented in this monograph that documents these organisms as they occur in Arizona. Included are the Geastraceae and Lycoperdaceae, which are commonly known as earthstars and puffballs respectively. Astraeus hygrometricus, commonly called the false earthstar, is also present as this species closely resembles the true earthstar in the genus Geastrum and can easily be confused with them. Mycological literature describing earthstars and puffballs found in Arizona is scant. Long and Stouffer (1948) contributed twelve Arizona records of Geastrum species and cite their distribution. In his doctoral thesis, Demoulin (1972a) mentions four species of Lycoperdon as occurring in the state; however, distribution data within Arizona are not given. In addition to records from these publications, numerous specimens of Arizona puffballs and earthstars have been collected and deposited in herbaria by the few field mycologists who have worked in the state, namely W.H. Long, R.L. Gilbertson and J.S. States (figs. 106a-c). To supplement this material, specimens of gasteroid species have also been collected by the author over the course of three seasons in the field. The data from over 350 specimens are included in this study, and more than half of the 51 species presented here are reported from Arizona for the first time. Thorough descriptions and

26 new distribution data are given for poorly known taxa such as Holocotylon brandegeeanum and Calvatia cf. leiospora. In addition, Calvatia bicolor is reported for the first time from the United States. Over all, gasteroid species have been found in nearly all of Arizona s major biotic communities and a wealth of species diversity has been discovered. The Geastraceae and Lycoperdaceae have traditionally been placed within the class Gasteromycetes. Persoon s Synopsis Methodica Fungorum (1801) was established as the starting point for Gasteromycete nomenclature in 1910 at the second International Botanical Congress in Brussels and subsequently incorporated into the International Code of Botanical Nomenclature. However, Persoon did not recognize this class and alternatively placed members of this group within his class Angiocarpi in the order Dermatocarpi under the suborder Trichospermi. In Systema Mycologicum, Fries (1821) introduced the Gasteromycetes in his taxonomy for the fungi along with three other classes; Coniomycetes, Hymenomycetes and Hyphomycetes. Fries circumscribed his class of stomach fungi primarily on the closed nature of their basidiocarps, Fungus totus clausus, in centro sporidia collagens. Being influenced at that time by the German romantic philosophy, he proposed that the development of the Gasteromycetes is determined by the reproductive force (nisum reproductivum), which gives rise to fungi, working in conjunction with the element of heat (Ainsworth 1976). Based on anatomical data, workers have suggested since the early nineteenth century that the Gasteromycetes are a polyphyletic class (Hibbet et al. 1997). By the early twentieth century, systematic mycologists began to produce alternative taxonomies, based on evolutionary theories, which included members of the Gasteromycetes within 4

27 the Hymenomycetes. In 1934, for example, Heim placed the gasteromycetaceous family Hydnangiaceae within the Agaricales (Bessey 1950). Throughout the middle of that century, several evolutionary series were proposed that recognized relationship between certain taxa within the Gasteromycetes and other groups such as the Boletales and Agaricales (Singer 1962). With the close of the twentieth century, molecular phylogenetic analyses using sequence data from the nuclear genes encoding ribosomal RNA presented convincing evidence that the Gasteromycetes were indeed a polyphyletic assemblage (Hibbet et al. 1997). Here we shall refer to taxa within the Geastraceae and Lycoperdaceae, which were formerly placed within the Gasteromycetes, as gasteroid fungi. Hibbet et al. (1997) reported that members of the genera Geastrum and Sphaerobolus formed a monophyletic clade with the Phallales. Subsequent studies (Krüger et al. 2001, Binder and Bresinsky 2002) have confirmed the position of the Geastraceae within the Phallales. The Lycoperdaceae, on the other hand, form a separate gasteroid lineage derived from within the Agaricaeae. Molecular phylogenetic analyses continue to support the position that members of this family have arisen from within the Lepiotoid mushrooms (Hibbet et al. 1997, Krüger et al. 2001, Moncalvo et al. 2002, Vellinga 2004). It is also interesting to note that as early as Kreisel (1967a) suggested this relationship and pointed out similarities between Lepiota lycoperdoides and Lycoperdon species. Finally, Binder and Bresinsky (2002) offered evidence, based on nuclear large subunit rrna (LSU, 28S), that the gasteroid species Astraeus hygrometricus belongs in the Boletales and forms a clade with other members of the Sclerodermataceae. 5

28 Taxonomy for gasteroid taxa has changed quite drastically over the past two and a half centuries. For example, Linnaeus (1753) placed members of the Gasteromycetes, Myxomycetes, Pyrenomycetes, Tuberales, Elaphomycetales and Uredinales within Tournefort s genus Lycoperdon in the class Cryptogamia (Demoulin 1973a). Later, Persoon s (1801) concept of Lycoperdon would include only gasteroid Basidiomycetes. Species in Lycoperdon have consequently been segregated into other genera, such as Bovista, Bovistella, Calvatia, Handkea, Morganella and Vascellum based on macro and microscopic characters. The circumscriptions of many gasteroid taxa have become more stable in resent years with the development of technologies like the electron microscope, which has been used to discover ultrastructural characters that assist in producing more robust taxonomies. However, the advent of polymerase chain reaction (PCR), gene sequencing and molecular phylogenetic analysis has given rise to new taxonomic questions for this group. Recent studies (Krüger et. al. 2001, Krüger and Kreisel 2003) based on rrna sequence data indicates that the genus Lycoperdon is polyphyletic and questions the stability of the boundaries between the genera Bovista, Bovistella, Lycoperdon and Morganella. Although convincing arguments for new taxonomies based on molecular phylogenetic evidence can be made, taxonomic revisions should ultimately be based on multiple gene phylogenies as well as morphological and ultrastructure data. The goals of this study are threefold. First, species of fungi from the Geastraceae and Lycoperdaceae are described and their distribution recorded in order to help in establishing baseline data for fungal biodiversity in Arizona. Secondly, spore morphology data are gathered from numerous taxa presented in this study using SEM in 6

29 order to assist in elucidating natural relationships within these families. Thirdly, genomic DNA is extracted from several gasteroid species and the internal transcribed spacer (ITS) and 5.8S regions of the nuclear rrna gene are amplified and sequenced. These data are then used to produce more robust molecular phylogeny for the family Lycoperdaceae. 7

30 Table 1. Origin of records for Geastraceae species (with Astraeus hygrometricus) included in this study. Species Literature Herbarium New Collection Geastraceae Geastrum arenarium Lloyd G. campestre Morgan G. corollinum (Batsch) Hollós G. coronatum Pers. G. elegans Vittad. G. fimbriatum Fr. G. floriforme Vittad. G. fornicatum (Huds.) Hook. G. hieronymi Henn. G. kotlabae V.J. Stanek G. lageniforme Vittad. G. minimum Schwein. G. quadrifidum Pers.: Pers. G. rufescens Pers.: Pers. G. saccatum Fr. G. schmidelii Vittad. G. smardae V.J. Stanek G. smithii Lloyd G. striatum DC. G. triplex Jungh. G. xerophilum Long Myriostoma coliforme (Dicks.) Corda. Sclerodermataceae Astraeus hygrometricus (Pers.) Morgan 8

31 Table 2. Origin of records for Lycoperdaceae species included in this study. Species Literature Herbarium New Collection Lycoperdaceae Bovista aestivalis (Bonord.) Demoulin B. dermoxantha (Vittad.) Demoulin B. plumbea Pers.: Pers. Calvatia bicolor (Lév.) Kreisel C. booniana A.H. Sm. C. craniiformis (Schwein.) Fr. C. cyathiformis (Bosc) Morgan C. fragilis (Vittad.) Morgan C. cf. leiospora Morgan C. pachyderma (Peck) Morgan C. rugosa (Berk. & M.A.Curt.) D.A. Reid Disciseda arida Velen. D. candida (Schwein.) Lloyd D. cervina (Berk.) Hollós D. pedicellata (Morgan) Hollós Holocotylon brandegeeanum Lloyd Lycoperdon lividum Pers. L. marginatum Vittad. L. molle Pers.: Pers. L. perlatum Pers.: Pers. L. pulcherrimum Berk. & M.A. Curt. L. rimulatum Peck ex Trelease L. umbrinum Pers.: Pers. Morganella pyriformis (Schaeff.: Pers.) Kreisel & D. Krüger Mycenastrum corium (Guers. ex DC. & Lam.) Desv. Vascellum intermedium A.H. Sm. V. lloydianum A.H. Sm. V. texense A.H. Sm. 9

32 MATERIALS AND METHODS Methods Monography Macromorphological data are based on original descriptions of herbarium material or collections made in the field. Micromorphological data were gathered using an Olympus BH2 conventional bright field microscope. Illustrations of spores, capillitia and exoperidial cells were drawn using an Olympus U-DA drawing attachment. All material for microscopic examination was first infiltrated with 95% aqueous ethanol before being mounted on glass slides. Observations of characters, such as spore ornamentation and capillitial structure, were made from glebal material mounted separately in H 2 O and 3% aqueous KOH. Observations of paracapillitia were made on glebal material mounted in Lactophenol Cotton Blue. In addition, a small portion of the exoperidium was mounted in Melzer s reagent to observe structure of exoperidial hyphae and/or sphaerocysts. Measurements were made from material mounted in KOH at 1000x using an ocular reticle with units calibrated using an optical micrometer. All spore measurements include ornamentation, and spore statistics include: x, the arithmetic mean of the spore length by spore width (± SD) for n spores measured; Q, the quotient of spore length and spore width in any one spore, indicated as the range of variation in n spores measured; Q m the mean of Q values. Standard classical taxonomic and descriptive methodologies for gasteroid fungi such as those described by Calonge (1998) are used. The classification of biotic communities cited follow the system outlined in Biotic Communities: Southwestern United States and Northwestern Mexico (Brown and Lowe 1980, Brown 1994). Color

33 descriptions adhere to the nomenclature and codification system outlined in the Methuen Handbook of Colour (Kornerup and Wanscher 1967). Additional color names, indicated by quotations, follow the nomenclature given by Ridgway (1912). The taxa are presented alphabetically and taxonomy, for the most part, follows that outlined in the Ainsworth and Bisby's Dictionary of the Fungi (Kirk et al. 2001) and CABI Index Fungorum available online ( Abbreviations for the author s of fungal names are consistent with Brummit and Powell (1992) and subsequent additions found in the CABI Index of Fungi Supplement: Authors of Fungal Names available online ( Basidiospore ultrastructure Observations of spore ultrastructure were made on a F.E.I. - XL30 environmental scanning electron microscope (ESEM) in standard scanning mode, at 3-5 kv, with a spot size of 2.0 or 3.0. The spore morphology data were collected from digital micrographs produced on the ESEM. Spores were fixed in 2% aqueous glutaraldehyde after re-hydration from twenty-four to forty-eight hours in deionized H 2 O. After fixation, spores were washed in a PBS buffer solution and then subjected to a dehydration series from 70% to 100% ethanol. After dehydration, spores were critical-point-dried in 100% acetone at 73.8 bar at 31 C under CO 2 gas. Once removed from the critical-point-dryer, the spores were affixed to carbon coated aluminum specimen holders with poly-l-lysine and sputter coated with palladium-gold for 5 min. at 10 milliamps. DNA extraction For extraction, a small portion of mycelium ( mg) was taken from cultures grown on cornmeal agar (CMA) or mg of glebal material containing spores and capillitium was removed from the interior parts of dried herbarium 11

34 specimens. Fungal material was placed in a 2.0 ml microcentrifuge tube and ground using a small pestle. Genomic DNA was then extracted using Qiagen s DNeasy Plant Mini Kits and following standard protocol described in the kit handbook with buffers and spin columns provided. To the fungal material, 400 µl of AP1 buffer and 4.0 µl of RNase were added and then the tube was vigorously vortexed. This mixture was incubated for 10 min. at 65 o C and mixed periodically by inverting the tube in order to lyse the cells. Next, 130 µl of AP2 buffer was added to the lysate, incubated on ice for 5 min. and then centrifuged for 5 min. at maximum speed. The lysate was then added to the QUIshredder spin column with 2.0 ml collection tube and centrifuged for 2 min. at maximum speed. Flow-through was transferred to a new microcentrifuge tube and 450 µl of AP3/E buffer was added and mixed by pipetting to clear the lysate. Of that mixture, 650 µl was then added to a DNeasy mini spin column and centrifuged for 1 min. at 8000 rpm. The flow-through was discarded and the same procedure was then followed for the remainder of the mixture. After two washes with 500 µl of AW buffer and centrifugation first for 1 then secondly for 2 min., the extracted DNAs were eluted from the mini spin column in 100 µl of AE buffer heated to 65 o C. To precipitate the DNAs, 1/10 volumes of 3M sodium acetate were added in order to adjust the concentration of monovalent cations, the extracted DNAs were then precipitated in 100% ethanol and recovered by centrifugation. After being rinsed in 70% ethanol, the solution was again centrifuged, the supernatant removed, and the resultant pellet was allowed to air dry for 2-3 hours. The DNA pellet was then re-suspended in 25 µl of Te Buffer. 12

35 PCR amplification Polymerase chain reaction (PCR) was performed to amplify the internal transcribed spacers (ITS 1 and 2) in addition to the 5.8S regions of the nuclear ribosomal RNA gene using the ITS4 and ITS5 primer pairs. Each reaction tube theoretically contained the following volume of reagents prior to initiating the PCR reaction: 5.0 µl H 2 O, 2.5 µl 50% glycerol, 2.5 µl 10X Taq I buffer, 2.0 µl dntps (10 mm), 1.25 µl ITS4 primer (10 mm), 1.25 µl ITS5 primer (10 mm), 0.75 MgCl 2 (50 mm) and 0.1 µl Invitrogen Platinum Taq enzyme (2-5 units/ µl). From µl of DNA template was then added to the reaction reagents after being suspended in H 2 O to bring the final volume 10.0 µl. The PCR reactions were then carried out in a DYAD Peltier Thermal Cycler. After an initial denaturing at 92 o C for 2 min., 35 cycles were preformed at 92 o C for 45 sec. (denaturing), followed by incubation at 55 o C for 30 sec.(annealing), and then 72 o C for 1 min. (extension). After the cycling, a final extension at 72 o C for 7 min. was run, followed by indefinite storage at 4 o C. Agarose gel electrophoresis was then carried out on the cycle sequencing product to test for the presence and relative concentration of fungal DNA. For electrophoresis, the cycle sequencing product was then run on a 10% agarose gel in 0.5% TBE buffer at 60 V for min. The gels were stained in ethidium bromide for min. and digitally photographed under UV light. Concentrations of PCR product were measured on a Nanodrop ND-1000 spectrophotometer. Finally, ng of PCR product and 20 ng of primer where brought to 6.0 µl volume in 1.5 microcentrifuge tubes to prepare for sequencing. Automated sequencing was carried out at Arizona State University at the School of Life Sciences DNA Laboratory. 13

36 Phylogenetic analyses Complementary DNA sequences were compiled and manually edited on Sequecher 4.1 software (Gene Codes Corp.). Additional sequences were culled from GenBank ( and a table citing all sequences used in the study with their GenBank accession number are given in tables 5. Automatic computed alignments were performed on Clustal X (Thompson et al. 1997), which were then manually adjusted and edited using MacClade (Maddison & Maddison 1992). The final alignment generated in MacClade was then saved as a NEXUS file to facilitate phylogenetic analyses. A total of 39 sequences for the ITS and 5.8S region of the nrrna gene for the Lycoperdaceae were examined with parsimony and distance analyses using the computer program PAUP 4.0b10 (Swofford 2003). For the phylogenetic analyses, character states were unordered and equally weighted. Highly variable regions in the sequence alignment dataset (positions , , , and ) were excluded from the analyses, and gaps were treated as missing data. Phylogenetic trees generated were rooted using Chlorophyllum molybdites (Agaricales) as the out-group to the Lycoperdaceae because this taxon is outside of the family (Moncalvo et al. 2002). For the maximum parsimony analysis (MP) a heuristic search was performed with tree-bisection-reconnection (TBR) and MAXTREES set at 10,000. A consensus trees (50% majority rule) was then generated from the trees uncovered in the heuristic search to assist in assessing changes in tree topology. For the maximum likelihood analysis (ML) a heuristic search was performed with TBR and MAXTREES set at 10,000. To evaluate clade support a non-parametric bootstrap analysis was performed with 1000 replicates under the heuristic search criteria mentioned above. 14

37 Specimens examined and herbaria Over 350 specimens were examined for this study, and label data for each of these specimens and the herbarium of origin are cited in the Material examined section of each description. Many of the specimens are from the author s personal herbarium. Citations for these collections lack a reference to a herbarium of origin and are indicated by the prefix STB before the collection number. When a collection number was not available for a specimen, the accession number from the herbarium of origin is cited. Information regarding types for each of the species presented, such as the herbarium where the type specimen is housed and/or label data, is given under the Type section of each description. Type specimens that have been examined by the author are indicated by an exclamation mark (!) after the herbarium of origin and collection or accession number. In addition, the word Type appears following the herbarium of origin and collection or accession number in the Material examined section. Abbreviations of herbaria follow those set forth in the Index Herbariorum (Holmgren et al. 1990) with additional updates available online ( through the New York Botanical Garden. Herbaria cited in this study are as follows: Academy of Natural Sciences, Philadelphia, Pennsylvania, USA (PH) Ada Hayden Herbarium, Iowa State University, Ames, Iowa, USA (ISC)* Arizona State University Lichen Herbarium, Phoenix, Tempe, USA (ASU)* Botanischer Garten und Botanisches Museum Berlin-Dahlem, Zentraleinrichtung der Freien Universität Berlin, Berlin, Germany (B) 15

38 Farlow Herbarium of Cryptogamic Botany, Harvard University, Cambridge, Massachusetts, USA (FH) Gilbertson Herbarium, University of Arizona, Tucson, Arizona, USA (ARIZ)* Göteborg University, Göteborg, Sweden (GB) H.D. Thiers Herbarium, San Francisco State University, San Francisco, California, USA (SFSU)* Muséum National d Histoire Naturelle, Paris, France (PC) Mycological Collection, National Museum, Praha, Czech Republic (PRM) Nationaal Herbarium Nederland, Leiden University, Leiden, Netherlands (L) New York Botanical Garden, New York, New York, USA (NY) New York State Museum, Albany, New York, USA (NYS) Royal Botanic Garden, Kew, England, U.K. (K) Swedish Museum of Natural History, Stockholm, Sweden (S) Università degli Studi di Torino, Torino, Italy (TO) University of Copenhagen Herbarium, Copenhagen, Denmark (C) University of Iowa Herbarium, Iowa City, Iowa, USA (IA) University of Michigan Fungus Collection, Ann Arbor, USA (MICH)* University of North Carolina, Chapel Hill, North Carolina, USA (NCU) U.S. National Fungus Collections, Beltsville, Maryland, USA (BPI)* * Herbaria that made specimens available on loan. 16

39 ECOLOGY Brown (1994) outlined 27 major biotic communities (biomes) as occurring in the Southwestern United States and Northwestern Mexico, and describes a hierarchical, ecosystem based classification for these biotic communities. Prior to that publication, a map was produced (Brown and Lowe 1980) that excluded wetland biomes and has a slightly modified nomenclature, which is followed here. The map depicts 22 biomes with 6 subdivisions of the Sonoran desertscrub community for the southwestern United States and northwestern Mexico. Of these, 14 biomes with 2 subdivisions of the Sonoran desertscrub are included in Arizona (fig. 1). This study includes reports of puffballs and earthstars from a total of 9 of the biotic communities present in the state (tables 3 & 4). Interestingly, all of North America s major biome types occur in the Sonoran desert region (Phillips and Wentworth-Comus 2000). These biomes include xeric alpine tundra at extremely high elevations (above 3500 m); mesic subalpine and montane conifer forests at high elevations (ca m); arid adapted chaparral in the mid elevations (ca m); and xeric desert scrub in the lower elevations (below 1050 m). It is also notable that boundaries of 4 major desert systems, the Chihuahuan, Great Basin, Mojave and Sonoran, come in contact with some portion of the state. The higher elevations of the state are primarily covered in conifer forests. Petran (Rocky Mountain) subalpine conifer forest (above ca m) is characterized by tree species such as Picea engelmanii, Populus tremuloides and Pseudotsuga menziesii. This biome is fertile ground for many puffballs and earthstar species such as Morganella pyriformis, Geastrum quadrifidum and Vascellum lloydianum. Numerous gasteroid species, such Calvatia booniana, Lycoperdon perlatum and G. fimbriatum are found in

40 the Petran montane conifer forest (ca m), which is dominated by Pinus ponderosa. Bovista aestivalis, Disciseda cervina, Geastrum kotlabae and G. lageniforme are among the species found in the Great Basin conifer woodland (ca m), which is characterized by Juniperus spp. and Pinus edulis. The Madrean evergreen woodland spans the mid to higher elevations (ca m) and is found in the southern parts of the state. This biome contains various Quercus species, such as Q. emoryi and Q. oblongifolia. Unique earthstar species, such as Geastrum fornicatum and G. hieronymi, have been collected from this biome along with puffball species such as Calvatia cyathiformis and C. pachyderma. There are several biotic communities, which occupy the mid elevations in the state. Quercus turbinella is a common dominant shrub species in the interior or Arizona chaparral (ca m), in addition to Juniperus spp., Pinus edulis and Cercocarpus montanus. Geastrum minimum and Astraeus hygrometricus are commonly found in the interior chaparral, and the unmistakable puffball Lycoperdon pulcherrimum has also been reported from this biome. The Plains and Great Basin grasslands (above ca m) can also be found in the mid elevations. In these grasslands, species such as Andropogon spp., Bouteloua spp. and Sporobolus spp. can be found along side Opuntia spp., and gasteroid species such as L. lividum and C. craniiformis have also been collected in these biomes. Finally, the semidesert grassland (ca m) is characterized by grass species such as Aristida spp. and Bouteloua spp. as well as plant species such as Yucca spp., Prosopis spp., Opuntia spp. and Acacia spp. Gasteroid fungi such as G. campestre, C. rugosa and L. lividum and also be encountered in this biome. 18

41 In Arizona, the lower elevations (below ca m) are dominated by the Sonoran desertscrub. Vascular plant species such as Larrea tridentata, Carnegia gigantea, Cercidium spp. and Prosopis spp. characterize the Lower Colorado River Valley subdivision of the Sonoran desertscrub. The puffball Disciseda arida can be found in this subdivision along with the earthstars Geastrum floriforme and G. striatum. Many of the same vascular plant species can be found in the Arizona Upland subdivision of the Sonoran desertscrub. This biome is also host to several arid adapted earthstar and puffball species, such as G. arenarium, G. smithii, G. xerophilum, Calvatia cf. leiospora and D. pedicellata. Members of the Geastraceae and Lycoperdaceae have a widespread distribution and are commonly found in temperate, arid and tropical climates (Pegler et al. 1995). A large part of southern Arizona is occupied by the Sonoran Deserts, which is arid with considerable tropical influences, and the mid-elevations of the state are covered by large tracts of temperate forest (Brown 1994, Phillips and Wentworth-Comus 2000). Considering these factors, it is not surprising that a biologically diverse and species rich assortment of gasteroid fungi are present within the state. For the most part, the gasteroid species found in Arizona are saprobic, living off of dead plant material found in the soil. Some species are mycorrhizal, while this mutually beneficial symbiosis has been suggested but never verified in other gasteroid taxa. Most species within the Geastraceae are humicolous, while a few taxa are lignicolous and even termiticolous (Pegler et al. 1995). Sunhede (1989) cites several workers who have suggested that species within the Geastraceae are ectomycorrhizal with various conifer species. Possible ectomycorrhizal species include G. fimbriatum, G. 19

42 quadrifidum and G. coronatum. Sunhede also mentions a study by Lihnell (1939) of mycorrhizal fungi associated with Juniperus, where that researcher noted an association between J. communis and G. minimum. Lihnell, however, was not able to produce mycorrhizae under axenic conditions. Sunhede goes on to state, Judging from many ecological notes in the literature and my own field experience... I find it likely that the species of Geastrum, Myriostoma and Trichaster are saprophytes. One study (Agerer et al. 1998) does offer evidence of a mycorrhizal association between G. fimbriatum and Fagus sylvatica; however, the rhizomorphs are somewhat irregular and a Hartig net is not formed. It is generally held that the Lycoperdaceae are saprobic, being terricolous and humicolous in habit; however, a few species are lignicolous. The lignicolous species are found within the genus Morganella. Krüger and Kreisel (2003) recently transferred Lycoperdon pyriforme to that genus based on the lignicolous habit and phylogenetic analysis of rrna sequence data. Some members of this family, primarily within the genus Calvatia, are actively sought out by mycophagists. Their search is often amply rewarded as the edible species such as C. gigantea and C. booniana are occasionally found, which weight 20 lbs. or more. Calvatia gigantea is also a very prolific spore producer, and it has been estimated that larger gasterocarps of this species can produce upwards of 160 trillion spores (Bessey 1950). Fortunately, the majority of these spores will never reach maturity. Many species within the Sclerodermataceae, such as Pisolithus arhizus, are mycorrhizal. This family is represented in this study by a single species, Astraeus hygrometricus. This cosmopolitan and easily recognizable fungus is ectomycorrhizal, 20

43 associating with various tree species (Pegler et al. 1995). Calonge (1998) notes that A. hygrometricus is extremely flexible and able to adapt to a variety of ecological niches, fruiting throughout the year after periods of sufficient rain. This fact has been supported by my field observation here in Arizona; however, gasterocarps are normally found abundantly in wash areas, after they have become detached from their mycelium, rather than in close proximity to their mycorrhizal associate. 21

44 Kilometers Bioticdeci.shp ALPINE TUNDRAS AZ.UPLAND SONORAN DESERTSRUB CHIHUAHUAN DESERTSCRUB GREAT BASIN CONIFER WOODLAND GREAT BASIN DESERTSCRUB INTERIOR CHAPARRAL LOWR COLO.R. SONORAN DESERTSCRUB MADREAN EVERGREEN WOODLAND MOHAVE DESERTSCRUB PETRAN MONTANE CONIFER FOREST PETRAN SUBALPINE CONIFER FOREST PLAINS & GREAT BASIN GRASSLAND SEMIDESERT GRASSLAND SUBALPINE GRASSLAND W N S E Figure 1. Biotic communities present in Arizona 22

45 Table 3. The Geastraceae (with Astraeus hygrometricus) and their occurrence within various biotic communities. GBCW = Great Basin conifer woodland; IC = interior chaparral; MEW = Madrean evergreen woodland; PMCF = Petran montane conifer forest; PSCF = Petran subalpine conifer forest; PGBG = Plains and Great Basin grassland; SDS-AU = Arizona Upland division of the Sonoran desertscrub; SDS-LC = Lower Colorado division of the Sonoran desertscrub; SG = Semidesert grassland Taxon GBCW IC MEW PMCF PSCF PGBG SDS- AU SDS- LC SG Geastraceae Geastrum arenarium G. campestre G. corollinum G. coronatum G. elegans G. fimbriatum G. floriforme G. fornicatum G. hieronymi G. kotlabae G. lageniforme G. minimum G. quadrifidum G. rufescens G. saccatum G. schmidelii G. smardae G. smithii G. striatum G. triplex G. xerophilum Myriostoma coliforme Sclerodermataceae Astraeus hygrometricus 23

46 Table 4. The Lycoperdaceae and their occurrence within various biotic communities. GBCW = Great Basin conifer woodland; IC = interior chaparral; MEW = Madrean evergreen woodland; PMCF = Petran montane conifer forest; PSCF = Petran subalpine conifer forest; PGBG = Plains and Great Basin grassland; SDS-AU = Arizona Upland division of the Sonoran desertscrub; SDS-LC = Lower Colorado division of the Sonoran desertscrub; SG = Semidesert grassland Taxon GBCW IC MEW PMCF PSCF PGBG SDS- AU SDS- LC SG Lycoperdaceae Bovista aestivalis B. dermoxantha B. plumbea Calvatia bicolor C. booniana C. craniiformis C. cyathiformis C. fragilis C. cf. leiospora C. pachyderma C. rugosa Disciseda arida D. candida D. cervina D. pedicellata Holocotylon brandegeeanum Lycoperdon lividum L. marginatum L. molle L. perlatum L. pulcherrimum L. rimulatum L. umbrinum Morganella pyriformis Mycenastrum corium Vascellum intermedium V. lloydianum V. texense 24

47 MORPHOLOGY AND TAXONOMIC CHARACTERS It is now widely accepted that the gasteroid habit has evolved from Hymenomycete ancestors (Hibbet et al. 1997). This evolution was most likely driven by aridity and climatic conditions that are unfavorable to mushrooms which need moisture to facilitate forceful spore liberation (Thiers 1984). Gasteroid fungi have lost the ability to forcefully eject their spores from the basidium and are said to be non-ballistosporic or statismosporic. The numerous forms exhibited in the gasteroid fungi can be considered as experiments in spore liberation. And, being better suited to more arid conditions the gasteroid fungi have reached a peak of diversity and variation in warm, dry climates found throughout the world (Ingold 1971), such as are found in Arizona. Macromorphology Gasterocarp The fruiting bodies of gasteroid basidiomycetaceous fungi are known as gasterocarps. There is a great deal of gasterocarp variation found within the Geastraceae and Lycoperdaceae. Despite this variation, however, there is a common generalized structure found in all members of these families; that is a mass of spores and fertile mycelial tissue, known as the gleba, surrounded by one or more layers skin known as the peridia (sing. peridium). The majority of Geastraceae covered in this study, Geastrum for example, have an outer and inner peridium. As the gasterocarp matures, the outer peridium develops splits radiating from the apex. These splits form numerous rays, which then separate from the inner peridium. The inner peridium normally remains intact and surrounds the gleba. The resultant structure is a central fertile body, from which the spores dehisce,

48 surrounded by stellate rays of the outer peridium. In some species, the rays of the outer peridium remain close to the fertile body surrounding or covering it to some degree; however, in other species the rays peel away from the fertile body, inverting and lifting the fertile body off the surface of the ground. The stellate pattern of the rays has given rise to the common name of earthstars for members of this family. Astraeus hygrometricus, the sole representative in this study from the Sclerodermataceae, is nearly identical in regard to external structure; therefore, the common name of false earthstar has often been used for this species. The Lycoperdaceae are commonly called puffballs as they have a globose to ovoid shape in general and often puff out spores through an apical opening. Members of this family normally have two distinct peridia, although in some species these are fused to such a degree that they are difficult to distinguish separately. The variation in the outer peridium ranges from being nearly glabrous to having long warts or spines, which can be persistent or fugacious. The spores dehisce from the inner peridial layer through an orbicular opening or as the peridium cracks open or disintegrates. Peridia The outer peridium is termed the exoperidium, where as the inner peridium is known as the endoperidium. The exoperidium of A. hygrometricus and species in the Geastraceae is further distinguished by two distinct layers, the outer fibrous layer and the inner pseudoparenchymatous layer. The fibrous layer can be thick or thin and is described as being papery, coriaceous or rigid. The pseudoparenchymatous layer is often thick when fresh and later becomes thin upon drying. The alternative term fleshy layer has been applied by some authors. Coloration, persistence and the structure of this layer is noted to aid in identification. After the formation of the exoperidial rays, 26