ABSTRACT Title of Document: SYSTEMATICS OF THE GENUS COSMOSPORA (NECTRIACEAE, HYPOCREALES), AND COSPECIATION OF COSMOSPORA SPECIES WITH THEIR ASSOCIATED FUNGAL HOSTS. Cesar Samuel Herrera, Doctor of Philosophy, 2014 Directed By: Associate Professor, Priscila Chaverri, Plant Science and Landscape Architecture Cosmospora (in the broad sense; Nectriaceae, Hypocreales, Ascomycota) are fungi that parasitize other fungi, particularly fungi in the Xylariales (Ascomycota), or scale insects. Morphologically, these fungi are known for having one of the most simplest and smallest sexual fruiting bodies (<300 µm) among the Nectriaceae. The sexual spores are generally warted. The majority of Cosmospora species have acremonium- like or fusarium-like asexual states. The name Cosmospora is derived from the ornamentation in the sexual spores (Gr. kosmos = ornamented + Gr. spora = spore). The main goals of this dissertation were to revise Cosmospora sensu stricto, and to determine the evolutionary relationship between Cosmospora species and their associated fungal hosts. Additionally, Corallomycetella (Nectriaceae, Hypocreales, Ascomycota), a lineage basal to Cosmospora sensu lato, was revised as well. Molecular and classical taxonomic tools were used to revise the genera. A genus was recognized if the clade met the following criteria: 1) the clade was well supported, 2) the clade was associated with a unique asexual state, and 3) the clade was ecologically different. A species was recognized if the clade met the following criteria: 1) the clade was well supported in the majority of single gene trees, 2) the clade was morphologically different, and/or 3) the clade was ecologically different in regards to host. Cosmospora species were observed to be highly host specific. Thus, host was recognized as an important character to delineate species, and the host specificity led us to hypothesize that Cosmospora species and their associated hosts were cospeciation (i.e., their association was not random). Two new genera, nine new combinations, and eleven new species were described in the taxonomic work included in this dissertation. A significant global congruence was determined between the Cosmospora and host phylogenies. However, host-switch events seemed more abundant in the early lineages of the host, while cospeciation events seemed more common in more recent lineages of the host. This phylogenetic signature is consistent with pseudocospeciation, but it could not be confirmed given that divergence estimates could not be estimated. SYSTEMATICS OF THE GENUS COSMOSPORA (NECTRIACEAE, HYPOCREALES), AND COSPECIATION OF COSMOSPORA SPECIES WITH THEIR ASSOCIATED FUNGAL HOSTS. By Cesar Samuel Herrera Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2014 Advisory Committee: Dr. Priscila Chaverri, Chair Dr. Charles F. Delwiche Dr. Charles Mitter Dr. Maile C. Neel Dr. Amy Y. Rossman © Copyright by Cesar Samuel Herrera 2014 ii Preface This dissertation contains an introductory chapter, four original articles/chapters, and a concluding chapter. Each original article is presented in manuscript format. The first three original articles represent taxonomic work for Pseudocosmospora C. Herrera & P. Chaverri, Corallomycetella Henn., and Cosmospora Rabenh., respectively, and have similar methodologies. The fourth original article is a coevolutionary study between cosmospora-like fungi and their associated fungal hosts. A single reference section containing all the cited literature throughout the dissertation is presented at the end. iii Dedication To my dad, Alfonso Herrera, who despite his humble upbringing and the countless adversities he faced as an immigrant, was able to support his sons’ dreams. iv Acknowledgements I am grateful to many people for their support. I am particularly grateful to my advisor Dr. Priscila Chaverri for her guidance in completing my research. Drs. Priscila Chaverri, Amy Y. Rossman, and Gary J. Samuels collectively taught me how to collect specimens of the Nectriaceae. Additionally, Gary J. Samuels showed me how handle fresh samples of the Nectriaceae once we had them in the lab as well as how to handle herbarium specimens. Dr. Amy Y. Rossman’s comments on my manuscripts have made me a better writer. I could not be here at this stage in my life without the mentoring of Dr. Chun-Juan Wang at the State University of New York: College of Environmental Science and Forestry (Syracuse, NY) as an undergraduate student. I am grateful to her for sharing her knowledge about the fungi, and teaching me new skills that would allow me to be successful in the mycological world. I thank the many people that helped us with collecting trips: A. Romero and R. Sanchez in Argentina; O. Liparini Pereira, G. Barata, D. Lustosa in Brazil; C. Mendez in Costa Rica; P. Johnston in New Zealand; Luis Mejia in Panama, and T. Iturriaga in Venezuela. I also thank many people that have generously provided us with fresh specimens and/or isolates: Paul Diederich, Yuuri Hirooka, Christian Lechat, Kadri Põldmaa, and Keith A. Seifert. I gratefully acknowledge the assistance of the curators and their staff of the herbaria from which specimens were generously loaned. These herbaria include: U.S. National Fungus Collection (BPI); Royal Botanic Gardens, Kew (K); William and Lynda Steere Herbarium, New York Botanical Garden (NY); v the New Zealand Fungal and Plant Disease Collection (PDD); and Herbarium of the Botany Department, Swedish Museum of National History (S). I thank the Latin American Studies Center (LASC) at the University of Maryland and the North American Mycological Association (NAMA) for the funding of a collecting trip to Panama. I also thank the American Society of Naturalists (ASN) for the partial support to attend the 2013 Workshop on Molecular Evolution at Woods Hole, MA; the Mycological Society of America (MSA), the Fungal Environmental Sampling and Informatics Network (FESIN), and The Graduate School (University of Maryland) for the funding to travel and present at the annual Mycological Society of America meetings. My research was funded by the National Science Foundation (NSF) PEET grant DEB-0731510 and the Department of Plant Science and Landscape Architecture (University of Maryland). vi Table of Contents Preface........................................................................................................................... ii Dedication .................................................................................................................... iii Acknowledgements ...................................................................................................... iv Table of Contents ......................................................................................................... vi List of Tables ............................................................................................................. viii List of Figures .............................................................................................................. ix Introduction ................................................................................................................... 1 CHAPTER SUMMARIES ............................................................................................... 5 Chapter 1: Pseudocosmospora, a new genus to accommodate Cosmospora vilior and related species ............................................................................................................... 8 ABSTRACT .............................................................................................................. 8 INTRODUCTION .................................................................................................... 9 MATERIALS AND METHODS ............................................................................ 11 Teleomorph and anamorph morphological characterization ............................. 11 DNA Extraction, PCR, and Sequencing .............................................................. 13 Phylogenetic Analyses ........................................................................................ 14 RESULTS ............................................................................................................... 16 TAXONOMY ......................................................................................................... 17 Key to species of Pseudocosmospora ................................................................. 19 DISCUSSION ......................................................................................................... 34 Genus Concept .................................................................................................... 34 Species Concept .................................................................................................. 37 CHAPTER 2: REVISION OF THE GENUS CORALLOMYCETELLA WITH CORALLONECTRIA GEN. NOV. FOR C. JATROPHAE (NECTRIACEAE, HYPOCREALES) ............................................ 54 ABSTRACT ............................................................................................................ 54 INTRODUCTION .................................................................................................. 55 MATERIALS AND METHODS ............................................................................ 56 Herbarium specimens and cultures .................................................................... 56 Morphological characterization ......................................................................... 56 DNA extraction, PCR, and sequencing ............................................................... 57 Phylogenetic analyses ......................................................................................... 59 RESULTS ............................................................................................................... 61 DISCUSSION ......................................................................................................... 64 Genus concepts ................................................................................................... 64 Species concepts.................................................................................................. 65 The incorrect application of a name: “Nectria mauritiicola” ........................... 67 vii TAXONOMY ......................................................................................................... 69 Chapter 3: Phylogenetic and taxonomic revision of the Cosmospora viliuscula species complex: Cosmospora species that grow on xylariaceous fungi. ............................. 100 ABSTRACT .......................................................................................................... 100 INTRODUCTION ................................................................................................ 100 MATERIALS AND METHODS .......................................................................... 102 Herbarium specimens and cultures .................................................................. 102 Morphological characterization ....................................................................... 103 DNA extraction, PCR, and sequencing ............................................................. 104 Phylogenetic analyses ....................................................................................... 105 Species Recognition .......................................................................................... 106 RESULTS ............................................................................................................. 107 Phylogenetic analyses ....................................................................................... 107 Morphological studies ...................................................................................... 108 DISCUSSION ....................................................................................................... 109 Cosmospora Rabenh. ........................................................................................ 109 Species recognition ........................................................................................... 110 TAXONOMY ....................................................................................................... 112 Key to species of Cosmospora sensu stricto ..................................................... 112 Chapter 4: Pseudocospeciation of the mycoparasite Cosmospora with their associated fungal hosts. .............................................................................................................. 172 ABSTRACT .......................................................................................................... 172 INTRODUCTION ................................................................................................ 172 METHODS ........................................................................................................... 175 Cosmospora phylogeny ..................................................................................... 175 Host phylogeny.................................................................................................. 176 Cophylogenetic analyses ................................................................................... 177 RESULTS ............................................................................................................. 178 Phylogenetic analyses ....................................................................................... 178 Distance-based analyses ................................................................................... 179 Tree reconciliation analyses ............................................................................. 180 DISCUSSION ....................................................................................................... 180 Conclusions ............................................................................................................... 195 Bibliography ............................................................................................................. 198 viii List of Tables CHAPTER 1: TABLE I.I. ...............................................................................................................40 SUPPLEMENTARY TABLE I.I. ............................................................................41 CHAPTER 2: TABLE 2.1 ...............................................................................................................84 TABLE 2.2 ...............................................................................................................88 TABLE 2.3 ...............................................................................................................88 CHAPTER 3: TABLE 3.1. ............................................................................................................144 TABLE 3.2. ............................................................................................................149 TABLE 3.3. ............................................................................................................150 SUPPLEMENTAL TABLE 3.1.............................................................................151 CHAPTER 4: Table 4.1. ...............................................................................................................185 Table 4.2. ...............................................................................................................187 ix List of Figures CHAPTER 1: FIG. 1.1. ..................................................................................................................45 FIG. 1.2. ..................................................................................................................46 FIG. 1.3. ..................................................................................................................47 FIG. 1.4. ..................................................................................................................48 FIG. 1.5. ..................................................................................................................50 FIG. 1.6. ..................................................................................................................51 FIG. 1.7. ..................................................................................................................52 FIG. 1.8. ..................................................................................................................53 CHAPTER 2: FIG. 2.1. ..................................................................................................................89 FIG. 2.2. ..................................................................................................................90 FIG. 2.3. ..................................................................................................................91 FIG. 2.4. ..................................................................................................................92 FIG. 2.5. ..................................................................................................................93 FIG. 2.6. ..................................................................................................................94 FIG. 2.7. ..................................................................................................................96 FIG. 2.8. ..................................................................................................................98 CHAPTER 3: FIG. 3.1. ................................................................................................................152 FIG. 3.2. ................................................................................................................153 FIG. 3.3. ................................................................................................................154 FIG. 3.4. ................................................................................................................155 FIG. 3.5. ................................................................................................................156 FIG. 3.6. ................................................................................................................157 FIG. 3.7. ................................................................................................................158 FIG. 3.8. ................................................................................................................159 FIG. 3.9. ................................................................................................................160 FIG. 3.10. ..............................................................................................................161 FIG. 3.11. ..............................................................................................................162 FIG. 3.12. ..............................................................................................................163 FIG. 3.13. ..............................................................................................................164 FIG. 3.14. ..............................................................................................................165 SUPPLEMENTAL FIG. S3.1. ...............................................................................166 SUPPLEMENTAL FIG. S3.2. ...............................................................................167 SUPPLEMENTAL FIG. S3.3. ...............................................................................168 SUPPLEMENTAL FIG. S3.4. ...............................................................................169 SUPPLEMENTAL FIG. S3.5. ...............................................................................170 SUPPLEMENTAL FIG. S3.6. ...............................................................................171 x CHAPTER 4: FIG. 4.1. ................................................................................................................188 FIG. 4.2. ................................................................................................................189 FIG. 4.3. ................................................................................................................191 FIG. 4.4. ................................................................................................................192 FIG. 4.5. ................................................................................................................193 FIG. 4.6. ................................................................................................................194 1 Introduction This dissertation is a contribution to fungal systematics. Systematics is the science of biological diversity, specifically the science that discovers, describes, and classifies all organisms. Taxonomy and nomenclature are part of systematics. Taxonomy deals with the classification of species, and nomenclature provides the principles and rules to name species and other taxa (reviewed in Schuh & Brower 2009). In this dissertation, a classification reflecting phylogenetic relationships (phylogeny) was sought. Fungi remain a poorly known group of organisms. Fungal diversity is estimated conservatively at 1.5 million species; however, only 5% of fungal species have been identified and studied (Hawksworth 1991, 2001). Although the number of described species has risen exponentially since the first edition of the Dictionary of the fungi (reviewed in Blackwell 2011), there is much fungal diversity to be discovered. Hence, taxonomic work, such are the first three articles in this dissertation, is important to discover new species and to increase our knowledge of the world’s biota. The fungal system studied in this dissertation was Cosmospora Rabenh. (sensu lato; Nectriaceae, Hypocreales, Ascomycota). Fungi in the Hypocreales are characterized by brightly colored, soft-textured, ostiolate ascomata (sexual fruiting bodies); unitunicate asci; and hyaline to golden-yellow/golden-brown ascospores (sexual spores; Rogerson 1970; Rossman et al. 1999). Hypocrealean asexual states are 2 numerous, but they generally have phialidic conidiogenesis (or conidium development; Samuels and Seifert 1987). There are seven families in the Hypocreales: Bionectriaceae, Clavicipitaceae, Cordycipitaceae, Hypocreaceae, Nectriaceae, Niessliaceae, and Ophiocordycipitaceae. The Nectriaceae have generally superficial, red to purple, uniloculate ascomata that change color in 3% KOH and lactic acid and have non-disarticulating ascospores. Members of the Nectriaceae are unlike species of other major hypocrealean families such as the Hypocreaceae that have disarticulating ascospores and from the Bionectriaceae that have white to brown ascomata that do not change color in 3% KOH and lactic acid (Rossman et al. 1999). Higher-level phylogenetic studies have confirmed the Nectriaceae to be a distinct monophyletic family (Rehner & Samuels 1995; Rossman et al., 2001; Castlebury et al. 2004). Cosmospora (sensu lato) have reddish, small-sized (<300 microns), and pear-shaped sexual fruiting bodies (perithecia). The asci are unitunicate, cylindrical to clavate, and usually have an apical ring. Each ascus contains eight, uniseriately arranged sexual spores (ascospores). The ascospores are ellipsoid to ellipsoid-fusiform, one-septate, yellow-brown and warted at maturity (Samuels et al. 1991; Rossman et al. 1999). The morphology of the sexual state is highly conserved, and it is the main reason that at one time there were 73 species classified under Cosmospora (sensu lato; http://www.indexfungorum.org). However, Cosmospora was thought to be polyphyletic given the range of asexual states (anamorphs). With the use of molecular data, the genus was restricted to cosmospora-like fungi with an acremonium-like asexual state (Gräfenhan et al. 2011). Many monophyletic genera were revived or 3 created to accommodate the remaining species: Chaetopsina Rambelli, Nectricladiella Crous & Schoch, Fusicolla Bonord., Kryptocosmospora Hirooka et al., Macroconia (Wollenw.) Gräfenhan et al., Microcera Desm., Pseudocosmospora C. Herrera & P. Chaverri,, Stylonectria Höhn., and Volutella Fr. (Schoch et al. 2000, Gräfenhan et al. 2011, Luo and Zhang 2010, 2012, Herrera et al. 2013). Most cosmospora-like fungi are parasites of other fungi (mycoparasites) including Cosmospora sensu stricto. Tsuneda (1982) first described the attack by cosmospora- like mycoparasites. Species of Cosmospora penetrate the fruiting bodies of the host, and slowly consume the fleshy insides of the fungal host with its vegetative hyphae. This growth of Cosmospora on their hosts is slow, perhaps to ensure an extended period of nutrient uptake. The fungal host is able to mature, but is prevented from releasing its ascospores. Ultimately, the host’s fleshy insides are completely replaced by vegetative hyphae of the Cosmospora species. The mycoparasitic attack ends with the formation of perithecia directly on the surface of the host’s fruiting bodies, while simultaneously consuming its own vegetative hyphae for the production of perithecia (Tsuneda 1982). An exception to the fungicolous habit of cosmospora-like fungi is Microcera, which is entomo-parasitic on scale insects (Gräfenhan et al. 2011). Porcelli & Frisullo (1998) suggested that insect dispersal might be the primary dispersal mechanism for this group of fungi; asexual spores (conidia) are dispersed via crawlers. It suggests that the attack by Microcera species is slow, but ultimately it will kill the scale insect carrying conidia. Species such as M. larvarum only affects female scale insects, which are not allowed to reproduce. The bodies of infected scale insects are 4 completely colonized with hyphae, and appear as “waxy mummies.” Sporodochia (fructifications of the asexual state) and perithecia are formed on the dead mummified scale insects; the sporodochia appear as a whitish cylindrical stalk and an orange globular head (Porcelli & Frisullo 1998). Additionally, some species of Microcera have been shown to be lichenicolous (Bills et al. 2009; unpublished data). Species of cosmospora-like fungi are of economic importance. Species of Microcera have the potential to become biocontrol agents against scale insects, and have been shown to contain pharmaceutically active secondary metabolites. Studies have shown high mortality rates of scale insects caused by isolates of Microcera in vitro conditions (Ganassi et al. 2000; Cozzi et al. 2002). An antidiabetic secondary metabolite (aquastatin A), which could be useful against type 2 diabetes and obesity, was extracted from a species of Microcera isolated from intertidal sediments (Seo et al. 2009). Antifungal metabolites called parnafungins were extracted from the M. larvarum species complex. Parnafungins have been shown to have potent and broad- spectrum growth inhibitory activity against important clinical fungal pathogens such as Aspergillus fumigatus and Candida albicans (Bills et al. 2009). It is possible that other cosmospora-like fungi could have similar properties to Microcera. Species of Pseudocosmospora are parasites of species of Eutypa Tul. & C. Tul. and Eutypella (Nitschke) Sacc. (Herrera et al. 2013), which include some important plant pathogens. For example, Eutypa dieback of grapevine is caused by Eutypa lata (Pers.) Tul. & C. Tul., and is responsible for significant economic losses in the wine industry (Siebert 2001). Finding a Pseudocosmospora species that could be used as a biocontrol agent against plant diseases caused by Eutypa and Eutypella species is likely. 5 Chapter Summaries The goal of the first chapter is to determine the identity of Cosmospora vilior (Starbäck) Rossman & Samuels. Cosmospora vilior was a confused species as result of the conservative morphology of Cosmospora species. The name was applied to Cosmospora species that grow on xylariaceous fungi (fungi growing on the fruiting bodies the Xylariaceae) and had a dark-green colony on cornmeal-dextrose agar (CMD; Samuels et al. 1991). However, the holotype specimen of C. vilior was determined to grow on a Eutypella species. A recently collected specimen resembling the holotype specimen of C. vilior was selected as epitype. An epitype is a specimen that supplements the holotype specimen (i.e., it provides information on missing characters such as DNA barcodes). Cosmospora vilior clustered with other Cosmospora species that grew on Eutypa and Eutypella species. The clade was determined to be distinct from other cosmospora-like genera, and described as a new genus Pseudocosmospora C. Herrera & P. Chaverri. The following characters unite Pseudocosmospora species: the asexual state (acremonium-like), the colony (salmon- pink in potato-dextroxe agar), and the hosts. Ten species were included in Pseudocosmospora including P. vilior (Starbäck) C. Herrera & P. Chaverri (≡ C. vilior). The first chapter has been published as Herrera et al. (2013a). The second chapter is a taxonomic revision of Corallomycetella Henn. (Nectriaceae, Hypocreales, Ascomycota). Corallomycetella is basal to Cosmospora (in the broad sense, excluding Chaetopsina and Volutella; Hirooka et al. 2011). Two species of Corallomycetella were included in Rossman et al. (1999), and are plant pathogens of tropical trees such as rubber trees (Hevea brasiliensis Müll.Arg.). Corallomycetella 6 species are characterized by the root-like structures (rhizomorphs) produced in culture and in nature. The ascospores are reported to be smooth in C. repens (Berk. & Broome) Rossman & Samuels and roughened in C. jatrophae (Möller) Rossman & Samuels. However, a recent collection had striated ascospores, but otherwise was similar to Corallomycetella species. This collection led us to re-examine Corallomycetella, and determine if this unusual specimen represented a new species. Striated ascospores (surface view) were determined to be an overlooked character in C. repens. Corallomycetella repens was determined to comprise two species, and C. jatrophae was determined to be unrelated to Corallomycetella. Corallonectria C. Herrera & P. Chaverri was described to accommodate C. jatrophae. Additionally, we found sequences in GenBank that have been labeled as “Nectria mauritiicola,” a synonym of C. repens (sensu Rossman), but are actually sequences of Sarocladium kiliense (Grütz) Summerb. The second chapter has been published as Herrera et al. (2013b). The third chapter is taxonomic revision of Cosmospora viliuscula species complex. Cosmospora viliuscula grows on other fungi, particularly on the fruiting bodies of the Xylariaceae. It was previously confused with C. vilior. Apart from the host, C. viliuscula is characterized by the dark-green colony produced on CMD (Samuels et al. 1991). Up to now, it has been thought to consist of a single species, but the phylogenetic results revealed that Cosmospora viliuscula was a species complex. Each well-supported clade was host specific and regarded as a species. Cosmospora viliuscula was restricted to Cosmospora species growing on Kretzschmaria cf. deusta (in tropical forests). Seven new species and one new combination were described. 7 Additionally, the sexual states of C. arxii a (W. Gams) Gräfenhan & Schroers and C. khandalensis (Thirum. & Sukapure) Gräfenhan & Seifert were described for the first time. The fourth chapter seeks to determine the evolutionary relationship between Cosmospora species and their associated fungal hosts. During the taxonomic revision of these fungi (chapter three), it was observed that these species demonstrated a high degree of host-specificity, which suggested that their association could not be random. It was hypothesized that Cosmospora species and their associated fungal hosts had cospeciated. The phylogenies of Cosmospora and their hosts were determined to be congruent. However, there was only a global congruence. The only host-parasite links that could be considered cospeciation events occurred in more recent evolutionary lineages of the host. Host-switches seemed to be occur more frequently in the early lineages of the host. This suggests that the host-switch events must have been very conservative (i.e., host-switches between closely related hosts) that mimic the phylogenetic signal of cospeciation. 8 Chapter 1: Pseudocosmospora, a new genus to accommodate Cosmospora vilior and related species C.S. Herrera, A.Y. Rossman, G.J. Samuels, and P. Chaverri. 2013. Mycologia 05(5): 1287–1305. Reprinted with permission of Mycologia. ©The Mycological Society of America. ABSTRACT Cosmospora sensu Rossman accommodated nectroid fungi with small, reddish, smooth, thin-walled perithecia but recently was found to be polyphyletic and has been segregated into multiple genera. Not all cosmospora-like fungi have been treated systematically. Some of these species include C. vilior and many specimens often labeled as “Cosmospora sp.” The objectives of this research were to establish the identity of C. vilior through epitypication using a recent collection that agrees with the type specimen in morphology, host and geography, and to determine its phylogenetic position within Cosmospora sensu lato and the Nectriaceae. A multilocus phylogeny was constructed based on six loci (ITS, LSU, MCM7, rpb1, tef1, and tub) to estimate a phylogeny. Results from the phylogenetic analyses indicated that C. vilior forms a monophyletic group with other cosmopora-like fungi that have an acremonium-like anamorph and that parasitize Eutypa and Eutypella (Ascomycota, Sordariomycetes, Xylariales, Diatrypaceae). The group is 9 phylogenetically distinct from other previously segregated genera. A new genus, Pseudocosmospora is described to accommodate the type species, P. eutypellae, and nine additional species in this clade. INTRODUCTION Cosmospora sensu Rossman (Nectriaceae, Hypocreales, Ascomycota; Gräfenhan et al. 2011) was erected to accommodate nectroid fungi with small, reddish, KOH+, smooth, thin-walled, laterally collapsing when dry, non- or weakly stromatic perithecia (Samuels et al. 1991, Rossman et al. 1999). These fungi have been reported throughout the world, but they are assumed to have greater diversity in warm temperate and tropical regions. In addition, they tend to have a higher diversity in recently disturbed stands (1–2 years old) compared to early successional stands (25– 27 years old) and old-growth stands in tropical forests (Chaverri and Vílchez 2006). In that study, frequently collected species in recently disturbed stands, where newly killed woody substrates and herbaceous debris are prevalent, were members of Chaetopsinectria Lou & Zhuang and Volutellonectria Lou & Zhang, two genera segregated from Cosmospora sensu Rossman (Luo and Zhuang 2010, 2012). Many species of Cosmospora sensu Rossman are parasites of their fungal hosts (see Tsuneda 1982). Among genera segregated from Cosmospora sensu Rossman, some members of Cosmospora sensu stricto grow on basidiomycetes or xylariaceous hosts, species of Dialonectria (Sacc.) Cooke occur on Diatrype Fr. (Diatrypaceae), and Microcera Desm. parasitize scale insects (Gräfenhan et al. 2011). The generic name Cosmospora has been a source of much taxonomic 10 confusion. Rabenhorst (1862) described this genus that was later reduced to a subgenus of Nectria (Fr.) Fr. by Saccardo (1883). Much later, it was synonymized with Dialonectria (Moravec 1954), which had been elevated from a subgenus of Nectria to generic rank by Cooke (1884). Rossman et al. (1999) resurrected the generic name Cosmospora based on priority. The group has also been referred to as Nectria subgenus Dialonectria or the ‘Nectria episphaeria-group’ (Booth 1959, Samuels et al. 1991, Rossman et al. 1999). Early on, the group was presumed to be polyphyletic given its range of anamorphs and ecological niches (Samuels et al. 1991), and at some point, there were about 70 species classified under Cosmospora (www.indexfungorum.org). The polyphyly of Cosmospora was confirmed by recent phylogenetic studies (Zhuang and Zhuang 2006, Luo and Zhuang 2008, Samuels et al. 2009, Gräfenhan et al. 2011). Following the genus-for-genus concept, i.e. the delimitation of a genus based on the correlation of the teleomorph to its corresponding anamorph (Rossman 1993), Cosmospora was segregated into new or revived genera that correlate roughly with the anamorphs: Chaetopsinectria, Cyanonectria Samuels & P. Chaverri, Nectricladiella Crous & Schoch, Fusicolla Bonord., Macroconia (Wollenw.) Gräfenhan et al., Microcera, Stylonectria Höhn., and Volutellonectria (see Schoch et al. 2000, Samuels et al. 2009, Luo and Zhuang 2010, 2012, Gräfenhan et al. 2011). With the change to one scientific name for each species as directed in the International Code of Nomenclature for algae, fungi, and plants (ICN) (McNeill et al. 2012), Chaetopsinectria and Volutellonectria are considered synonyms of the older genera Chaetopsina Rambelli and Volutella Fr. Cosmospora vilior was described as Nectria vilior by Starbäck (1899) with the 11 diagnosis “Peritheciis discretis, superficialibus, ovoideis, coccineis...Hab. in fungillo valsaceo.” Traditionally, the name has been applied to collections of cosmospora-like fungi having short, coarsely warted ascospores occurring on black stromata, particularly those of the Xylariales (Weese 1916, Samuels et al. 1990, Samuels et al. 1991). Nectria vilior has been reported to have a wide tropical and temperate distribution (Samuels et al. 1990). Re-examination of the type specimen of C. vilior revealed that its associated host is a species of Eutypella (Nitschke) Sacc. (Diatrypaceae). Our recent molecular analyses suggest that true C. vilior is unrelated to species of Cosmospora that occur on xylariaceous fungi, hereafter referred to as the C. viliuscula species complex. Species of the C. vilior complex occur only on species of Eutypella (Diatrypaceae) while C. viliuscula and related species are restricted to xylariaceous fungi. The present paper deals with the phylogenetic and taxonomic reassessment of the Cosmospora vilior and similar taxa. The objectives of this research are: (i) to establish the identity of C. vilior and stabilize the name using epitypification, (ii) to elucidate the phylogenetic placement of C. vilior and related species within Cosmospora sensu Rossman and in the Nectriaceae, (iii) to describe a new genus, Pseudocosmospora, to accommodate C. vilior and related species, and (iv) to describe new species within Pseudocosmospora including the type P. eutypellae. MATERIALS AND METHODS Teleomorph and anamorph morphological characterization Herbarium specimens were borrowed from the U.S. National Fungus Collections 12 (BPI), the William and Lynda Steere Herbarium, New York Botanical Garden (NY), and the Linnean Herbarium, Swedish Museum of Natural History (S). Fresh specimens were collected on trips to Argentina, Brazil, Costa Rica, France, and USA. For the characterization of the teleomorph, the following observations were made for perithecia: shape, size (length and width), color, ornamentation, and habit, e.g. perithecia being solitary or gregarious, immersed in substrata or superficial, stromatic or non-stromatic, and collapsing laterally or not when dry. Reaction to 3% w/v potassium hydroxide (KOH) and 100% lactic acid was observed for the perithecial wall. Sections of perithecia (ca. 11 µm in thickness) were made with the aid of a freezing microtome. Measurements of continuous characters (e.g. length and width) were made with Scion Image software beta 4.0.2 (Scion Corp., Frederick, Maryland) and summarized by descriptive statistics (e.g., minimum, maximum, mean and standard deviation). Cultures were obtained from the culture collection at USDA, ARS, Systematic Mycology and Microbiology Laboratory (SMML). Additional cultures were obtained by isolating single ascospores from freshly collected samples with the aid of a micromanipulator and grown in cornmeal dextrose agar (CMD; Difco™ cornmeal agar + 2% w/v dextrose + antibiotics). Morphological observations of the colony were made by growing three pseudoreplicates of each isolate on CMD and Difco™ potato dextrose agar (PDA) in an incubator that alternates 12h/12h between fluorescent light and darkness at 25 C. Cultural morphology is described based on strains grown on PDA; cultures on CMD exhibit little variability. Colony color is described using the color terms in Rayner (1970). Culture growth was measured 13 weekly for two weeks. The anamorph was observed by cutting an agar block of a culture grown in synthetic nutrient-poor agar (SNA; Nirenberg 1976) under the same conditions mentioned above, covering it with a cover-slip, and examining it by light microscopy (Olympus BX50; Olympus, Tokyo, Japan). Measurements of continuous characters were made and analyzed as described above. DNA Extraction, PCR, and Sequencing Hirooka et al. (2010) described the DNA extraction protocol used here. Briefly, the isolates were grown in Difco™ potato dextrose broth (PDB), and the mycelial mat was harvested after a week of growth. DNA was extracted with PowerPlant® DNA Isolation Kit (MO BIO Laboratories Inc., Solana Beach, California). Six partial loci were amplified. These loci are internal transcribed spacer (ITS; primers: ITS5 and ITS4; White et al. 1990), large subunit nuclear ribosomal DNA (LSU; primers: LROR and LR5; Vilgalys and Hester 1990), MCM7 (a DNA replication licensing factor; primers: Mcm7-709for & Mcm7-1348rev; Schmitt el al. 2009), RNA polymerase II subunit one (rpb1; primers: Crpb1a & rpb1c; Castlebury et al. 2004), translation elongation factor 1-α (tef1; primers: Tef1-728 and Tef1-986; Carbone and Kohn 1999), and β-tubulin (tub; O’Donnell and Cigelnik 1997). The PCR reaction mixture (25 µL total volume) consisted of 12.5 µL GoTaq®Green Master Mix 2X (Promega Corporation, Madison, Wisconsin), 1.25 µL for the forward and reverse primers each (10 mM), 1.0 µL of dimethyl sulfoxide (DMSO; Sigma- Aldrich, St. Louis, Missouri), up to 5.0 µL genomic DNA template, and RNAse-free water to complete the total volume. PCR reactions were carried out in an Eppendorf 14 Mastercycler thermocycler (Eppendorf, Westbury, New York) under the cycle conditions listed in TABLE I. I. PCR products were cleaned with ExoSAP-IT® (USB Corp., Cleveland, Ohio). Clean PCR products were sequenced at the DNA Sequencing Facility (Center for Agricultural Biotechnology, University of Maryland, College Park, Maryland) and McLAB DNA sequencing services (San Francisco, California). Sequences were assembled and edited with Sequencher 4.9 (Gene Codes, Madison, Wisconsin). Sequences were deposited in GenBank (SUPPLEMENTARY TABLE I. I.). Phylogenetic Analyses Two separate phylogenetic analyses were performed on two separate data sets as described below. The first data set contained a reduced number of isolates of Cosmospora vilior and related taxa as well as species of other cosmospora-like fungi to elucidate their phylogenetic placement in the Nectriaceae. The second data set contained all isolates of Cosmospora vilior and related taxa to determine their relationships. ITS-LSU, MCM7, rpb1, tef1, and tub sequences were aligned with MAFFT 6 (Katoh 2008), and manually edited, if necessary, in Mesquite 2.75 (Maddison and Maddison 2011). Gaps (insertions/deletions) were treated as missing data. Alignments were deposited in TreeBASE (http://www.treebase.org; accession no. S14038). Maximum likelihood (ML) and Bayesian (BI) analyses were performed on each of the datasets of individual loci first and then on the concatenated dataset. CONCATEPILLAR 1.4 (Leigh et al. 2008) was used to determine whether loci could 15 be analyzed by concatenating the datasets or whether loci should be analyzed separately. Loci were concatenated if the p-value was greater than the default α-level of 0.05, which indicated that the null hypothesis (i.e., congruence of loci) could not be rejected. For both ML and BI analyses, jModeltest (Guindon and Gascuel 2003, Posada 2008) was used to infer the models of nucleotide substitution for each locus. Default settings in jModeltest were used: 11 substitution schemes with equal or unequal base frequencies (+F) and invariable sites (+I) and/or rate variation among sites (+G). The base tree for likelihood calculations was ML optimized. Once likelihood scores were calculated, the models were selected according to the Akaike Information Criterion (AIC). Maximum likelihood (ML) analyses were performed with GARLI v2.0 (Genetic Algorithm for Rapid Likelihood Inference; Zwickl 2006) by submitting the job via the GARLI web service at http://www.molecularevolution.org (Bazinet and Cummings 2011), which uses a grid computing system (Cummings and Huskamp 2005) associated with The Lattice Project (Bazinet and Cummings 2008). Fifty independent search replicates were performed to search for the best tree. The starting tree was generated using a fast ML stepwise-addition algorithm. Two thousand bootstrap replicates were used for bootstrap analysis. Bayesian analyses were performed in MrBayes v3.2.1 (Ronquist et al. 2012). A majority rule consensus tree was generated by running four chains for 10,000,000 Markov Chain Monte Carlo generations, sampling trees every 100th generation, and discarding the first 25% of the sampled trees as burn-in. Tracer version 1.5 (Rambaut and Drummond 2007) was 16 used to confirm whether the negative log likelihoods had reached convergence. RESULTS Phylogenetic analyses: phylogenetic placement of C. vilior and related species within Cosmospora sensu Rossman.—The analysis performed in CONCATEPILLAR failed to reject the null hypothesis of congruence among loci (P =0.08). Therefore, all loci were concatenated. The concatenated matrix included 22 ingroup isolates that formed five major groups plus two outgroup taxa (Corallomycetella repens and Pseudonectria pachysandricola). It consisted of 3591 base pairs of which 970 were parsimony-informative, 318 were parsimony-uninformative, and 1858 were invariable sites. The topologies of the generated phylogenetic trees in both ML and BI were congruent. The negative log likelihoods for the phylogenetic trees were −16460.154 and −16513.115, respectively. The best tree (ML) is shown (FIG. 1.1). Cosmospora vilior, C. joca and related species formed a highly supported clade (94% BP, 100% PP). This clade is related to Dialonectria, Cosmospora sensu stricto and an orphan group that includes C. flavoviridis, C. obscura, and C. stegonsporii. These clades of cosmospora-like fungi were highly supported as well (>70% BP, >95% PP), but the inner nodes connecting these clades were poorly supported. Basal to all of these groups is Microcera, another segregate genus of cosmospora-like fungi. Phylogenetic analyses: relationship among C. vilior and related species.—The null hypothesis of congruence among loci (P =0.11) was not rejected in CONCATERPILLAR, and therefore, the five loci were concatenated to estimate a phylogeny. The concatenated matrix included 25 isolates belonging to the ingroup 17 and two outgroup taxa (C. repens and M. larvarum). The concatenated matrix consisted of 3353 bp of which 651 were parsimony-informative, 456 were parsimony- uninformative, and 1785 invariable sites. The tree topologies generated with ML and BI were congruent with one another. The log likelihoods for these two analyses were −23024.3191 and −23055.9286, respectively. The best tree generated with ML is shown (FIG. 1.2). The combined analyses of Cosmospora vilior and related species revealed that there were as many as 16 independently evolving lineages (= putative species). Clade I is a complex of species whose hosts are Eutypella species. Three species are recognized within this clade, which include C. vilior and two species described below (Pseudocosmospora eutypellae and P. rogersonii). Sister to clade I is P. eutypae (described below), whose host is a species of Eutypa Tul. & C. Tul. Sister to clade II (P. eutypae + clade I) is clade III, which comprises two monotypic species, C. joca and P. metajoca (described below). Cosmospora joca is associated with a species of Biscogniauxia Kuntze (Xylariaceae), while P. metajoca is associated with a species of Eutypa. All clades corresponding to recognized species received maximum BP and PP support (with one exception). TAXONOMY Pseudocosmospora C. Herrera & P. Chaverri, gen. nov. MycoBank MB 802432 Type species: Pseudocosmospora eutypellae C. Herrera & P. Chaverri 18 Etymology: “Pseudo” from Greek referring to the morphological similarity to both the teleomorphic and anamorphic states of Cosmospora sensu stricto. Teleomorph: Stroma absent. Perithecia superficial or slightly immersed in fungal host stroma, scattered to gregarious, subglobose to obpyriform with a blunt papilla, generally less than 250 μm high, soft-textured, smooth-walled, scarlet, KOH+ blood- red, LA+ yellow, collapsing laterally when dry, uniloculate. Perithecial surface cells forming textura angularis. Perithecial wall generally 20–30 μm thick, of two regions; outer region of cells forming textura globulsa to t. angularis; inner region of cells forming textura prismatica. Asci unitunicate, cylindrical to narrowly clavate, increasing in size as ascospores mature, without a conspicuous apical ring, with eight spores arranged uniseriately. Ascospores ellipsoidal, 1-septate, slightly constricted at septum, yellow-brown, verrucose, sometimes appearing smooth at maturity. Anamorph in culture: After 21 d at room temperature on PDA, colony surface crustose with no aerial mycelium or cottony with aerial mycelium, rosy-buff, pale- luteous, or salmon-pink. Sporulation on SNA usually abundant, arising directly from agar surface. Anamorphic state acremonium-like to verticillium-like; conidiophores generally simple, unbranched, sometimes verticillately branched, rarely densely aggregated. Phialides monophialidic, cylindrical, hyaline. Conidia ellipsoidal, ovoid, or reniform, smooth, sometimes guttulated, non-septate, hyaline. Habitat: On stromata of diatrypaceous fungi, particularly species of Eutypa and Eutypella, rarely on species of Biscogniauxia. Distribution: Asia, Africa, Europe, North America, Oceania, South America, possibly 19 cosmopolitan. Notes: Pseudocosmospora is similar to Cosmospora sensu stricto in its cosmospora- like teleomorph and acremonium-like anamorph, although they differ in cultural characteristics and host preference. Pseudocosmospora is most common on diatrypaceous fungi except for P. joca, which occurs on a Biscogniauxia sp. The latter does not belong among the known hosts of Cosmospora sensu stricto, although these attack xylariaceous fungi as well as polypores. In general, species of Pseudocosmospora have pinkish-colored colonies, while species of Cosmospora sensu stricto have olivaceous-green colonies on PDA. Phylogenetically, Pseudocosmospora appears to be closely related to Dialonectria. Both occur on diatrypaceous fungi, although they attack different genera. The genera also differ in their anamorphic state; Dialonectria has a fusarium-like anamorph. Key to species of Pseudocosmospora 1. On Biscogniauxia (Xylariaceae)…………………………………………….P. joca 1. On Eutypa or Eutypella (Diatrypaceae)…………………………………………...2 2. On Eutypa………………………………………………………………………….3 2. On Eutypella…………………………………………………………………….....4 3. Ascospores smooth, 6.3–8.7 μm long; colony rosy buff, 6–15 mm diam after 14 d at 25 C on PDA………………………………………………..……….P. eutypae 3. Ascospores verrucose, 7.7–11.9 μm long; colony salmon-pink, 24–25 mm diam after 14 d at 25 C on PDA…………………………...………………..P. metajoca 20 4. Perithecia with a discoidal apex…………………………………………………...5 4. Perithecia with a blunt apex…………………………………………………..…...6 5. Fungal host on Alnus sp.; ascospores smooth, 9–10.4 μm……....……..P. pithoides 5. Fungal host on Espeletia sp.; ascospores verrucose, 11–14 μm long…………………………………………..……………….P. pseudepisphaeria 6. Conidiophores branching, becoming densely ramulose (fasciculate) on SNA……………………………………………………………….……....P. triqua 6. Conidiophores not branching or sparingly branched on SNA………………..…...7 7. Colonies pale-luteous on PDA; conidia reniform, with two guttules at opposite ends, 3.4–7.4 μm long……………………………………….………………P. vilior 7. Colonies white to salmon-pink on PDA………………………………..……….....8 8. Colonies white on PDA; ascospores smooth, 10–15 μm long…………………………………………………………….. P. metepisphaeria 8. Colonies salmon-pink on PDA…………………………………..………………...9 9. Ascospores verrucose, 7.1–12.5 μm long; colonies 7.5–20 mm diam after 14 d at 25 C on PDA; conidia oblong to ellipsoidal, with two guttules at opposite ends, 3.1–6.2 μm long………………………………………………….…...P. eutypellae 9. Ascospores smooth, 7.9–12.2 μm long; colonies 18–27.5 mm diam after 14 d at 25 C on PDA; conidia oblong to ellipsoidal, without guttules, 2.9–5.5 μm long………………………………………………..……………..…...P. rogersonii Pseudocosmospora eutypae C. Herrera & P. Chaverri, sp. nov. FIG. 1.3. 21 Mycobank MB 802433 Holotype: FRANCE, Poitou-Charentes, Saint George de Rex (Marais Poitevin), on Eutypa sp., 26 Apr. 2011, C. Herrera (C.H. 11-01), BPI 884164, ex-holotype culture CBS 133961. Etymology: In reference to its fungal host, Eutypa. Teleomorph: Perithecia solitary, superficial, nonstromatic, subglobose with a discoidal apex, collapsing laterally when dry, scarlet, smooth, 171–200 × 150–183 μm (mean = 182 × 165; SD 16.1, 16.5; n = 3). Asci cylindrical to slightly clavate, with eight spores arranged uniseriately, 54–66 × 5.5–7 μm (mean = 59 × 6; SD 5.4, 0.7; n = 4). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, smooth, hyaline, 6.3–8.7 × 3.1–4.1μm (mean = 7.8 × 3.6; SD 0.7, 0.3; n = 30). Anamorph: Colonies 6–15 mm diam (mean = 11.4; SD 4.1; n = 5) after 14 d. at 25 C on PDA, cottony with rosy-buff aerial mycelium, reverse concolorous. Sporulation on SNA usually abundant, arising directly from agar surface. Anamorphic state acremonium-like; conidiophores generally simple, unbranched. Phialides monophialidic, cylindrical, collarette not flared, hyaline, length 36–53 μm (mean = 41.3; SD 4.1; n = 13), width at base 1.5–2.0 μm (mean = 1.8; SD 0.2; n = 13), width at tip 1–1.3 μm (mean = 1.1; SD 0.1; n = 13). Conidia oblong, unicellular, smooth, hyaline, 4.6–6.7 × 1.2–2.1 μm (mean = 5.7 × 1.7; SD 0.6, 0.2; n = 30). Habitat: On Eutypa cf. lata (Diatrypaceae) on bark. 22 Distribution: France, United Kingdom. Additional isolates examined: UNITED KINGDOM, on Crataegus sp., 1958, S. Francis, culture IMI 73016. Notes: Pseudocosmospora eutypae occurs on Eutypa cf. lata and has small and smooth ascospores. Pseudocosmospora metajoca is the only other species of Pseudocosmospora on Eutypa, but it has longer and verrucose ascospores. Pseudocosmospora eutypellae C. Herrera & P. Chaverri, sp. nov. FIG. 1.4. Mycobank MB 802434 Holotype: USA, Maryland, Beltsville, on Eutypella sp., on dead twigs of unidentified tree, 7 Oct. 2008, Y. Hirooka (Y.H. 08-17), BPI 884165, ex-holotype culture CBS 133966 = A.R. 4562. Etymology: In reference to its fungal host, Eutypella. Teleomorph: Perithecia gregarious, slightly immersed in host stromata, subglobose with a blunt apex to obpyriform, collapsing laterally, scarlet, smooth, 143–303 × 108– 205 μm (mean = 202 × 150; SD 39, 22.7; n = 22). Asci cylindrical to slightly clavate, eight-spored, uniseriately arranged, 63–78.6 × 5.9–7.9 μm (mean = 71.6 × 6.7; SD 4.5, 0.6; n = 27). Ascospores ellipsoid to fusiform, equally two celled, slightly verrucose, yellow-brown, 7.1–12.5 × 3.6–5.6 μm (mean = 9.9 × 4.5; SD 0.9, 0.4; n = 136). Anamorph: Colonies 7.5–20 mm diam (mean = 12.6, SD 3.4, n = 18) after 14 d. at 25 23 C on PDA, sometimes crustose, with or without aerial mycelium, buff, rosy-buff, or salmon-pink, reverse concolorous. Sporulation on SNA usually abundant, arising directly from agar surface, sometimes from lateral pegs. Anamorphic state acremonium-like to verticillium-like; conidiophores simple, unbranched, or branched, becoming densely branched. Phialides monophialidic, cylindrical, collarette not flared, hyaline, length (3.8–) 7.8–15.1 (–18.5) μm (mean = 10.2; SD 3.0; n = 29), width at base 0.9–2.3 μm (mean = 1.3; SD 0.3; n = 29), width at tip 0.7–1.2 μm (mean = 0.9; SD 0.14; n = 29). Conidia oblong to ellipsoidal, unicellular, with two guttules at opposite ends, smooth, hyaline, 3.1–6.2 × 1.0–2.4 μm (mean = 4.2 × 1.5; SD 0.6, 0.2; n =1 80). Habitat: On Eutypella sp. (Diatrypaceae) on bark. Distribution: France and U.S.A. Additional specimens and isolates examined: FRANCE, Oloron, Forêt de Bugangue, on Eutypella sp., on bark of Robinia pseudoacacia (?), 17 May 1993, F. Candoussau & J.D. Rogers (F. 262), BPI 802567, culture CBS 128986 = G.J.S. 93-15; USA, Kentucky, Clermont, Bernheim Arboretum and Research Forest, on Eutypella sp., on dead branch of unidentified tree, 27 June 2010, Y. Hirooka, BPI 884169, culture CBS 133977 = G.J.S. 10-248; Maryland, Frederick County, Cunningham Falls State Park, on Eutypella sp., on Rhus typhina, 26 Aug. 2007, L. Vasilyeva, BPI 878454, culture CBS 129430 = A.R. 4453; Pennsylvania, Greensburg, on Eutypella sp., Aug. 2008, J. Plitschke, culture CBS 133965 = A.R. 4527; West Virginia, Grafton, on Eutypella sp., on bark of unidentified tree, 26 June 2010, Y. Hirooka, BPI 884168, culture CBS 24 133960 = C.H. 10-02. Notes: Pseudocosmospora eutypellae is most closely related and similar to P. rogersonii, but can be distinguished from the latter by the ornamentation of its ascospores. Pseudocosmospora eutypellae has verrucose ascospores, while P. rogersonii has smooth ascospores. Pseudocosmospora joca (Samuels) C. Herrera & P. Chaverri, comb. nov. FIG. 1.5. Mycobank MB 802435 Basionym: Nectria joca Samuels, Mycol. Pap. 164: 21. 1991. ≡ Cosmospora joca (Samuels) Rossman & Samuels, Stud. Mycol. 42: 122. 1999. Teleomorph: Perithecia gregarious, superficial, nonstromatic, subglobose with a minute papilla, collapsing laterally, scarlet at first, becoming blood red, darker at apex, smooth, 375–384 × 317–349 μm (mean = 380.2 × 336.3; SD 4.9, 16.9; n = 3). Asci cylindrical to clavate, eight-spored, uniseriately arranged, 90.7–112.6 × 8.8–11.1 μm (mean = 102.2 × 9.8; SD 8.5, 0.7; n = 9). Ascospores ellipsoid, equally two- celled, one-septate, constricted at septum, verrucose, yellow-brown, 10.9–14 × 6.4– 7.7 μm (mean = 12.8 × 7; SD 0.8, 0.3; n = 30). Anamorph: Colonies 4 mm diam (n = 3) after 14 d. at 25 C on PDA, crustose, salmon-pink to orange colony, reverse concolorous. Rarely sporulating on SNA. Anamorphic state acremonium-like; conidiophores generally simple, unbranched. Phialides monophialidic, cylindrical, hyaline, length 14.3–24.2 µm (mean = 18.8; SD 25 5.0; n = 3), width at base 1.6–2.4 µm (mean = 2.0; SD 0.4; n = 3), width at tip 1.1 (n = 3). Conidia oblong, unicellular, smooth, hyaline, 3.0–5.5 × 1.3–2.1 µm (mean = 4.1 × 1.6; SD 0.6, 0.2; n = 30). Habitat: On Biscogniauxia sp. on bark. Distribution: Argentina and Brazil. Holotype: BRAZIL, Amazonas, Pico Rondon, Km 211 on Perimetral Norte, ca 3h walk from FUNAI post toward summit, 01°32’N, 02°48’W, on Biscogniauxia sp., 25 Mar. 1984, G.J. Samuels (1094), Pipoly & Guedes, INPA (not seen), ISOTYPES BPI 802606, NY 00671973 (not seen). Epitype designated herein: ARGENTINA, Río Negro Province, San Carlos de Bariloche, Luma forest, on Biscogniauxia sp., on very rotten wood, 15 Apr. 2011, A. Romero, BPI 884175, ex-epitype culture CBS 133967 = A.R. 4779. Notes: The application of the name is restricted here to species of Pseudocosmospora on Biscogniauxia. The isotype and the designated epitype both occur on species of Biscogniauxia. The colony and the anamorph are similar to the morphology described in the original description although the perithecia and ascospores of the epitype are larger than those reported in the literature. Pseudocosmospora metajoca C. Herrera & P. Chaverri, sp. nov. FIG. 1.6. Mycobank MB 802436 Holotype: NEW ZEALAND, North Island, Mt. Williams, on Eutypa sp., on dead 26 woody branch of Beilschmiedia tawa, 7 Mar. 2009, A.Y. Rossman & P. Chaverri (P.C. 952), BPI 879088, ex-holotype culture CBS 133968 = A.R. 4576. Etymology: ‘Meta’ from Greek meaning adjacent and ‘joca’ in reference to the fact that it was originally classified as C. joca, and later found to be phylogenetically close to C. joca. Teleomorph: Solitary or gregarious, superficial, nonstromatic, subglobose with a discoidal apex, some collapsing laterally, scarlet, smooth, 222–251 × 204–213 μm (mean = 236 × 208; n = 2). Asci clavate, eight-spored, uniseriately arranged, 62.2– 69.2 × 5.9–7.2 μm (mean = 65.5 × 6.4; SD 2.5, 0.5; n = 5). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, slightly verrucose, yellow-brown, 7.7–11.9 × 3.3–5.3 µm (mean = 8.9 × 4.3; SD 0.9, 0.5; n = 29). Anamorph: Colonies 24–25 mm diam (mean = 24.5; SD 0.5; n = 3) after 14 d. at 25 C on PDA, slightly cottony, pale salmon-pink, reverse concolorous. Sporulation on SNA usually abundant, arising directly from agar surface. Anamorphic state acremonium-like; conidiophores generally simple, unbranched. Phialides monophialidic, cylindrical, collarette not flared, hyaline, length 26–49 μm (mean = 37.3; SD 5.7; n = 9), width at base 2.1–2.8 μm (mean = 2.4; SD 0.2; n = 9), width at tip 1–1.3 μm (mean = 1.2; SD 0.1; n = 9). Conidia oblong to ellipsoidal, unicellular, guttulated, smooth, hyaline, 3.8–6.1 × 1.6–3.1 μm (mean = 4.8 × 2.1; SD 0.6, 0.3; n = 30). Habitat: On Eutypa sp. (Diatrypaceae) on dead branch of Beilschmiedia tawa. 27 Distribution: New Zealand. Notes: Pseudocosmospora metajoca was originally identified as C. joca based on its occurrence on what was thought to be a stroma of a Biscogniauxia species and its salmon-pink culture on PDA. On close examination of the specimen, the host was found to be a species of Eutypa. The colony of P. metajoca has a faster growth rate than that of P. joca. In addition, P. metajoca has much smaller perithecia and ascospores compared to P. joca. Pseudocosmospora metajoca differs from P. eutypae, the other Pseudocosmospora on Eutypa, by having verrucose ascospores. Pseudocosmospora metepisphaeria (Samuels) C. Herrera & P. Chaverri, comb. nov. Mycobank MB 802437 Basionym: Nectria metepisphaeria Samuels, Mycol. Pap. 164: 29. 1991. ≡ Cosmospora metepisphaeria (Samuels) Rossman & Samuels, Stud. Mycol. 42: 123. 1999. Anamorph: Acremonium-like Habitat: On Eutypella sp. (Diatrypaceae) on unidentified bark. Distribution: Venezuela (known only from the type collection) Holotype: VENEZUELA, Drt. Federale, vic. Macarao, on Eutypella sp., on unidentified bark, 21 Jun. 1971, K.P. Dumont (VE 335), J.H. Haines, G. Morillo & E. Moreno, VEN (not seen), ISOTYPE NY. Notes: The isotype specimen was studied and determined to occur on a Eutypella sp. 28 Based on this host, it can be predicted that C. metepisphaeria would fall within the Pseudocosmospora clade. In addition to the host, the reported acremonium-like anamorphic state supports the placement of this species in Pseudocosmospora. Unique to this species is its smooth ascospores, (10–)11–14(–15) µm long and the white, crustose colony on PDA, reverse brown (Samuels et al. 1991). A culture no longer exists. Pseudocosmospora pithoides (Ellis & Everh.) C. Herrera & P. Chaverri, comb. nov. Mycobank MB 802438 Basionym: Nectria pithoides Ellis & Everh., Proc. Acad. Nat. Sci. Philad. 43: 247 (1891). Anamorph: Unknown Habitat: On an Eutypella sp. (Diatrypaceae) on bark of dead alder. Distribution: British Columbia (known only from the type collection). Holotype: CANADA, British Columbia, on bark of dead alder, May 1889, J. Macoun (122), NY 00927939. Notes: The holotype specimen of Nectria pithoides was examined and determined to agree with the concept of Pseudocosmospora in regard to the host, which appears to be a Eutypella species. The perithecia have a prominent discoidal apex, which according to the description, gives an impression of being barrel-shaped (pithos from Greek = barrel). No asci were observed. The ascospores are ellipsoidal, one-septate, slightly constricted at the septum, smooth, 9–10.4 × 4.1–4.5 µm (mean = 9.7 × 4.4; 29 SD 0.5, 0.1; n = 8). Pseudocosmospora pseudepisphaeria (Samuels) C. Herrera & P. Chaverri, comb. nov. Mycobank MB 802439 Basionym: Nectria pseudepisphaeria Samuels, Mycol. Pap. 164: 34. 1991. ≡ Cosmospora pseudepisphaeria (Samuels) Rossman & Samuels, Stud. Mycol. 42: 124. 1999. Anamorph: Acremonium-like. Habitat: On Eutypella sp. (Diatrypaceae) on branch of Espeletia sp. Distribution: Venezuela (known only from the type collection). Holotype: VENEZUELA, Merida, Parque Nacional Sierra Nevada, near Apartaderos, E. of Laguna Mucubaji, Laguna Negra, on Eutypella sp., on Espeletia sp., 18 Jul. 1971, K.P. Dumont (VE 2277), J.H. Haines, G.J. Samuels & A. Revas, NY 01013169. Notes: Based on our examination of the holotype specimen, the fungal host of C. pseudepisphaeria is a Eutypella sp. The fungal host and the reported acremonium-like anamorphic state support the placement of C. pseudepisphaeria in the genus Pseudocosmospora. Unique to this species are the discoidal perithecial apices, its verrucose, (11–) 11.2–13(–14) µm long ascospores, and its white to pale salmon- colored colony (Samuels et al. 1991). A culture no longer exists. Pseudocosmospora rogersonii C. Herrera & P. Chaverri, sp. nov. FIG. 1.7. 30 Mycobank MB 802440 Holotype: USA, New York, Dutchess County, Pawling, Pawling Nature Reserve, on Eutypella sp., 6–8 Oct. 1990, G.J. Samuels & C.T. Rogerson, BPI 1107121, ex- holotype culture CBS 133981 = G.J.S. 90-56. Etymology: In honor of Clark T. Rogerson for his work on the Hypocreales that has guided all of us. Teleomorph: Perithecia gregarious, slightly immersed in host stromata, subglobose with a blunt papilla, collapsing laterally, scarlet, smooth, 163–245 × 131–180 μm (mean = 193 × 152; SD 37, 21; n = 7). Asci broadly cylindrical to narrowly clavate, eight-spored, uniseriately arranged, 54-69 × 5.7-8.4 μm (mean = 63 × 6.7; SD 4.9, 0.7; n = 12). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, smooth, yellow-brown, 7.9–12.2 × 3.3–4.9 μm (mean = 9.6 × 4.1; SD 0.9, 0.3; n = 86). Anamorph: Colonies 18–27.5 mm diam (mean = 22.4; SD 3.4; n = 8) after 14 d. at 25 C on PDA, crustose, rosy-buff to salmon-pink, reverse concolorous. Sporulation on SNA usually abundant, arising directly from agar surface. Anamorphic state acremonium-like; conidiophores generally simple, unbranched. Phialide cylindrical, smooth, straight, collarette not flared, hyaline, length 6.8–29.4 μm (mean = 12.3; SD 5; n = 30), width at base 1.0–2.3 μm (mean = 1.5; SD 0.3; n = 30), width at tip 0.7– 1.2 μm (mean = 0.9; SD 0.13; n = 30). Conidia oblong to ellipsoidal, unicellular, smooth, hyaline, 2.9–5.5 × 1.1–2.6 μm (mean = 3.8 × 1.6; SD 0.6, 0.3; n = 89). 31 Habitat: On Eutypella sp. (Diatrypaceae) on bark. Distribution: USA. Additional specimens and isolates examined: USA, New York, Dutchess County, Pawling, Pawling Nature Reserve, on Eutypella sp., 6–8 Oct. 1990, G.J. Samuels & C.T. Rogerson, BPI 1107120; New York, Huguenot, YMCA Greenkill Retreat enter, on Eutypella sp., 26 Sept. 2009, C. Herrera (C.H. 09-02), BPI 884167, culture = G.J.S. 09-1384; New York, Painted Post, Watson Homestead Conference and Retreat Center, on Eutypella sp., on dead branch of Fagus grandifolia, 17 Sept. 2010, C. Herrera (C.H. 10-11), BPI 884166, culture CBS 133978 = G.J.S. 10-296; New York, Painted Post, Watson Homestead Conference and Retreat Center, on Eutypella sp., on dead branch of Fagus grandifolia, 17 Sept. 2010, C. Herrera (C.H. 10-12), BPI 884170, culture CBS 133979 = G.J.S. 10-297. Notes: Pseudocosmospora rogersonii is closely related to P. eutypellae, but differs conspicuously in the ornamentation of its ascospores. Pseudoscosmospora rogersonii has smooth ascospores in contrast to P. eutypellae, which has verrucose ascospores. Pseudocosmospora triqua (Samuels) C. Herrera & P. Chaverri, comb. nov. Mycobank MB 802441 Basionym: Nectria triqua Samuels, Mycol. Pap. 164: 40. 1991. ≡ Cosmospora triqua (Samuels) Rossman & Samuels, Stud. Mycol. 42: 125. 1999. 32 Anamorph: Acremonium-like. Habitat: On Eutypella sp. (Diatrypaceae) on unidentified bark. Distribution: French Guiana (known only from the type collection). Holotype of Nectria triqua: FRENCH GUIANA, Upper Marouini River, vic. roche Koutou, 02°55’N, 54°04’W, elev. 400 m., on Eutypella sp., on unidentified bark, 17 Aug. 1987, G.J. Samuels (5818), J.-J. de Granville, L. Allorge, W. Hahn, M. Hoff, NY 01013269. Notes: Examination of the holotype revealed that the host is a Eutypella sp., which suggests that C. triqua should be placed in the genus Pseudocosmospora. Additionally, the reported anamorphic state is similar to that of P. vilior and P. eutypellae in having branching conidiophores branch that terminate with multiple phialides. Cultural morphology in PDA was not reported in the description of Nectria triqua (Samuels et al. 1991). The culture no longer exists. The ascospores are verrucose and (6.8–) 7.8–9.7 (–10.5) µm long. Pseudocosmospora vilior (Starbäck) C. Herrera & P. Chaverri, comb. nov. FIG. 1.8. Mycobank MB 802442 Basionym: Nectria vilior Starbäck, Bih. Kongl. Svenska Vet.-Acad. Handl. 25(3,1): 28. 1899. ≡ Cosmospora vilior (Starbäck) Rossman & Samuels, Stud. Mycol. 42: 126. 1999. 33 Teleomorph: Perithecia gregarious, slightly immersed in host stromata, subglobose with blunt apex, collapsing laterally, scarlet, smooth, 195–224 × 136–183 μm (mean = 213 × 164; SD 9.1, 14.7; n = 10). Asci cylindrical to clavate, eight-spored, uniseriately arranged, 59–81 × 5.3–11.0 μm (mean = 69 × 7.6; SD 6.2, 1.7; n = 18). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, slightly verrucose, yellow-brown, 8.3–13.0 × 4.1–6.4 µm (mean = 10.2 × 5.2; SD 1.0, 0.5; n = 90). Anamorph: Colonies 23–65 mm diam (mean = 49; SD 16.9; n = 8) after 21 d. at 25 C on PDA, cottony with pale luteous aerial mycelium, reverse concolorous. Sporulation on SNA usually abundant, arising directly from agar surface; acremonium-like to verticillium-like; conidiophores simple and unbranched at first, becoming densely branched. Phialides cylindrical, smooth, straight, collarette not flared, hyaline, length 5.9–19.5 μm (mean = 12.9; SD 3.4; n = 9), width at base 1.1–1.8 μm (mean = 1.5; SD 0.2; n = 29), width at tip 0.7–1.3 μm (mean = 1.0; SD 0.2; n = 29). Conidia reniform, unicellular, smooth, with two guttules at opposite ends, hyaline, 3.4–7.4 × 1.1–2.3 μm (mean = 4.8 × 1.7; SD 0.7, 0.3; n = 90). Habitat: On Eutypella sp. (Diatrypaceae) on bark. Distribution: Argentina and Brazil. Holotype: BRAZIL, Rio Grande do Sul, Santo Angelo pr. Cachoaira, on Eutypella sp., 12 Jan. 1893, Gustav Malme (114), S F46424. Epitype designated herein: ARGENTINA, Misiones Province, Iguazú Biological 34 Station, on Eutypella sp., 25 Apr. 2011, A.Y. Rossman, C. Salgado, A. Romero, R. Sanchez, BPI 884176, ex-epitype culture CBS 133971 = A.R. 4810. Additional specimens and isolates examined: ARGENTINA, Tucuman Province, Tucuman, on Eutypella sp., on standing dead branch of Piper tucumanum, 19 Apr. 2011, A. Romero, BPI 884174, culture CBS 133970 = A.R. 4771; BRAZIL, Bahia, Igrapiúna, on Eutypella sp., 12 Aug. 2010, P. Chaverri (P.C. 1246), O. Liparini Pereira, D. Pinho, A. Luiz Firmino, BPI 884172, culture CBS 133963. Notes: An epitype was needed to establish an anamorph for P. vilior and to determine its phylogenetic placement. The epitype was selected based on the relatively close proximity to the collecting site of the holotype. The application of the name is restricted to species of Pseudocosmospora on Eutypella from South America that have pale-luteous colonies on PDA. However, it is recognized here that P. vilior consists of a species complex. DISCUSSION Genus Concept The generic concept Cosmospora sensu stricto is based on its type Cosmospora coccinea Rabenh., which has Verticillium olivaceum W. Gams as its anamorph. Although the anamorph bears the name Verticillium Nees, the anamorphic state is acremonium-like (single phialide, unbranched) to verticillium-like (branching into multiple phialides). Accepted species in Cosmospora sensu stricto have an acremonium-like anamorph, and it is the character that circumscribes the genus (Gräfenhan et al. 2011). Conidiophore branching is not unique to the anamorph of C. 35 coccinea as this is also observed in some anamorphs in the Cosmospora viliuscula species complex. Pseudocosmospora (described above) is recognized as a new genus based on the one-to-one genus concept suggested by Rossman (1993) to accommodate C. vilior and related species. The one-to-one genus concept has been used extensively in the Ascomycota to delimit genera (e.g. Gräfenhan et al. 2011, Luo and Zhang 2010, 2012). Briefly, this genus concept suggests that a genus should be circumscribed based on the correlation of its teleomorph to its unique anamorph state and vice versa. The groups circumscribed based on this concept are monophyletic and often supported by ecological traits (e.g. Gräfenhan et al. 2011, Luo and Zhang 2010, 2012). In Gräfenhan et al. (2011), the reported hosts for members of Cosmospora sensu stricto were basidiomycetes (e.g., Fomitopsis P. Karst., Inonotus P. Karst. and Stereum Hill ex Pers.) and xylariaceous fungi (e.g., Hypoxylon Bull.). Microcera and Dialonectria species have fusarium-like anamorphs, Microcera species are parasites of scale insects, and the lectotype species of Dialonectria, D. episphaeria, is reported on Diatrype stigma (Hoffm.) Fr. (Diatrypaceae; Booth 1959). The host of Dialonectria ullevolea Seifert & Gräfenhan has not been identified, but it is predicted here that the host will be a diatrypaceous fungus. The orphan clade consisting of C. flavoviridis (Fuckel) Rossman & Samuels, C. stegonsporii Rossman, Farr & Akulov, and C. obscura Lowen has not been taxonomically revised, and may require generic recognition. Species in this clade have a fusarium-like anamorphs, but little is known about their fungal hosts. Only the host of C. stegonsporii, Stegonsporium pyriforme (Hoffm.: Fr.) Corda (Diaporthales, Sordariomycetes), has been identified to species. 36 It is possible that all fungal hosts of species in this clade are members of the Diaporthales. The one-to-one genus concept is ideal for the circumscription of genera in the Ascomycota because it forces the study of the holomorph and not only the teleomorph or anamorph. Such view is crucial in shifting to one name (Norvell 2011). Discarding information of either the teleomorph or anamorph in order to favor one generic hypothesis over the other may result in para- or polyphyletic groups. For example, a weak case could be made to group Cosmospora, Dialonectria, Pseudocosmospora and the orphan clade that consists of C. flavoviridis, C. stegonsporii and C. obscura into one genus because they have a cosmospora-like teleomorph and occur generally on Sordariomycetes. However, when the anamorphs are superimposed on the phylogeny, a paraphyletic group is formed with two groups having acremonium-like anamorphs and the remaining two groups fusarium-like anamorphs. It suggests that the teleomorp state is probably a symplesiomorphic character (or ancestral), while the anamorp represents a synapomorphic character (derived). Moreover, segregation of the discussed genera is supported by specialization to different host taxa. The cosmospora-like teleomorphic state of Pseudocosmospora was correlated here to an acremonium-like anamorph. Our phylogeny (FIG. 1.2) demonstrates that Pseudocosmospora (BP 100%, PP 100%) is not congeneric with Cosmospora s.str., the only other group of cosmospora-like fungi with an acremonium-like anamorph (Hirooka et al. 2010, Gräfenhan et al. 2011). The two groups differ primarily by their cultural characteristics. In general, Pseudocosmospora produces pinkish colonies, 37 while Cosmospora sensu stricto, produces olivaceous-green colonies on PDA. Members of each genus considered in this study occur only on a particular group of host fungi. Pseudocosmospora is reported here to occur primarily on Eutypa and Eutypella species (Diatrypaceae) with the exeption of Cosmospora joca (Samuels) Rossman & Samuels, whose host is a species of Biscogniauxia. The genus Dialonectria also occurs on diatrypaceous fungi but has a fusarium-like anamorphic state as do species in the genus Microcera that occur primarily on insects. Species Concept The Genealogical Concordance Phylogenetic Species Recognition was used to delimit species boundaries (GCPSR; Taylor et al. 2000). According to this operational species concept, putative species are clades that are concordant across all single gene trees. The morphological species recognition was also used to support the species inferences made when applying GCPSR. Hence, inferred species may be associated with unique morphological features that set them apart from other closely related species. Another species concept that could be useful in determining additional characters to delimit species is the ecological species concept. According to this species concept, ecological niches or adaptive zones can be used to delimit species (reviewed in de Queiroz 2007). Host, an ecological niche, could be a character specific to a particular Pseudocosmospora species. However, host identification of Eutypa and Eutypella to species was not possible based on morphology alone. Identification of the fungal host based on DNA sequences would resolve this 38 problem. Moreover, analyzing DNA sequences of cosmospora-like fungi and their associated fungal hosts would allow testing the hypothesis of cospeciation. Evidence for cospeciation would provide independent evidence for the delimitation of species in this genus. A problem of GCPSR is that it requires multiple individuals per species. By definition a clade is formed by a minimum of two individuals per species (reviewed in Vinuesa 2010). In the case of this paper, only a single collection was made for many of the lineages, and it left us with a dilemma on how to deal with the many singletons present in the phylogeny (FIG. 1.2). It was decided to use the rule of rarity (reviewed in Lim et al. 2012) to recognize a singleton, Pseudocosmospora metajoca (described below), as a species. This species is morphologically and ecologically distinct from species recognized with GCPSR and other singletons. Pseudocosmospora metajoca occurs on an Eutypa sp. on Beilschmiedia tawa (A.Cunn.) Kirk (Lauraceae), which is a broadleaf tree native to New Zealand, and has verrucose ascospores. Also supporting the view that P. metajoca is a distinct species is the relatively long branch length, which indicates that there have been multiple substitutions per site since its segregation. Cosmospora vilior represents a case where morphology is insufficient to distinguish closely related species. This species is characterized by its relatively fast growing, pale luteous colony on PDA. However, the clade probably represents a species complex given the highly supported subclade that consists of the strains AR 4771 and PC 1246. The species complex may consist of up to three species, but the selected epitype strain, AR 4810, is considered closer to the true C. vilior based on 39 geographical proximity to the original collecting site of the type specimen and its host. Phylogenetic placement of Cosmospora joca.—The phylogenetic placement of Cosmospora joca, the host of which is a Biscogniauxia sp., is puzzling considering that other members of Pseudocosmospora have an Eutypa or Eutypella species (Diatrypaceae) as their host. Two potential explanations for this observation are i) that the Biscogniauxia sp. represents the ancestral host for Psedocosmospora species, or ii) that the Biscogniauxia host of C. joca represents an independent host shift. Given that Pseudocosmospora species have diatrypaceous and xylariaceous hosts and assuming the first view, Pseudocosmospora could represent a link in the divergence from Cosmospora sensu stricto to Dialonectria (or vice-versa). Phylogenies of the fungal hosts have placed the Diatrypaceae as a sister clade to Xylariaceae (Moster et al. 2004, Tang et al. 2009), and these cosmospora-like fungi could have tracked their hosts faithfully as they diverged. Host specificity is not uncommon in the Hypocreales (e.g., species of Cordyceps sensu lato are known to be host specific to insect species; Sung et al. 2007). Coevolution/cospeciation analyses are needed to test these hypotheses. 40 TA BL E I.I . Lo ci u se d in th e ph yl o ge n et ic a n a ly se s Co mb ine d 35 91 97 0 31 8 18 58 33 53 65 1 45 6 178 5 Tu b TP M3 uf+ I+G 58 0 20 4 90 19 9 TIM 3+ I+G 54 7 159 92 213 Bt ub -T I, B tu b- T2 (O ’Do nn ell & C ige lni k 1 99 7) 55 °C , 3 0 s, 35 x Te f1 Tr N+ I+G 36 5 23 6 42 25 HK Y+ G 28 0 172 64 0 te f1- 72 8, te f1- 98 6 (C ar bo ne & Ko hn 19 99 ) 66 °C , 5 5 s , 9 x 5 6 °C , 5 5 s , 3 5x Rp b1 TIM 2+ I+G 69 2 23 5 80 28 2 TIM 2+ I+G 64 2 211 70 25 3 cr pb 1a, rp b1c (C as tle bu ry et al . 2 00 4) 50 °C , 2 m in, 40 x Mc m7 Tr N+ I+G 57 0 20 1 29 32 4 Tr N+ I+G 56 1 44 16 6 32 7 mc m7 -7 09 for , m cm 7- 13 48 re v (S ch mi tt et al. 20 09 ) 56 °C , 5 0 s, 38 x LS U TIM 2+ I+2 13 84 94 77 10 28 TIM ef+ I+G 13 23 65 64 99 2 IT S5 , IT S4 (W hit e e t a l. 19 90 ) 53 °C , 1 mi n, 40 x ITS LR 5, LR OR (V ilg aly s n .d. ) Lo cu s Nu cle ot ide su bs tit ut ion m od els Inc lud ed si te s Ph ylo ge ne tic all y i nf or ma tiv e s ite s Un inf or ma tiv e p oly mo rp hic si te s Inv ar iab le sit es Nu cle ot ide su bs tit ut ion m od els Inc lud ed si te s Ph ylo ge ne tic all y i nf or ma tiv e s ite s Un inf or ma tiv e p oly mo rp hic si te s Inv ar iab le sit es Pr im er s u se d ( re fe re nc e) PC R pr ot oc ol: A nn ea lin g t em p. & cy cle s Cosmospora sensu lato dataset Pseudocosmospora dataset 41 SU PP LE ME NT AR Y T AB LE I.I . I so lat es an d a cc es sio n n um be rs us ed in th e p hy log en eti c a na lys es . Ge ne Ba nk Ac ce ss ion N o. tub K C2 91 90 2 K C2 91 90 3 K C2 91 90 1 JF 83 28 38 H M 48 45 89 K C2 91 90 5 K C2 91 90 8 K C2 91 90 7 K C2 91 90 6 K C2 91 90 4 K C2 91 93 2 tef 1 - K C2 91 85 8 K C2 91 82 8 JF 83 25 17 H M 48 45 15 K C2 91 84 1 K C2 91 84 3 K C2 91 84 9 K C2 91 85 1 K C2 91 83 6 K C2 91 84 2 rp b1 K C2 91 86 3 K C2 91 86 4 K C2 91 86 2 JF 83 27 63 G Q5 06 02 0 K C2 91 86 6 K C2 91 86 9 K C2 91 86 8 K C2 91 86 7 K C2 91 86 5 K C2 91 89 2 mc m7 K C2 91 82 1 K C2 91 82 4 K C2 91 79 2 K C2 91 79 5 - JN 99 33 22 JN 99 33 26 K C2 91 81 3 K C2 91 81 5 K C2 91 80 4 - LS U K C2 91 78 5 K C2 91 78 8 K C2 91 75 5 JF 83 26 79 G Q5 05 99 0 JN 93 98 26 JN 93 98 24 K C2 91 77 7 K C2 91 77 9 K C2 91 76 5 K C2 91 77 1 ITS K C2 91 74 7 K C2 91 71 9 K C2 91 71 8 JF 83 25 94 H M 48 45 37 JN 99 56 27 JN 99 56 29 K C2 91 73 2 K C2 91 74 8 K C2 91 73 1 K C2 91 74 4 Co un try U n ite d K in gd o m Ja pa n U kr ai n e Fr en ch G u ia n a G er m an y A u st ra lia U SA N ew Ze al an d Fr en ch G u ia n a Cz ec h R ep u bl ic U SA Su bs tra te/ Ho st B ra n ch pa rt ia lly su bm er ge d in st re am Tw ig Ti lia co rd a ta B ar k In o n o tu s n o du lo su s K re tz ch m a ri a sp . H yp o xy lo n fra gi for m e H yp o xy lo n bo ve i Xy la ri a sp . Ti lia sp . D ia tr yp e st ig m a He rb ar ium N o. - - B PI 87 82 74 B PI 88 10 71 B PI 80 27 29 B PI 87 89 94 - PD D N Y - - Iso lat e N o. IM I 3 38 17 3 M A FF 24 14 84 A . R . 43 85 , CB S 12 23 05 A . R . 45 47 , CB S 12 38 26 A . R . 27 41 , CB S 11 40 50 G . J.S . 09 - 41 1 G . J.S . 10 - 24 7 G . J.S . 83 - 19 7, CB S 12 40 32 G . J.S . 86 - 31 5 CB S 10 24 33 G . J.S . 10 - 19 3, C. H . 10 - 01 Sp ec ies “ Co sm os po ra ” fla vo v iri di s “ Co sm os po ra ” ob sc u ra “ Co sm os po ra ” st eg on sp or ii Co ra llo m yc et el la re pe n s Co sm os po ra co cc in ea Co sm os po ra v ili u sc u la Co sm os po ra v ili u sc u la Co sm os po ra v ili u sc u la Co sm os po ra v ili u sc u la Co sm os po ra v iri de sc en s D ia lo n ec tr ia ep isp ha er ia 42 SU PP LE ME NT AR Y T AB LE I.I . C on tin ue d. Ge ne Ba nk Ac ce ss ion N o. tub K C2 91 93 3 K C2 91 93 4 K C2 91 93 1 - K C2 91 93 7 K C2 91 93 6 K C2 91 93 5 K C2 91 92 5 - JF 83 28 39 K C2 91 90 9 K C2 91 91 2 tef 1 K C2 91 84 8 - K C2 91 83 9 K C2 91 85 0 K C2 91 85 5 K C2 91 85 7 K C2 91 83 2 K C2 91 83 7 K C2 91 85 6 JF 83 25 93 K C2 91 82 9 K C2 91 83 0 rp b1 K C2 91 89 3 K C2 91 89 0 K C2 91 89 1 K C2 91 89 6 K C2 91 89 7 K C2 91 89 5 K C2 91 89 4 K C2 91 88 4 K C2 91 88 5 JF 83 27 64 K C2 91 87 0 K C2 91 87 1 mc m7 K C2 91 81 2 - K C2 91 80 7 K C2 91 81 4 K C2 91 82 0 K C2 91 82 3 K C2 91 79 8 K C2 91 80 5 K C2 91 82 2 K C2 91 79 3 K C2 91 79 4 K C2 91 79 6 LS U K C2 91 77 6 K C2 91 76 8 K C2 91 76 9 K C2 91 77 8 K C2 91 78 4 K C2 91 78 7 K C2 91 75 9 K C2 91 76 6 K C2 91 78 6 JF 83 26 80 K C2 91 75 6 K C2 91 75 7 ITS K C2 91 74 3 K C2 91 74 1 K C2 91 74 2 K C2 91 75 3 K C2 91 75 4 K C2 91 75 2 K C2 91 75 1 K C2 91 73 5 K C2 91 73 6 JF 83 25 95 K C2 91 72 0 K C2 91 72 1 Co un try U SA U SA B el gi u m N ew Ze al an d Pu er to Ri co Ja pa n N ew Ze al an d Fr an ce G re at B rit ai n U SA U SA U SA Su bs tra te/ Ho st D ia tr yp ac ea e D ia tr yp ac ea e D ia tr yp e bu lla ta sc al e in se ct sc al e in se ct H em ib er le si a la ta n ia e (S ca le in se ct ) sc al e in se ct Eu ty pa sp . U n kn o w n Eu ty pe lla sp . Eu ty pe lla sp . Eu ty pe lla sp . He rb ar ium N o. - - - - B PI 74 83 93 - - B PI 88 41 64 - B PI 87 84 54 - B PI 88 41 65 Iso lat e N o. G . J.S . 10 - 29 8, C. H . 10 - 14 C. H . 11 - 06 G . J.S . 09 - 11 97 G . J.S . 83 - 19 8 G . J.S . 98 - 50 M A FF 24 14 82 A . R . 45 80 C. H . 11 - 01 IM I 7 30 16 A . R . 44 53 , CB S 12 94 30 A . R . 45 27 A . R . 45 62 Sp ec ies D ia lo n ec tr ia sp . D ia lo n ec tr ia sp . D ia lo n ec tr ia sp . M icr oc er a co cc op hi la M icr oc er a co cc op hi la M icr oc er a co cc op hi la M icr oc er a la rv ar u m Ps eu do co sm os po ra eu ty pa e Ps eu do co sm os po ra eu ty pa e Ps eu do co sm os po ra eu ty pe lla e Ps eu do co sm os po ra eu ty pe lla e Ps eu do co sm os po ra eu ty pe lla e 43 SU PP LE ME NT AR Y T AB LE I.I . C on tin ue d. Ge ne Ba nk Ac ce ss ion N o. tub K C2 91 91 0 K C2 91 91 1 H M 48 48 78 K C2 91 92 4 K C2 91 92 3 K C2 91 91 7 K C2 91 91 6 K C2 91 91 4 K C2 91 91 5 K C2 91 92 2 K C2 91 92 9 tef 1 K C2 91 84 5 K C2 91 84 4 H M 48 48 49 - K C2 91 83 1 K C2 91 84 6 K C2 91 84 7 K C2 91 84 0 K C2 91 85 2 - K C2 91 83 5 rp b1 K C2 91 87 3 K C2 91 87 2 G Q5 06 03 5 K C2 91 88 7 K C2 91 88 6 K C2 91 87 6 K C2 91 87 7 K C2 91 87 5 K C2 91 87 8 K C2 91 88 1 K C2 91 88 8 mc m7 K C2 91 80 9 K C2 91 80 8 K C2 91 81 7 K C2 91 80 1 K C2 91 79 7 K C2 91 81 0 K C2 91 81 1 - K C2 91 81 6 K C2 91 79 9 K C2 91 80 3 LS U K C2 91 77 3 K C2 91 77 2 G Q5 06 00 6 K C2 91 76 2 K C2 91 75 8 K C2 91 77 4 K C2 91 77 5 K C2 91 77 0 K C2 91 78 0 K C2 91 76 0 K C2 91 76 4 ITS K C2 91 72 3 K C2 91 72 2 H M 48 48 56 K C2 91 74 6 K C2 91 74 5 K C2 91 72 7 K C2 91 72 8 K C2 91 72 6 K C2 91 72 9 K C2 91 72 4 K C2 91 74 0 Co un try U SA U SA Fr an ce A rg en tin a N ew Ze al an d U SA U SA U SA U SA A rg en tin a A rg en tin a Su bs tra te/ Ho st Eu ty pe lla sp . Eu ty pe lla sp . Eu ty pe lla sp . Bi sc o gn ia u xi a sp . Eu ty pa sp . Eu ty pe lla sp . Eu ty pe lla sp . Eu ty pe lla sp . Eu ty pe lla sp . Eu ty pe lla sp . Eu ty pe lla sp . He rb ar ium N o. B PI 88 41 68 B PI 88 41 69 B PI 80 25 67 B PI 88 41 75 B PI 87 90 88 B PI 88 41 66 B PI 88 41 70 B PI 88 41 67 B PI 11 07 12 1 B PI 88 41 73 B PI 88 41 77 Iso lat e N o. G . J.S . 10 - 29 4, C. H . 10 - 02 G . J.S . 10 - 24 8, C. H . 10 - 10 G . J.S . 93 - 15 , CB S 12 89 86 A . R . 47 79 A . R . 45 76 G . J.S . 10 - 29 6, C. H . 10 - 11 G . J.S . 10 - 29 7, C. H . 10 - 12 G . J.S . 09 - 13 84 G . J.S . 90 - 56 A . R . 47 68 A . R . 48 26 Sp ec ies Ps eu do co sm os po ra eu ty pe lla e Ps eu do co sm os po ra eu ty pe lla e Ps eu do co sm os po ra eu ty pe lla e Ps eu do co sm os po ra joc a Ps eu do co sm os po ra m et ajo ca Ps eu do co sm os po ra ro ge rs on ii Ps eu do co sm os po ra ro ge rs on ii Ps eu do co sm os po ra ro ge rs on ii Ps eu do co sm os po ra ro ge rs on ii Ps eu do co sm os po ra sp . Ps eu do co sm os po ra sp . 44 SU PP LE ME NT AR Y T AB LE I.I . C on tin ue d. Ge ne Ba nk Ac ce ss ion N o. tub K C2 91 91 9 K C2 91 92 1 K C2 91 92 0 K C2 91 93 0 K C2 91 91 3 K C2 91 91 8 K C2 91 92 6 K C2 91 92 8 K C2 91 92 7 JF 83 29 09 tef 1 K C2 91 83 8 K C2 91 85 3 K C2 91 85 4 - K C2 91 85 9 K C2 91 86 0 K C2 91 83 3 K C2 91 83 4 K C2 91 86 1 JF 83 25 44 rp b1 K C2 91 88 2 K C2 91 88 3 K C2 91 88 0 K C2 91 88 9 K C2 91 87 4 K C2 91 87 9 K C2 91 89 8 K C2 91 90 0 K C2 91 89 9 JF 83 27 91 mc m7 K C2 91 80 6 K C2 91 81 8 - K C2 91 81 9 K C2 91 82 5 K C2 91 82 6 K C2 91 80 0 K C2 91 80 2 K C2 91 82 7 - LS U K C2 91 76 7 K C2 91 78 1 K C2 91 78 2 K C2 91 78 3 K C2 91 78 9 K C2 91 79 0 K C2 91 76 1 K C2 91 76 3 K C2 91 79 1 JF 83 27 15 ITS K C2 91 72 5 K C2 91 74 9 K C2 91 75 0 K C2 91 73 3 K C2 91 73 9 K C2 91 73 0 K C2 91 73 4 K C2 91 73 7 K C2 91 73 8 JF 83 26 58 Co un try Fr an ce U ga n da U ga n da U SA Ja pa n Ja pa n A rg en tin a A rg en tin a B ra zi l U SA Su bs tra te/ Ho st Eu ty pe lla sp . Eu ty pe lla sp . Eu ty pe lla sp . U n kn o w n U n kn o w n U n kn o w n Eu ty pe lla sp . Eu ty pe lla sp . Eu ty pe lla sp . Pa ch ys a n dr a sp . He rb ar ium N o. B PI 88 41 71 B PI 73 77 06 B PI 73 77 08 - - - B PI 88 41 74 B PI 88 41 76 B PI 88 41 72 B PI 87 99 36 Iso lat e N o. C. H . 11 - 02 G . J.S . 95 - 14 1 G . J.S . 95 - 14 3 G . J.S . 96 - 21 6, CB S 13 39 84 M A FF 24 14 99 M A FF 24 15 31 A . R . 47 71 A . R . 48 10 P. C. 12 46 A . R . 45 92 , CB S 12 86 74 Sp ec ies Ps eu do co sm os po ra sp . Ps eu do co sm os po ra sp . Ps eu do co sm os po ra sp . Ps eu do co sm os po ra sp . Ps eu do co sm os po ra sp . Ps eu do co sm os po ra sp . Ps eu do co sm os po ra v ili or Ps eu do co sm os po ra v ili or Ps eu do co sm os po ra v ili or Ps eu do n ec tr ia pa ch ys an dr ico la FIG. 1.1. Phylogenetic placement of sensu Rossman based on a dataset. Best tree generated with ML analysis ( indicate Maximum Likelihood bootstrap (ML BP)/Bayesian posterior probabilities (BI PP). 45 C. vilior and related species within Cosmospora combined 5-loci (ITS-LSU, MCM7, rpb1, tef1 −16460.154). Values at branches , and tub) FIG. 1.2. Phylogenetic relationship combined 5-loci (ITS-LSU, MCM7, with ML analysis (−23024.3191 bootstrap (ML BP)/Bayesian posterior probabilities (BI P 46 of C. vilior and related species based on a rpb1, tef1, and tub) dataset. Best tree ). Values at branches indicate Maximum Likelihood P). generated 47 FIG. 1.3. Pseudocosmospora eutypae. (A) Perithecia on natural substrata. Scale bar = 200 μm. (B) Perithecium in 3% KOH. Scale bar = 100 μm. (C) Median section of perithecium. Scale bar = 100 μm. (D) Perithecial surface cells. Scale bar = 100 μm. (E) Asci. Scale bar = 10 μm. (F) Ascospore. Scale bar = 10 μm. (G) Cultures after 3 wks at 25 C on PDA. Scale bar = 10 mm. (H) Phialide. Scale bar = 10 μm. (I) Conidia. Scale bar = 10 μm. 48 49 FIG. 1.4. Pseudocosmospora eutypellae. (A, B) Perithecia on natural substrata. A. Scale bar = 600 μm. B. Scale bar = 200 μm. (C) Perithecium in 3% KOH. Scale bar = 100 μm. (D) Median section of perithecium. Scale bar =100 μm (E) Perithecial surface cells. Scale bar = 100 μm. (F) Asci. Scale bar = 10 μm. (G). Ascopore. Scale bar = 10 μm. (H, I) Cultures after 3 wks at 25 C on PDA. Scale bars = 10 mm. (J, K) Phialides. Scale bars = 10 μm. (L) Lateral phialidic pegs. Scale bar = 10 μm. (M) Conidia. Scale bar = 10 μm. 50 FIG. 1.5. Pseudocosmospora joca. (A, B) Perithecia on natural substrata. A. Scale bar = 2 mm. B Scale bar = 200 μm. (C) Asci. Scale bar = 10 μm. (D) Ascospores. Scale bar = 10 μm. (E) Cultures after 3 wks at 25 C on PDA. Scale bar = 10 mm. (F) Phialide. Scale bar = 10 μm. (G). Conidia. Scale bar = 10 μm. 51 FIG. 1.6. Pseudocosmospora metajoca. (A, B) Perithecia on natural substrata. A. Scale bar = 2 mm. B. Scale bar = 200 μm. (C) Perithecium in 3% KOH. Scale bar = 100 μm. (D) Median section of perithecium. Scale bar = 100 μm. (E) Asci. Scale bar = 10 μm. (F) Ascospores. Scale bar = 10 μm. (G) Cultures after 3 wks at 25 C on PDA. Scale bar = 10 mm. (H, I) Phialides. Scale bars = 10 μm. (J) Conidia. Scale bar = 10 μm. 52 FIG. 1.7. Pseudocosmospora rogersonii. (A, B) Perithecia on natural substrata. A. Scale bar = 2 mm. B. Scale bar = 200 μm. (C) Perithecium in 3% KOH. Scale bar = 100 μm. (D) Perithecial surface cells. Scale bar = 100 μm. (E) Ascus. Scale bar = 10 μm. (F) Ascospores. Scale bar = 10 μm. (G, H) Cultures after 3 wks at 25 C on PDA. Scale bar = 10 mm. (I) Phialide. Scale bar = 10 μm. (J) Conidia. Scale bar = 10 μm. Scale bars: (A) 2 mm, (B) 200 μm, (C, D) 100 μm, (E,F, I–J) 10 μm, (G, H) 10 mm. 53 FIG. 1.8. Pseudocosmospora vilior. (A, B) Perithecia on natural substrata. A. Scale bar = 2 mm. B. Scale bar = 200 µm. (C) Perithecium in 3% KOH. Scale bar = 100 µm. (D) Asci. Scale bar = 10 µm. (E) Ascospore. Scale bar = 10 µm. (F) Cultures after 3 wks at 25 C on PDA. Scale bar = 10 mm. (G) Phialides. Scale bar = 10 µm. (H) Conidia. Scale bar = 10 µm. 54 Chapter 2: Revision of the genus Corallomycetella with Corallonectria gen. nov. for C. jatrophae (Nectriaceae, Hypocreales) C.S. Herrera, A.Y. Rossman, G.J. Samuels, C. Lechat and P. Chaverri. 2013. Mycosystema 32(3): 518–544. Reprinted with permission of Mycosystema. ABSTRACT The genus Corallomycetella (Ascomycota, Sordariomycetes, Hypocreales, Nectriaceae) has been defined to include red nectrioid fungi associated with rhizomorphs in nature and culture. With the recent collection of an unusual specimen having striated ascospores, the genus was re-examined using this and previously obtained cultures. A multilocus tree was constructed based on three loci (ITS, mcm7, β-tubulin) to determine phylogenetic relationships. Our results indicate that Corallomycetella repens sensu lato forms two clades associated with biogeography. Corallomycetella repens sensu stricto is restricted to specimens from Asia while C. elegans is resurrected for specimens from Africa and America. Minute striations in the ascospores are an overlooked character in species of Corallomycetella. Corallomycetella jatrophae is related to Neonectria sensu lato and unrelated to C. 55 repens and C. elegans; thus, a new genus, Corallonectria, is described to accommodate this species. Corallonectria is characterized by furfuraceous perithecia and synnematous fusarium-like anamorph. INTRODUCTION The genus Corallomycetella Henn. is recognized for two species having large, orange-red to red, smooth to scurfy ascomata arranged in caespitose clusters, and smooth to roughened ascospores (Rossman et al. 1999). These species occur primarily in tropical regions. Based on the reddish, KOH+ ascomata this genus is placed in the Nectriaceae, Hypocreales. One species, C. repens (Berk. & Broome) Rossman & Samuels, has a synnematal asexual state with red, rhizomorph-like strands at the base that has been referred to as Rhizostilbella hibisci (Pat.) Seifert. The reddish rhizomorph-like strands are also produced in culture as well as ellipsoid, non- septate conidia each with a truncate base. This species causes a number of diseases, specifically ‘violet root rot’ of Theobroma cacao L., root rot of Carica papaya L., and ‘stinking root disease’ of several tropical woody plants (Booth & Holliday 1973). Corallomycetella jatrophae (A. Møller) Rossman & Samuels has a similar looking ascomatal state with a reddish synnematal asexual state that produces large, fusiform, multi-septate conidia. A specimen collected in French Guiana has striate ascospores although otherwise is similar to C. repens. As part of a study to determine if this unusual specimen is a distinct species, the phylogenetic placement of Corallomycetella within the Nectriaceae was investigated. Previous studies had suggested that it was basal to the 56 genus Cosmospora Rabenh., which has recently been shown to be polyphyletic (Gräfenhan et al. 2011). MATERIALS AND METHODS Herbarium specimens and cultures Fresh specimens of Corallomycetella sensu Rossman et al. (1999) were collected on trips to Brazil, Costa Rica, French Guiana, and Gabon (Kadri Põldmaa). Cultures were obtained by isolating single asci or ascospores and grown in cornmeal dextrose agar (CMD; Difco™ cornmeal agar + 2% w/v dextrose + antibiotics). Dried specimens were deposited at the U.S. National Fungus Collections (BPI), Beltsville, Maryland, USA. Cultures were deposited at Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands (Table 1) from where additional fungal strains were obtained. Herbarium specimens of Corallomycetella were borrowed from the U.S. National Fungus Collections (BPI), Farlow Reference Library and Herbarium of Cryptogamic Botany (FH), Royal Botanic Gardens Kew (K), and William and Lynda Steere Herbarium, New York Botanical Garden (NY). Morphological characterization The macro-morphology of the teleomorph was observed using a stereoscope (Olympus SZX12 Olympus, Tokyo, Japan). The color, shape, size, ornamentation, and habit of the perithecia were characterized. To observe their internal structures, the perithecia were rehydrated in 3% KOH and the centrum isolated on a glass slide and 57 covered with a coverslip. Microscopic characters, e.g. asci and ascospores, were observed with a compound microscope (Olympus BX50; Olympus, Tokyo, Japan). The color reaction of the perithecial wall was observed using 3% KOH and 100% lactic acid (LA). Sections of perithecia (ca. 10µm in width) were made with the aid of a freezing microtome. To observe colony morphology strains were grown on Difco™ potato dextrose agar (PDA) in an incubator that alternates between fluorescent light and darkness (12h/12h) at 25°C. Two replicates with two pseudoreplicates were grown for each isolate. Culture growth was measured weekly for two weeks. Colony color is described using the terms in Rayner (1970). To observe the mononematous anamorph isolates were grown in synthetic nutrient-poor agar (SNA; Nirenberg 1976) under the conditions described above. A block of agar was cut, placed on a microscope slide, covered with a coverslip, and examined by light microscopy (Olympus BX50; Olympus, Tokyo, Japan). Measurements of continuous characters, e.g. length and width, were made with Scion Image software beta 4.0.2 (Scion Corp., Frederick, Maryland), and summarized by descriptive statistics, e.g., minimum, maximum, mean and standard deviation. DNA extraction, PCR, and sequencing The detailed DNA extraction protocol is described in Hirooka et al. (2010). Briefly, the strains were grown in Difco™ potato dextrose broth (PDB) for one week, and the mycelial mat harvested for DNA extraction. DNA was extracted with PowerPlant® 58 DNA Isolation Kit (MO BIO Laboratories Inc., Solana Beach, California). DNA of Corallomycetella jatrophae (P.C. 1300) was amplified directly from the several centra of the perithecia because the isolate did not survive −80°C storage. The centra were isolated in antibiotics, transferred to a microcentrifuge tube with 10µm RNAse- free water, incubated for 10min at 65°C, and homogenized using a micropestle. The sample was centrifuged, and 5µm of the supernatant was transferred to a new tube. DNA was amplified with Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare Bio-Sciences Corp., Piscataway, New Jersey) following the manufacturer’s instructions. Four partial loci were amplified. These loci include ITS ribosomal DNA (ITS; White et al. 1990) and three protein coding regions: α-actin (act; Samuels et al. 2006), mcm7 (a DNA replication licensing factor; Schmitt et al. 2009), and β-tubulin (tub; O’Donnell and Cigelnik 1997). The PCR reaction mixture (25µL total volume) consisted of 12.5µL GoTaq® Green Master Mix 2X (Promega Corporation, Madison, Wisconsin), 1.25µL 10mmol/L forward primer, 1.25µL 10mmol/L reverse primer, 1.0µL of dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, Missouri), 2.0µL genomic DNA template, and 7µL of sterile RNAse-free water. PCR amplifications were carried out in an Eppendorf Mastercycler thermocycler (Eppendorf, Westbury, New York) under the cycle conditions listed in Table 2. PCR products were cleaned with ExoSAP-IT® (USB Corp., Cleveland, Ohio). Clean PCR products were sequenced at the DNA Sequencing Facility (Center for Agricultural Biotechnology, University of Maryland, College Park, Maryland) and McLAB DNA sequencing 59 services (San Francisco, California). Sequences were assembled and edited with Sequencher 4.9 (Gene Codes, Madison, Wisconsin). Sequences were deposited in GenBank (Table 2.1). Phylogenetic analyses A multiple sequence alignment for each locus was performed in the MAFFT v.6 web service (http://mafft.cbrc.jp/alignment/server/; Katoh 2008) with the E-INS-i alignment strategy. Alignments were manually edited in in Mesquite 2.75 (Maddison ＆ Maddison 2011). CONCATEPILLAR 1.4 (Leigh et al. 2008) was used to determine which loci could be concatenated and analyzed to generate a phylogeny. Loci were concatenated if the p-value was greater than the default α-level of 0.05, which indicated that the null hypothesis, i.e. congruence of loci, could not be rejected. JModeltest (Guindon and Gascuel 2003; Posada 2008) was used to infer the model of nucleotide substitution for each locus. Default settings in jModeltest were used: 11 substitution schemes with equal or unequal base frequencies (+F), and with/without invariable sites (+I) and/or rate variation among sites (+G). The base tree for likelihood calculations was ML optimized. Once likelihood scores were calculated, the models were selected according to the Akaike Information Criterion (AIC). Maximum likelihood (ML) analyses were performed with GARLI v2.0 (Genetic Algorithm for Rapid Likelihood Inference; Zwickl 2006) in the GARLI web service (http://www.molecularevolution.org; Bazinet and Cummings 2011), which uses a 60 grid computing system associated with The Lattice Project (Cummings and Huskamp 2005; Bazinet and Cummings 2008). Fifty independent search replicates were performed to generate the starting tree and search for the best tree with a fast ML stepwise-addition algorithm. Two thousand bootstrap replicates were used in the bootstrap analysis. Bayesian analyses were performed in MrBayes v3.2.1 (Ronquist et al. 2012). A majority rule consensus tree was generated by running four chains for 10,000,000 Markov Chain Montecarlo generations sampling trees every 100th generation, and discarding the first 25% of the sampled trees as burn-in. Tracer version 1.5 (Rambaut and Drummond 2007) was used to confirm whether the negative log likelihoods had reached convergence. Species recognition in Corallomycetella sensu stricto was based on genealogical concordance phylogenetic species recognition (GCPSR; Taylor et al. 2000), 95% connection limit in statistical parsimony networks (Posada & Crandall 2001; Templeton 2001), and genealogical sorting index (gsi; Cummings et al. 2008). Haplotype networks based on statistical parsimony were generated for each locus and the combined multilocus dataset in TCS v1.21 (Clement et al. 2000). Haplotypes are joined by an edge only if the edge has a probability of parsimony greater than or equal to 95% (default settings in TCS). The gsi is a statistic that measures genealogical exclusivity of a group of individuals/species on a rooted tree with 0=polyphyly and 1=monophyly. The gsi was implemented in the gsi web interface (http://www.genealogicalsorting.org/) with single locus trees generated with GARLI (as described above) and 10,000 permutations to test statistical significance of the gsi 61 value (P<0.05). The ensemble statistic (gisT) was estimated from a multi-tree file containing all single locus trees. RESULTS The analysis performed in CONCATEPILLAR rejected the null hypothesis of congruence among all loci (P-value < 0.001), and for this reason act was analyzed separately (Fig. 2.1). The analysis determined that only ITS, mcm7, and tub were congruent (P-value=0.3), and therefore these loci could be concatenated. The concatenated matrix consisted of 2,304 base pairs of which 799 were parsimony- informative, 236 were parsimony-uninformative, and 345 were invariable (Table 2.2). The topologies of the generated phylogenetic trees in both ML and BI were congruent. The negative log likelihoods for the phylogenetic trees were −17491.8148 and −17588.5333, respectively. The best tree (ML) is shown in Figure 2.2. The combined analyses of species in Corallomycetella sensu Rossman et al. (1999) revealed that these taxa consisted of three clades, although distantly related, i.e. the genus is polyphyletic. One clade is composed of Corallomycetella repens sensu Rossman et al. (1999) (100% BP, 100% PP), and allied with Cosmospora sensu stricto, Dialonectria (Sacc.) Cooke, Fusicolla Bonord., Macroconia (Wollenw.) Gräfenhan et al., and Microcera Desm. (94.5% PP). Corallomycetella repens sensu Rossman et al. (1999) clustered into two well- supported subclades that correlated with geographic origin (Fig. 2.2). One subclade is known only from Asia (99.6% BP, 100% PP), and represented in Fig. 2.2 by isolates from India, Java, and Sri Lanka. In single gene trees, this clade was present in act, 62 ITS, and mcm7, but only act and mcm7 strongly supported this clade (Fig. 2.3). This subclade is recognized below as C. repens sensu stricto. The second subclade of Corallomycetella repens sensu Rossman et al. (1999) is known from tropical regions in the western hemisphere and Africa. It is well supported (87.9% BP, 100% PP; Fig. 2.2) and referred to as C. elegans (see Taxonomy section). It is present and strongly supported in act, ITS, and tub single gene trees (Fig. 2.3). Within the C. elegans clade, ITS was the only locus supporting the monophyly of specimens from America, while tub was the only locus strongly supporting the monophyly of specimens from Africa (Fig. 2.3). TCS analyses resolved two segregate haplotype networks of Corallomycetella repens sensu Rossman et al. (1999; Fig. 2.4). The smaller haplotype network comprised two haplotypes from Asia, and the larger one included four haplotypes from tropical America and Africa. Within the larger haplotype network, eight polymorphic sites separated two small subgroups that showed geographic congruence. In single gene haplotype networks, only the analysis of tub reconstructed two separate haplotype networks as seen in the combined haplotype network, although ITS and mcm7 had a relatively high number of mutational differences between the “American” and “African” subgroups (five and six, respectively; Fig. 2.5). Gsi analyses did not support the monophyly of Corallomycetella repens sensu Rossman et al. (1999). The ensemble gsiT value was 1, but not significant (p-value = 0.075). Complete sorting was not observed in any of the subclades. The ensemble gsiT values for the C. repens sensu stricto and C. elegans subclades were 0.928 (p-value = 63 <0.001) and 0.735 (p-value = 0.001). The ensemble gsiT values for the “American” and “African” populations of C. elegans were 0.928 (p-value = <0.001) and 0.735 (p- value = 0.001), respectively. Gsi values for each locus are reported in Table 2.3. The second species included in Corallomycetella sensu Rossman et al. (1999), C. jatrophae, formed a well-supported clade distinct from that genus (100% BP, 100% PP, Fig. 2.2). This species is closely allied with Ilyonectria P. Chaverri & Salgado, Neonectria Wollenw., and Viridispora Samuels & Rossman, although this inner node is not well supported in the combined analysis (87% PP, Fig. 2.2). Act strongly supports a clade comprising C. jatrophae, Ilyonectria, and Neonectria (100% PP, Fig. 1). Given the distinctive morphology of C. jatrophae and the lack of affinity with any known genus, this species is placed in a new genus, Corallonectria (described below). Three isolates identified as “Nectria” mauritiicola (Henn.) Seifert & Samuels, a name considered a synonym of Corallomycetella repens (Rossman et al. 1999), were distinct from all isolates referred to as Corallomycetella. The ITS sequence of these isolates are identical (Max. Ident.=100% in Blast®; blast.ncbi.nlm.nih.gov/) to the sequence of CBS 122.29, ex-type culture of Sarocladium kiliense (Grütz) Summerb. (76% BP, 82% PP; Fig. 2.2), and related to other species of Sarocladium W. Gams & D. Hawksw. (100% BP, 100% PP; Fig. 2.2). 64 DISCUSSION Genus concepts In the prior taxonomic revision by Rossman et al. (1999), Corallomycetella was based on C. repens (neotype of C. heinsenii, designated by Rossman et al. 1999). The genus included nectrioid fungi with scurfy to furfuraceous perithecia with rhizomorphs, roughened or smooth ascospores, and synnematous, fusarium-like or Rhizostilbella anamorphs. The circumscription of Corallomycetella was revised based on the genus-for-genus concept (Rossman 1993) in which a teleomorphic fungal genus correlates with its unique anamorph. Using this concept teleomorphic and anamorphic genera are observed to be monophyletic (e.g., Chaverri et al. 2008, 2011; Gräfenhan et al. 2011; Luo and Zhuang 2010, 2012). Our phylogenetic analyses of molecular sequence data reveal that Corallomycetella sensu Rossman et al. (1999) consists of two distantly related major clades (Fig. 2.2). The clade that comprises Corallomycetella sensu stricto is related to Cosmopora sensu Rossman (94.5% PP) as previously shown by Hirooka et al. (2012). The anamorph of Corallomycetella sensu stricto is a synnematous Rhizostilbella. The genus is also characterized by scurfy perithecia that develop from rhizomorphs or at the base of synnemata, and produce finely striated ascospores that appear roughened in median section because the outer wall is often sinuous. Corallomycetella jatrophae forms a second clade of Corallomycetella sensu Rossman et al. (1999), but it is most closely related to neonectria-like fungi (92% PP; Fig. 2.2). Although the exact relationship is not well resolved (low node support), the topology 65 of this subclade is similar to that reported by Chaverri et al. (2011) in which the inner nodes are well supported due to the higher number of concordant loci used in the combined analyses. Corallomycetella jatrophae is characterized by its synnematous fusarium-like anamorph, which is unique among neonectria-like fungi. Synnematous fusarium-like anamorphs are also observed in Atractium Link and Microcera (Gräfenhan et al. 2011), but these genera appear to be unrelated to C. jatrophae. Representatives of Atractium were not included in our phylogeny, but Gräfenhan et al. (2011) showed that the type, A. stilbaster Link and a second species of Atractium form a distinct genus allied with Pseudonectria and Volutella. The genus Atractium is not included the major clade of neonectria-like fungi. Microcera and allies (Cosmospora sensu Rossman et al. 1999) are closely related to Corallomycetella (94.5% PP; Fig. 2.2). This suggests that the synnematous fusarium-like anamorph has been independently derived three separate times. Based on these results, the clade previously regarded as Corallomycetella jatrophae is segregated into a new monotypic genus Corallonectria (described below). Species concepts The species previously referred to as Corallomycetella repens sensu Rossman et al. (1999) is recognized in this study to comprise two species, namely C. repens sensu stricto and C. elegans, based on our multi-method approach (GCPSR, connection of haplotypes with ≥95% parsimony probability, and gsi). In addition to these criteria the two species are supported by morphological and biogeographical differences. Corallomycetella repens is restricted to isolates from South and Southeast Asia (Fig. 2.2), while isolates from Africa and America are recognized as C. elegans. 66 Corallomycetella repens is circumscribed in the strict sense based on our phylogenetic results. The clade is well supported in the combined analyses (99.6% BP, 100% PP; Fig. 2.2). The ensemble gsi value obtained for this clade (0.902, p- value = 0.004; Table 2.3) suggested that it is approximating monophyly (i.e., complete sorting). The clade is present in all single locus trees except tub, but only supported in act and ITS (Fig. 2.3). Under GCSPR (Taylor et al. 2000) the clade could not be recognized probably because complete lineage sorting has not occurred. This criterion seems too conserved, and we decided to follow the criterion used by Pringle et al. (2005), which recognizes a species if the clade is well supported in the majority of single gene trees. The segregate network of C. repens haplotypes also suggests that C. repens is not conspecific with C. elegans. A segregate network occurs when the observed mutational differences exceed the maximum number of mutational connections, i.e. the 95% parsimony probability cutoff, between haplotypes. Segregate parsimony networks have been associated to correspond to species boundaries (reviewed in Hart & Sunday 2007). The case for Corallomycetella elegans is slightly more complicated, but also conforms to the species recognition used in this study. The clade is well supported (87.9% BP, 100% PP; Fig. 2.2), and exhibits a moderate genealogical exclusivity across single gene trees (gsiT=0.838, p-value=0.001; Table 2.3). Monophyly of the clade was supported by ITS and tub single gene trees. Conflicting with the monophyly of this clade is the monophyly of the “American” isolates supported by the ITS locus, while tub supported the monophyly of these isolates for ones from Africa (Fig. 2.3). Under the applied criterion of GCPSR (Pringle et al. 2005), these 67 populations cannot be segregated into species because only a single gene supports its monophyly. Under the 95% parsimony probability, these subgroups were not separated into their own haplotype networks. Thus, this method also supports the hypothesis that isolates from Africa and America are conspecific. As mentioned above, the recognized species of Corallomycetella are correlated to geographic origin. It is possible that the observed distribution of Corallomycetella species may be the result of vicariance. Supporting this hypothesis are the plant hosts of Corallomycetella, some of which are native to the same geographic region. For example, C. elegans parasitizes Hevea brasiliensis Müll. Arg. (Table 2.1), which is native to the Americas. It could explain why haplotypes from Africa are conspecific with those in America (Fig. 2.3). African haplotypes could be the result of anthropogenic introductions from America into plantations of H. brasiliensis in Africa. Vicariance has been used to explain the geographic distribution of species of the biotrophic fungus Cyttaria Berk. and their hosts in the plant genus Nothofagus Blume (Peterson et al. 2010). The incorrect application of a name: “Nectria mauritiicola” In our revision of the genus Corallomycetella, an isolate labeled CBS 400.52, initially identified as Nectria mauritiicola, a synonym of C. repens, was determined to be unrelated to the genus Corallomycetella sensu Rossman et al. (1999). ITS sequences labeled Nectria mauritiicola and retrieved from GenBank were identical to those for CBS 400.52. The source of the GenBank sequences was listed as human blood, which cast doubt on the identity of these fungi because specimens of Corallomycetella are 68 reported to be plant pathogens (reviewed in Booth and Holliday, 1973). Additionally, the observed anamorph of CBS 400.52 was acremonim-like, unlike the anamorph of species of Corallomycetella (described below). The herbarium specimen of CBS 400.52 listed as IMI 44310 was not observed, and we could not determine whether the name was correctly applied to the original specimen/isolate. Examination of the type specimen of Nectria mauritiicola revealed that this name is correctly considered a synonym of Corallomycetella elegans (see Taxonomy section). Thus, the name Nectria mauritiicola was incorrectly applied to CBS 400.52 and the other sequences in GenBank. The sequences of CBS 400.52 and others retrieved from GenBank were found to be identical to that of CBS 122.29, ex-type culture of Sarocladium kiliense (≡ Acremonium kiliense). The acremonium-like anamorph produced by CBS 400.52 also suggests this species. Therefore, the sequences labelled “Nectria mauritiicola” in GenBank are determined to be conspecific with S. kiliense (76% BP; Fig. 2.2). They are incorrectly annotated creating confusion in the scientific community especially those who depend on the database for identification. Novicki et al. (2003) and Jang et al. (2012) are examples of clinical literature where the incorrect identification was assigned, although Novicki et al. (2003) questioned the validity of the GenBank name because their samples did not cluster with CBS 313.72 (a true Corallomycetella repens included in our study). This example calls for a method to change annotations in GenBank and highlights the importance of taxonomic studies to correct these crucial errors. 69 TAXONOMY Corallomycetella Henn., Hedwigia 43: 245. 1904. Generic type: Corallomycetella heinsenii [as heinesii] (Henn.) Henn. (≡ Corallomyces heinsenii Henn., Bot. Jahrb. Syst. 23: 358. 1897). [= Corallomyces Berk & M.A. Curtis, J. Acad. Nat. Sci. Philadelphia, Ser. 2, 2: 289. 1853, non Fr. 1849. — Type: C. elegans Berk. & M.A. Curtis] = Rhizostilbella Wolk, Mycol. Zentbl. 4: 237. 1914. Generic type: R. hibisci (Pat.) Seifert (≡ Stilbum hibsci Pat., = R. rubra Wolk, Mycol. Zentbl. 4: 237. 1914 fide Seifert, 1985). Perithecia solitary to gregarious, associated with reddish rhizomorphs or synnemata, obpyriform, scarlet to blood-red, KOH+ blood-red, LA+ yellow, slightly scurfy, uniloculate. Perithecial surface cells forming textura angularis. Perithecial wall of one region of cells forming textura angularis, becoming narrow, compressed towards the centrum, 50–70µm thick. Asci narrowly clavate, apex with a ring, with eight- ascospores arranged uniseriately. Ascospores ellipsoid, smooth, one-septate, constricted at septum, thick-walled, outer wall sometimes sinuous, appearing rough (optical section), finely striated (surface view), yellow-brown. Anamorph synnematous Rhizostilbella. Synnemata on natural substrata solitary or gregarious, 2–5 caespitose, arising laterally or as terminal extension of the rhizomorphs or directly from the substratum, cylindric-capitate, subulate-capitate, cylindrical, slender to robust, straight, curved or sinuous, unbranched or once or twice 70 branched, hirsute, pale luteous to luteous, KOH+ livid red to purple, LA+ yellow. Marginal hyphae echinulate to verrucose, pale luteous, KOH+ livid red, with clavate terminal cells, covering entire surface of stipe. Conidiophores unbranched, or once simple monochasial or monoverticillate. Phialides cylindrical, terminal, lateral and terminal, collarettes not flared, periclinal thickening conspicuous. Conidial mass white to yellow, subglobse. Conidia ellipsoidal, ovoidal with a truncate base, non- septate, smooth-walled, hyaline. Habitat: On bark and roots of decaying or living (diseased) tropical trees, and also isolated from soil. Distribution: Africa, Asia, America (pantropical). Notes: Species of Corallomycetella are unique nectriaceous fungi in that they have a synnematous Rhizostilbella anamorph. Corallomycetella is similar to Corallonectria in that species in these genera produce rhizomorphs in PDA, but Corallonectria has a synnematous, fusarium-like anamorph. Corallomycetella elegans (Berk. & M.A. Curtis) C. Herrera & P. Chaverri, comb. nov. Fig. 2.6 MycoBank No. MB803107 Basionym: [Corallomyces elegans Berk. & M.A. Curtis, J. Acad. Nat. Sci. Philadelphia, Ser. 2, 2: 239. 1853, genus illeg., Art. 53]. = Stilbum hibsci Pat., J. Bot. Paris 1891: 320 fide Seifert, 1985. ≡ Rhizostilbella hibisci (Pat.) Seifert, Stud. Mycol. 27: 162. 1985. 71 [= Corallomyces heinsenii Henn., Bot. Jahrb. Syst. 23: 538. 1897, genus illeg., Art. 53]. ≡ Corallomycetella heinsenii (Henn.) Henn., Hedwigia 43: 245. 1904. [= Corallomyces elegans var. camerunensis Henn., Bot. Jahrb. Syst. 22: 76. 1897, genus illeg., Art. 53]. [≡ Corallomyces camerunensis (Henn.) Henn., Bot. Jahrb. Syst. 23: 538. 1897, genus illeg., Art. 53]. [= Corallomyces berolinensis Henn., Verh. Bot. Vereins Prov. Brandenburg 40: 153. 1898, genus illeg., Art. 53]. = Nectria coccinea (Pers : Fr.) Fr. var. platyspora Rehm, Ann. Mycol. 7: 137. 1900. ≡ Nectria platyspora (Rehm) Weese, in Höhn. & Weese, Ann. Mycol. 8: 464. 1910. [= Corallomyces mauritiicola Henn., Hedwigia 43: 244. 1904, genus illeg., Art. 53]. ≡ Nectria mauritiicola (Henn.) Seifert & Samuels, Stud. Mycol. 27: 161. 1985. = Rhizostilbella rubra Wolk, Mycol. Zentbl. 4: 237. 1914 fide Seifert, 1985. Anamorph: synnematous Rhizostilbella. Teleomorph: Perithecia solitary to gregarious, associated with reddish rhizomorphs and/or synnemata, obpyriform, scarlet, KOH+ blood-red, LA+ yellow, with concolorous scurf, 512–940(–1073) × 309–634 µm (mean = 711 × 486; SD 127, 70; n 72 = 30). Asci narrowly clavate, apex with a ring, with eight-ascospores arranged uniseriately, (123–)145–211 × 8.6–14.3µm (mean=162×11; SD 21, 1.6; n=24). Ascospores ellipsoid, smooth, one-septate, constricted at septum, thick walled, outer wall sometimes sinuous, appearing rough (optical section), finely striated (surface view), 13.7–22.1×5.8–9.2µm (mean=16.8×7.7; SD 1.5, 0.7; n = 110). Culture and anamorph: Colonies 42–69 mm diam (mean=54; SD 7; n=34) after 14d. at 25°C on PDA. Colony surface with synnemata forming near the inoculum or scattered, aerial mycelium white to pale-luteous, cottony to velvety; below aerial mycelium greenish olivaceous; sometimes agar discoloring yellow-green; reverse isabelline at center becoming buff toward colony edge, with isabelline dichotomously branching rhizomorphs immersed in agar. Synnemata cylindrical-capitate, cylindrical, slender to robust, straight, hirsute, orange to luteous, KOH+ livid red, 876– 2,536×248–621µm (mean=1,394×424; SD 545, 108; n=19). Marginal hyphae of synnemata, septate, echinulate, covering entire surface of stipe, with clavate terminal cells, 15–29×7.6–9.9 (mean=21×8.5; SD 5.1, 0.8; n=7). Conidiophores unbranched or once simple monochasial or monoverticillate. Phialides cylindrical, slightly tapering towards tip, hyaline. Conidial mass buff-colored. On SNA, conidiophores simple, unbranched, acremonium-like. Phialides cylindrical, slightly tapering towards tip, collarettes not flared, periclinal thickening conspicuous, hyaline, length 27–81µm (mean=55; SD 11.8; n=81), width at base 2.2–4.2µm (mean=3.3; SD 0.4; n=81), width at tip 1.5–2.8µm (mean=2.1; SD 0.3; n=81). Conidia ellipsoidal, ovoidal with a truncate base, non-septate, smooth-walled, hyaline, 13–26(–28.4) × 7–13 µm (mean=19×10; SD 2.5, 1.1; n=270). 73 Habitat: On bark and roots of decaying or living (diseased) tropical trees, and also isolated from soil. Distribution: Brazil, Colombia, Costa Rica, French Guiana, Nicaragua, Panama, Venezuela (Samuels 1973; Samuels & Dumont, 1982; Rossman et al., 1999), DR Congo, Gabon, Guadeloupe, Ivory Coast, Jamaica, Liberia. Holotype of Corallomyces elegans: Suriname, on bark, Holotype ex herb. Schw. in herb. Berkeley (K; Neotype of Corallomycetella heinsenii designated in Rossman et al. 1999). Epitype of Corallomyces elegans designated herein: French Guiana, Région de Saül, Layon des Eaux Claires, on bark of unidentified tree, 2 May 2008, C. Lechat (CLL8064), Epitype BPI 881071, ex-epitype culture CBS 123826 = AR 4547. Additional type specimens examined: Brazil, Rio Grande do Sul, Saô Leopoldo, on bark, Oct. 1907, S.J. Rick (exsiccati no. 1813; NY, Isotype of Nectria coccinea var. platyspora); Estado de Amazonas, Rio Jurua, Miry, on Mauritia flexuosa, July 1901 (E. Ule Herbarium Brasiliense no. 2837, Holotype of Corallomyces mauritiicola); Cameroon, on bark, J.R. Jungner (FH, Holotype of Corallomyces elegans var. camerunensis). Additional specimens and isolates examined: Brazil, Bahia, Igrapiúna, on dead bark, 12 Sep. 2010, P. Chaverri (PC 1261), O. Liparini Pereira, D. Pinho, A. Luiz Firmino, BPI 884207, culture CBS 134440; Estado do Pará, Belterra, Maguari, Floresta Nacional do Tapajós, elev. 28 m., S2°46'59.6'', W55°01'38.9'', on dead tree of Hevea, 7 May 2011, O. Liparini Pereira & P. Chaverri (PC 1307), BPI 884208, culture CBS 74 134441. Colombia, Puerto Japon, Rio Peneya, Caqueta, on bark, 25 Jul. 1973–28 Jul. 1973, Y. Doi, BPI 842181 = TNS-F-224942 = TNS.D-1720. Costa Rica, isolated from rhizosphere of Musa sapientum, R.D. Goos (No. 1127), culture CBS 276.60 = ATCC 14043 = IMI 84360; Heredia Province, La Selva Biological Station, succession plots 2-3 years old, N10° 25' 23.5" W84° 00' 04.8", on dying base of Musaceae, 16 Mar. 2010, P. Chaverri (PC 1123), BPI 881540; Limón, Santa Rita de Pococí, on canker of Tectona grandis, M. Arguedas, BPI 748185; Heredia Province, La Selva Biological Station, Sendero Tres Rios and Camino Experimental Norte, N10°26’7.3” W84°00’31.4”, alt. 64 m., on unidentified Liana, 17 Mar. 2010, P. Chaverri (PC 1166), BPI 881547, culture CBS 131288 = G.J.S. 10-133; Heredia Province, La Selva Biological Station, Sendero Tres Rios and Camino Experimental Norte, N10°26’7.3” W84°00’31.4”, alt. 64 m., on unidentified palm, 17 Mar. 2010, P. Chaverri (PC 1169), BPI 882353, culture CBS 131289 = G.J.S. 10-134. DR Congo, stem base, INEAC, Afd. Phyto, No. 1191, culture CBS 275.60. French Guiana, Cayenne. Montagne Cacao, Piste Coralie, 4°32'0''N 52°25'0''W, on bark, 14 Feb. 1988, A. Rossman & C. Feuillet (3142), BPI 1107213. Gabon, Crystal Mountains National Park, on dead bark, 30 Apr. 2009, K. Põldmaa (GAB 18), TU 107818 = BPI 879302, culture CBS 125531 = A.R. 4659. Guadeloupe, Sentier de la Cascade, Vauchelet, Gorges de la Riviere, alt. 600 m., on bark, 6 Jan. 1996, J. Vivant, BPI 744460. Ivory Coast, IRCA Plantation, near Abidjan, Hevea brasiliensis, 1968, J.J. Guillaumin, culture CBS 119.84 = IMI 135503. Jamaica, Surrey, St. Andrew Parish, on decaying seed pod, 12 Jan. 1971, RP Korf (CUP-MJ824), JR Dixon, DP Dumont, RW Erb, DH Pfister, DR Reynolds, AY Rossman, GJ Samuels, NY. Liberia, 75 Cavalla, Harbel, Hevea brasiliensis, 25 Nov. 1963, J. Schreurs (No. 72), culture CBS 379.64. Venezuela, Territorio Federal Amazonas, Neblina Base Camp on Rio Baria (= Rio Mawarinuma), left bank, downstream from camp, alt. 140 m., on undetermined substrate, 18 Feb. 1985, A. Rossman (A.R. 2175), BPI 552587; Edo. Barinas, Caimital Forest, La Montana. Ca. 5 km NE of Caimital Village and ca. 10 km NE of intersection with Barinas-Opisbo Rd., dry secondary forest, alt. 800 m., 8°35'N 70°15'W, on bark, 25 Nov. 1990, G.J. Samuels, B. Hein, S. M. Huhndorf (7501), BPI 744870. Notes: Corallomycetella elegans and C. repens are indistinguishable morphologically, except for the synnemata produced in PDA. The synnemata of C. elegans can attain a height of up to 2,500 µm as described by Seifert (1985). The synnemata of C. repens only reaches 600 µm and are cushion-shaped. Corallomycetella elegans is apparently restricted to the tropical Western Hemisphere and Africa. Corallomycetella repens (Berk. & Broome) Rossman & Samuels, Stud. Mycol. 42: 113. 1999. Fig. 2.7 Basionym: Sphaerostilbe repens Berk. & Broome, J. Linn. Soc., Bot. 14: 114. 1875. = Stilbum hibisci Pat., J. Bot., Paris 1891: 320. 1891. ≡ Rhizostilbella hibisci (Pat.) Seifert, Stud. Myco. 27: 162. 1985. = Rhizostilbella rubra van der Wolk, Mycol. Centralbl. 4: 237. 1914. = Stilbum incarnatum Wakker, Ziekten van het Suikerriet op Java, Leiden, p. 197. 76 1898. = Stilbum incarnatum var. dioscoreae Sacc., Boll. Orto Bot. Regia Univ. Napoli 6: 63. 1918. = Cephalosporium kashiense R.Y. Roy & G.N. Singh, Curr. Sci. 37: 535. 1968. ≡ Acremonium kashiense (R.Y. Roy & G.N. Singh) W. Gams, Cephalosporium- artige Schimmelpilze (Hyphomycetes) p. 138. 1971. Anamorph: synnematous Rhizostilbella. Teleomorph: Perithecia solitary to gregarious, associated with reddish rhizomorphs and/or synnemata, obpyriform, scarlet, KOH+ blood-red, LA+ yellow, covered with scurfs, 386–659×264–367µm (mean= 548×318; SD 112, 34; n=6). Asci narrowly clavate, apex with a ring, with eight-ascospores arranged uniseriately, 194–228×16– 20µm (mean =213×18; SD 12.9, 1.4; n=10). Ascospores ellipsoid, smooth, one- septate, constricted at septum, thick walled, outer wall sometimes sinuous, appearing rough (optical section), finely striated (surface view), yellow-brown, 15–21.5×6.7– 8.8µm (mean=18 ×7.8; SD 2.2, 0.7; n=20). Culture and anamorph: Colonies 42–61mm diam (mean=52; SD 6.5; n=12) after 14d. at 25°C on PDA. Colony surface with white aerial mycelium, cottony to velvety; conidial masses buff colored, slimy, pionnotal produced by synnemata near the inoculum; agar discoloring yellow-green; reverse isabelline with conspicuous isabelline dichotomously branching rhizomorphs immersed in agar. Surface of sterile colonies with white aerial mycelium, cottony, reverse saffron. In culture, synnemata 77 cushion-shaped, orange to salmon, KOH+ livid red, 403–523×520–594µm (mean=450×552; SD 64, 38; n=3). Hyphae of synnemata, septate, echinulate. Conidiophores unbranched or once simple monochasial or monoverticillate. Phialides monophialidic, cylindrical, hyaline. On SNA, conidiophores simple, unbranched, acremonium-like. Phialides cylindrical, collarettes not flared, periclinal thickening conspicuous, hyaline, length 39–79µm (mean=53; SD 12.9; n=10), width at base 2.5– 3.8µm (mean=3.1; SD 0.5; n=10), width at tip 1.8–2.7µm (mean=2.3; SD 0.2; n=10). Conidia ellipsoidal to ovoidal with a truncate base, nonseptate, smooth-walled, hyaline, 13–19 × 7–11µm (mean=16×9; SD 1.5, 0.9; n=30). Habitat: On bark and roots of decaying or living (diseased) tropical trees, and also isolated from soil. Distribution: China, India, Indonesia, and Sri Lanka. Holotype of Sphaerostibe repens: Sri Lanka (Ceylon), Peradeniya, on decaying wood of Artocarpus integrifolia, August, Holotype Herb. Berkeley (K), no. 1005. Additional specimens and isolates examined: China, alt. 650m., on bark, 01 OCT. 1993, Y. Doi (93-10), BPI 802510. India, Uttar Pradesh, Varanasi, isolated from rhizosphere of Linum usitatissimum, Jan. 1967, G.N. Singh, culture ITCC 1330=CBS 313.72=IMI 132119 [ex-holotype of Cephalosporium kashiense]. Indonesia, Java, Bogor, on bark of Carica papaya, 16 May 1955, A. Kurnadi, BPI 631169; Java, on root of Carica papaya, Jun. 1948, K.B. Boedijn & J. Reitsma (No. B.R. 29/48), culture CBS 358.49. Sri Lanka, isolated from soil, 1963, O.S. Peries, culture IMI 101072=CBS 118.84. 78 Notes: Corallomycetella repens is only known from South to Southeast Asia. Morphologically, it is nearly indistinguishable from C. elegans, except for its short, cushion-shaped, synnemata in PDA. The observed synnema are from a single culture (CBS 118.84), which was isolated in 1963. One could question whether conidiomata development has changed from a stipitate synnemata to the cushion-shaped synnemata over the years. Fresh collections from Asia are needed to select an epitype in order to stabilize the name and determine the extent of morphological variation. Corallonectria C. Herrera & P. Chaverri, gen. nov. MycoBank No. MB803108 Generic type: Corallonectria jatrophae (A. Møller) C. Herrera & P. Chaverri Etymology: From Greek korallion=coral. Referring to the short, red stalk on which the perithecia develop and making reference to the genus Corallomycetella. Perithecia seated on a short red stalk, in caespitose clusters of 2 to several, ovoid to obpyriform, collapsing laterally or not collapsing when dry, scarlet, KOH+ blood-red, LA+ yellow, with a white to yellow furfuraceous coating of hyphae below apex; furfuraceous coating missing in age; apex acute, smooth, uniloculate. Perithecial surface cells forming textura angularis. Perithecial wall of one region of cells forming textura angularis, becoming narrow, compressed towards the centrum, 30– 40µm thick. Asci clavate, apex simple, with eight ascospores arranged biseriately. Ascospores fusiform-ellipsoid, sometimes reniform, 1-septate, often constricted slightly at septum, pale brown when discharged, smooth-walled. Anamorph synnematous, fusarium-like. 79 Habitat: On bark of decaying or living (diseased) tropical trees. Distribution: Tropical America and Greater Antilles. Notes: Corallonectria is similar to Corallomycetella in that it produces rhizomorphs on PDA. However, the anamorphic states are different. Corallomycetella has a synnematous Rhizostilbella anamorph, while Corallonectria has a synnematous fusarium-like anamorph. Corallonectria is also characterized by a white to yellow furfuraceous coating below the apex of the perithecia, and relatively large, pale- brown, and smooth ascospores. Corallonectria jatrophae (A. Møller) C. Herrera & P. Chaverri, comb. nov. Fig. 2.8 MycoBank No. MB803109 Basionym: [Corallomyces jatrophae A. Møller, Bot. Mitt. Tropen 9: 295. 1901, genus illeg., Art. 53]. ≡ Nectria jatrophae (A. Møller) Wollenw., Z. Parasitenk. (Berlin) 3: 498. 1931 ≡ Corallomycetella jatrophae (A. Møller) Rossman & Samuels, Stud. Mycol. 42: 114. 1999. = Nectria madeirensis Henn., Hedwigia 43: 244. 1904. [= Corallomyces caricae Henn., Hedwigia 43: 245. 1904, genus illeg., Art. 53]. = Macbridella amazonensis Bat., J.L. Bezerra & C.R. Almeida, An. XIV congr. Nac. Soc. Bot. Brasil, 1963: 118. 1964. 80 ≡ Nectria amazonensis (Bat., J.L. Bezerra & C.R. Almeida) Samuels, Canad. J. Bot. 51: 1278. 1973. Anamorph: synnematous, fusarium-like. Teleomorph: Perithecia seated on a short red stalk, in caespitose clusters of 2 to several, ovoid to obpyriform, 729–1308×447–748µm (mean=932×575; SD 171, 83; n=17), not collapsing or collapsing by lateral pinching, orange-red to scarlet, with a white to yellow furfuraceous coating below apex; apex acute, smooth, scarlet. Asci clavate, apex simple, with eight ascospores arranged biseriately, 93–128×10.3– 19.8µm (mean=108×15; SD 12.1, 3.1; n=9). Ascospores fusiform-ellipsoid, sometimes reniform, 1-septate, often constricted slightly at septum, pale brown when discharged, smooth, (26–)27–41.7×6.8–12.2 (mean=32×9; SD 2.9, 0.9; n=139). Culture and anamorph: Colonies 42–57mm diam (mean=48; SD 9.7; n=4) after 14d. at 25°C on PDA. Colony surface with white aerial mycelium, cottony to velvety; agar discoloring amber; synnemata produced near inoculum; agar discoloring amber; reverse saffron, with saffron dichotomously branching rhizomorphs immersed in agar. Synnemata cylindrical, slender to robust, straight or curved, rarely branching, appearing furfuraceous with loose, white hyphae, with a terminal cupulate capitulum (several with age), pale-luteous, KOH+ apricot to scarlet, 1,178–2,464×225–359µm (mean=1,698×286; SD 425, 43; n=10; taller with age). Conidiophores unbranched or once simple monochasial or monoverticillate. Phialides cylindrical, hyaline, length 9– 34µm (mean=22; SD 10.5; n=7), width at base 2.9–3.9µm (mean=3.3; SD 0.3; n=7), width at tip 1.9–2.7µm (mean=2.2; SD 0.3; n=7). Conidial mass forming inside 81 cupulate capitula, flame-shaped, luteous. Conidia fusarium-like, long-fusiform, slightly curving at the apical and basal ends, apical cell acute, basal cell pedicellate, hyaline, forming on PDA, not observed on SNA, 3–4(–5)-septate: 3-septate (68–)71– 84× 5.2–7.7µm (mean=77×6.5; SD 3.8, 0.6; n=30), 4-septate 73–90 × 5.0–7.5µm (mean = 80×6.6; SD 3.9, 0.6; n=30), 5-septate (75–)92–100×5.0–7.2µm (mean = 91 × 6.0; SD 8.4, 0.7; n=6). Habitat: On bark of decaying or living (diseased) tropical trees. Distribution: South and Central America (Belize, Brazil, Colombia, Costa Rica, French Guiana, Nicaragua, Panama, Puerto Rico, Venezuela; Rossman et al., 1999; Samuels, 1973; Samuels & Dumont, 1982). Lectotype of Corallomyces jatrophae designated herein: A plate of illustrations in the original paper of Corallomyces jatrophae, Lectotype (BPI-Stevenson Library) Møller 1901, Taf. I Figs. 21–28. Epitype of Corallomyces jatrophae designated herein: Puerto Rico, Cordillera Central, Guavate Picnic Area, off Rte. 184, alt. 500m., base of living tree, 25 Feb. 1996, G.J. Samuels (8120), H.-J. Schroers, D.J. Lodge, Epitype BPI 745232, ex- epitype culture CBS 913.96 = G.J.S. 96-18. Additional type specimens examined: Brazil, Manaus, on bark of unidentified plant, Batista, 20 Feb. 1961 (URM 22, Holotype of Macbridella amazonensis); Rio Jurua, Cacoeria, on dead stems of Carica sp., May 1901, Ule 2822 (FH, Isotype of Corallomyces caricae). Marmellos, Rio Madeira, on decaying bark, Mar. 1902, Ule 3115, Mycotheca Brasiliensis no. 69 (The Botanical Museum, University of 82 Copenhagen, Isotype of Nectria madeirensis). Additional specimens examined: Belize, Cayo Distr., Blue Hole National Park, Hermons trail, 19 Nov. 2001, L. Ryvarden (44387), BPI 843763. Brazil, Estado do Pará, Belterra, Maguari, Floresta Nacional do Tapajós, elev. 28m., S2°46'59.6'', W55°01'38.9'', on bark, 7 May 2011, O. Liparini Pereira & P. Chaverri (PC 1300), BPI 884209. French Guiana, Route de Belizon, track to Montage Tortue, 15km. from road N2, 52°20', 4°25', on bark of newly killed tree, 18 Feb. 1988, A.Y. Rossman & C. Feuillet (3222), BPI 1107291; ibid., A.Y. Rossman & C. Feuillet (3230B), BPI 1107295. Martinique, Precheur, Anse Couleuvre, on bark, 18 Aug. 2011, C. Lechat CLL MAR11044C (BPI 829340). Puerto Rico, Luquillo Mountains, Bisley Watershed in Valley left of Walkup Tower, below Landslide, Luquillo Experimental Forest, on tree trunk, 14 Mar. 1990, D.J. Lodge (PR 700), BPI 1109351. Venezuela, Edo. Bolivar, along Rio Caroni near Rapids Just below Uriman, elevation 393m., on bark, 11 Jan. 1955, J.A. Steyermark & J.J.Wurdack (80), BPI 552420; Chimanta Massif, Torono-Tepui, Estado Bolivar, rainforest Slopes above Base Camp, on bark, 24 Jan. 1955, J.A. Steyermark & J.J. Wurdack, BPI 1107269; Amazonas, Cerro de La Neblina, Valley at N. Base of Pico Phelps, Cloud Forest, alt. 1,000–1,250m, 00°49'N, 66°00'W, on bark, 12 Apr. 1984–13 Apr. 1984, G.J. Samuels (1297), BPI 1107268; Edo. Miranda, Parque Nacional Guatopo, trail between Agua Blanca and La Cruceta, alt. 500-600m., 10°3'N 66°26'W, on bark, 27 Nov. 1990–30 Nov. 1990, G.J. Samuels, B. Hein, S.M. Huhndorf (7570), BPI 744831. Illustrations. —Samuels (1973, Figs 10-13, as N. amazonensis); Wollenweber (1930, No. 684, as C. jatrophae). 83 Notes: Corallonectria jatrophae is the only species in the genus Corallonectria. It can be easily identified by furfuraceous perithecia on a short red stalk. On PDA, this species produces a luteous colony with rhizomorphs. Ideally an epitype from the same collecting region as the type would be designated, which it would make our isolate from Brazil (PC 1300) ideal. However, the isolate did not survive -80ᵒC storage. Our phylogeny demonstrated that isolates from Puerto Rico (CBS 913.96) and Brazil (PC 1300) are conspecific, and suggesting a broad biogeographic range. Based on the teleomorph, they are indistinguishable. Thus, the specimen from Puerto Rico is designated as epitype. 84 Ta bl e 2. 1 Is o la te s a n d a cc es sio n n u m be rs u se d in th e ph yl o ge n et ic a n a ly se s G en eB a n k A cc es sio n N o . tu b D Q1 90 59 1 G Q2 67 23 2 A Y 67 72 21 A Y 67 72 14 K C4 79 78 0 K C4 79 78 1 K C4 79 78 2 JF 83 28 38 K C4 79 78 3 K C4 79 77 5 K C4 79 77 6 K C4 79 77 8 K C4 79 77 9 K C4 79 77 7 K C4 79 78 8 K C4 79 78 7 K C4 79 78 4 m cm 7 - - - - K C4 79 76 9 K C4 79 76 1 K C4 79 76 2 K C2 91 79 5 K C4 79 76 3 K C4 79 76 8 K C4 79 76 4 K C4 79 76 6 K C4 79 76 7 K C4 79 76 5 - - K C4 79 77 0 a ct - - H M 35 28 81 H M 35 28 82 K C4 79 74 1 K C4 79 73 4 K C4 79 73 5 JF 83 24 40 K C4 79 73 6 K C4 79 73 3 K C4 79 73 0 K C4 79 73 2 K C4 79 73 1 K C4 79 73 7 K C4 79 74 5 K C4 79 74 4 K C4 79 73 8 IT S G Q2 80 54 6 G Q2 80 64 7 A Y 67 73 01 A Y 67 73 06 K C4 79 74 8 K C4 79 74 6 K C4 79 74 7 JF 83 25 94 K C4 79 74 9 K C4 79 75 2 K C4 79 75 0 K C4 79 75 4 K C4 79 75 1 K C4 79 75 3 K C4 79 75 9 K C4 79 75 8 K C4 79 75 5 C o u n tr y o f o ri gi n - - So u th A fri ca - Co st a R ic a Co st a R ic a Co st a R ic a Fr en ch G u ia n a B ra zi l Iv o ry Co as t G ab o n Li be ria B ra zi l Za ire B ra zi l Pu er to R ic o Sr i L an ka Su bs tr a te / H o st - - Vi tis vi n ife ra - M u sa sa pi en tu m Li a n a sp . D ec ay in g pa lm B ar k H ev ea br a si lie n sis H ev ea br a si lie n sis B ar k H ev ea br a si lie n sis B ar k St em ba se B ar k B as e o f l iv in g tr ee So il H er ba ri u m N o . B PI 88 15 47 B PI 88 23 53 B PI 88 10 71 B PI 88 42 08 B PI 87 93 02 B PI 88 42 07 B PI 88 42 09 B PI 74 52 32 Is o la te N o . CB S 11 48 13 CB S 12 52 61 , CM W 31 18 2 CB S 11 26 13 CB S 11 26 79 CB S 27 6. 60 G . J.S . 10 - 13 3, CB S 13 12 88 G . J.S . 10 - 13 4, CB S 13 12 89 A . R . 45 47 , CB S 12 38 26 P. C. 13 07 , CB S 13 44 41 CB S 11 9. 84 A . R . 46 59 , CB S 12 55 31 CB S 37 9. 64 P. C. 12 61 , CB S 13 44 40 CB S 27 5. 60 P. C. 13 00 G . J.S . 96 - 18 , CB S 91 3. 96 CB S 11 8. 84 Sp ec ie s Ca lo n ec tr ia a ci co la Ca lo n ec tr ia de n sa Ca m py lo ca rp o n fas ci cu la re Ca m py lo ca rp o n ps eu do fas ci cu la re Co ra llo m yc et el la el eg a n s Co ra llo m yc et el la el eg a n s Co ra llo m yc et el la el eg a n s Co ra llo m yc et el la el eg a n s Co ra llo m yc et el la el eg a n s Co ra llo m yc et el la el eg a n s Co ra llo m yc et el la el eg a n s Co ra llo m yc et el la el eg a n s Co ra llo m yc et el la el eg a n s Co ra llo m yc et el la el eg a n s Co ra llo m yc et el la jat ro ph a e Co ra llo m yc et el la jat ro ph a e Co ra llo m yc et el la re pe n s 85 Ta bl e 2. 1. C o n tin u ed G en eB a n k A cc es sio n N o . tu b K C4 79 78 5 K C4 79 78 6 H M 48 45 89 K C2 91 90 4 - H M 48 46 11 K C2 91 93 2 - EU 86 00 31 EU 86 00 32 A Y 67 72 42 H M 35 28 56 H M 05 41 09 JF 83 29 13 - - K C2 91 93 5 m cm 7 K C4 79 77 1 K C4 79 77 2 - K C2 91 80 4 - - K C4 79 77 3 - - - - - - - - - K C2 91 79 8 a ct K C4 79 74 0 K C4 79 73 9 G Q5 05 96 7 K C4 79 74 2 - G Q5 05 96 1 - - - - - H M 35 28 71 - JF 83 24 81 - - - IT S K C4 79 75 6 K C4 79 75 7 H M 48 45 37 K C2 91 73 1 H M 62 66 60 H M 48 45 58 K C2 91 74 4 H Q8 97 81 1 EU 86 00 58 EU 86 00 60 H M 36 43 02 H M 36 42 90 - JF 83 26 56 H Q8 97 81 0 H Q8 97 82 6 K C2 91 75 1 C o u n tr y o f o ri gi n In do n es ia In di a G er m an y Cz ec h Re pu bl ic - Fr an ce U SA - Co m o ro s Fr an ce - - - Ec u ad o r - - N ew Ze al an d Su bs tr a te / H o st Ca ric a pa pa ya So il In o n o tu s n o du lo su s Ti lia - B u x ac ea e D ia tr yp e st ig m a - - - - - - - - - sc al e in se ct H er ba ri u m N o . B PI 80 27 29 B PI 74 83 07 - Is o la te N o . CB S 35 8. 49 CB S 31 3. 72 A R 27 41 , CB S 11 40 50 CB S 10 24 33 CB S 12 55 54 G . J.S . 98 - 12 7, CB S 10 17 34 G . J.S . 10 - 19 3, C. H . 10 - 01 CB S 12 54 94 F- 22 3, 90 8 F- 16 7, 58 9 CB S 11 26 02 A . R . 25 53 , A TC C 20 88 37 st ra in 56 22 G JS 01 - 66 , CB S 12 64 41 CB S 10 00 01 CB S 12 54 95 A . R . 45 80 , CB S 13 39 64 Sp ec ie s Co ra llo m yc et el la re pe n s Co ra llo m yc et el la re pe n s Co sm o sp o ra co cc in ea Co sm o sp o ra vi rid es ce n s Cy a n o n ec tr ia bu xi Cy a n o n ec tr ia cy a n o st o m a D ia lo n ec tr ia ep isp ha er ia D ia lo n ec tr ia ep isp ha er ia Fu sic o lla a ce til er ea Fu sic o lla vi o la ce a Ily o n ec tr ia lir io de n dr i Ily o n ec tr ia ra di ci co la La n a to n ec tr ia fla vo la n a ta La n a to n ec tr ia flo cc u le n ta M a cr o co n ia le pt o sp ha er ia e M a cr o co n ia pa pi lio n a ce a ru m M ic ro ce ra la rv a ru m 86 Ta bl e 2. 1. C o n tin u ed G en eB a n k A cc es sio n N o . tu b EU 86 00 20 H M 48 46 06 JF 83 29 02 H M 35 28 57 H M 35 28 63 H M 48 48 86 JF 83 28 55 H M 48 45 93 A B 23 75 26 JF 83 29 11 - - - - K C4 79 78 9 - - m cm 7 - JN 99 33 35 JN 99 33 21 - - - - - - - - - - - K C4 79 77 4 - - a ct - H M 48 45 03 JF 83 25 06 H M 35 28 72 H M 35 28 79 - JF 83 24 64 H M 48 45 07 - JF 83 25 15 - - - - K C4 79 74 3 - - IT S EU 86 00 74 H M 48 45 48 JF 83 26 47 H M 36 42 91 H M 36 42 97 H M 48 48 67 JF 83 26 03 H M 48 45 44 A B 23 31 76 JF 83 26 61 A Y 56 69 95 A J6 21 77 5 A J5 57 83 0 A J5 58 11 4 K C4 79 76 0 A Y 21 44 39 H Q8 97 80 5 C o u n tr y o f o ri gi n U SA Fr an ce V en ez u el a - - Ca m er o o n A u st ria A u st ria Ja pa n U SA - G er m an y U SA R u ss ia En gl an d - - Su bs tr a te / H o st - D ea d tw ig s o f A es cu lu s sp . de ad ba rk o f t re e - - lia n a Pi n u s sy lv es tr is Be rb er is vu lg a ri s M yr ic a ru br a - - sk in in fe ct io n o f m an - hu m an bl o o d Fi cu s ca ric a - - H er ba ri u m N o . B PI 87 99 81 B PI 88 10 41 B PI 86 38 54 B PI 74 63 48 B PI 74 63 49 B PI 88 10 70 - - Is o la te N o . F- 26 7, 62 3 A . R . 44 77 , CB S 12 51 65 G . J.S . 09 - 13 29 A . R . 31 03 , CB S 12 51 33 A TC C 16 23 7 G . J.S . 01 - 20 6, CB S 10 98 76 A . R . 27 78 , CB S 12 51 30 A . R . 27 79 , CB S 11 50 34 TP PH 32 Y . H . 10 - 01 , CB S 12 91 58 CB S 39 9. 73 CB S 12 2. 29 N R R L 20 42 0 N H R C- FC 04 2 CB S 40 0. 52 CB S 34 6. 70 CB S 12 54 89 Sp ec ie s M ic ro ce ra ru br a N ec tr ia ci n n a ba rin a N ec tr ia ps eu do tr ic hi a N eo n ec tr ia fuc ke lia n a N eo n ec tr ia ra m u la ria e O ph io n ec tr ia tr ic ho sp o ra Pl eo n ec tr ia cu cu rb itu la Pl eo n ec tr ia la m yi Ru go n ec tr ia ru gu lo sa Ru go n ec tr ia ru gu lo sa Sa ro cl a di u m a tte n u a tu m Sa ro cl a di u m ki lie n se Sa ro cl a di u m ki lie n se as N ec tr ia m a u rit iic o la Sa ro cl a di u m ki lie n se as N ec tr ia m a u rit iic o la Sa ro cl a di u m ki lie n se as N ec tr ia m a u rit iic o la Sa ro cl a di u m st ric tu m St yl o n ec tr ia a pp la n a ta 87 Ta bl e 2. 1. C o n tin u ed G en eB a n k A cc es sio n N o . tu b - H M 35 28 60 H M 48 46 10 JF 83 29 12 - * K C4 79 73 0– K C4 79 78 9 se qu en ce s w er e pr o du ce d in th is st u dy . m cm 7 - - - - - a ct - H M 35 28 75 G Q5 05 95 9 G Q5 05 98 5 - IT S H Q8 97 83 1 H M 36 42 94 H M 48 45 59 JF 83 26 78 JN 04 98 38 C o u n tr y o f o ri gi n - - N ew Ze al an d Po rt u ga l - Su bs tr a te / H o st - - D a cr yd iu m cu pr es sin u m - - H er ba ri u m N o . Is o la te N o . D A O M 23 58 18 A . R . 43 24 , CB S 12 51 53 G . J.S . 83 - 15 6, CB S 11 24 64 A . R . 17 70 , CB S 12 51 23 CB S 10 27 97 Sp ec ie s St yl o n ec tr ia pu rt o n ii Th el o n ec tr ia di sc o ph o ra Th el o n ec tr ia w es tla n di ca Vi rid isp o ra a la ta Vi rid isp o ra di pa rie tis po ra 88 Table 2.2. Loci used in the phylogenetic analyses. Information on the primers, including base pairs, PCR protocols, and models of nucleotide substitution are indicated. Locus ITS act mcm7 tub Combined Included sites 561 584 576 583 2304 Phylogenetically informative sites 248 102 167 282 799 Uninformative polymorphic sites 48 38 73 77 236 Invariable sites 0 154 0 192 345 Nucleotide substitution models (coded as) TVM+G (nst = 6, rates = gamma) TIM1+I+G (nst = 6, rates = invgamma) TIM1+G (nst = 6, rates = gamma) HKY+I+G (nst = 2, rates = invgamma) Primers used (reference) ITS5, ITS4 (White et al. 1990) Tact1, Tact2 (Samuels et al. 2006) mcm7-709for, mcm7-1348rev (Schmitt et al. 2009) Btub-TI, Btub-T2 (O’Donnell & Cigelnik 1997) PCR protocol: Annealing temp. & cycles 53 °C, 1 min, 40x 65 °C, 30 s, 15x 48 °C, 30 s, 30x 56 °C, 50 s, 38x 55 °C, 30 s, 35x Table 2.3. Genealogical Sorting Index (gsi) and probability values for single gene trees and the ensemble of gene trees. Gene C. repens sensu Rossman et al. (1999) C. repens s. str. (Asian clade) C. elegans (African + American clades) C. elegans (African Clade) C. elegans (American Clade) gsi P gsi P gsi P gsi P gsi P act 1 0.071 1 0.003 0.675 0.001 0.711 0.001 0.711 <0.001 ITS 1 0.069 1 0.003 1 0.001 0.711 0.001 1 <0.001 mcm7 1 0.072 1 0.002 0.675 0.001 0.519 0.005 1 <0.001 tub 1 0.075 0.6061 0.014 1 0.001 1 <0.001 1 0.001 ALL (gsiT) 1 0.075 0.902 0.004 0.838 0.001 0.735 0.001 0.902 0.003 89 Fig. 2.1. Phylogenetic placement of Corallomycetella sensu Rossman et al. (1999) inferred from act. Best tree generated with ML analysis (−Ln = 2514.7837). Values at branches indicate Maximum Likelihood bootstrap (ML BP)/Bayesian posterior probabilities (BI PP). 90 Fig. 2.2 Phylogenetic placement of Corallomycetella sensu Rossman et al. (1999) based on a combined 3-loci (ITS, mcm7, and tub) dataset. Best tree generated with ML analysis (−Ln = 17491.8148). Values at branches indicate Maximum Likelihood bootstrap (ML BP)/Bayesian posterior probabilities (BI PP). 91 Fig. 2.3. Phylogenetic relationship of Corallomycetella repens sensu Rossman et al. (1999). Trees with the best log likelihood are presented for (A) act, (B) ITS, (C) mcm7, and (D) tub. Thicker lines indicate well-supported branches (>70% ML BP). Fig. 2.4. Multilocus haplotype network for Rossman et al. (1999). circle represents a haplotype; size of circle is proport Within each haplotype circle, geographic origins of isolates are proportionally represented as a pie chart. Empty circles represent intermediate haplotypes inferred by TCS. Each line segmen 92 Corallomycetella repens The network was constructed in TCS v1.21. Each colored ional to haplotype frequency. t represents a single mutation. sensu 93 Fig. 2.5. Haplotype networks of act, ITS, mcm7, and tub. The network was constructed in TCS v1.21. A) act; B) ITS; C) mcm7; D) tub. Each colored circle represents a haplotype; size of circle is proportional to haplotype frequency. Within each haplotype circle, geographic origins of isolates are proportionally represented as a pie chart. Empty circles represent intermediate haplotypes inferred by TCS. Each line segment represents a single mutation. 94 95 Fig. 2.6. Corallomycetella elegans. A, B: Perithecia on natural substrata; C: Perithecium in 3% KOH; D: Median section of perithecium; E: Perithecial surface cells; F: Asci; G: Ascospores (optical section); H: Ascospore (surface view); I: Synnemata on PDA; J: Conidia produced by synnemata; K: Conidium produced by mononematous anamorph; L: Synnema in 3% KOH; M: Marginal hyphae of synnema; N–O: Conidiophores/phialides of synnema; P: Mononematous anamorph; Q–S: Colonies after 2 wks at 25℃ on PDA. (T–U) Colony reverse after 2 wks at 25℃ on PDA. Scale bars: A–C, I=500µm; D=200µm; E, G, H, J, K, O=20µm; F, M, N, P=50µm; R–U=10mm. 96 97 Fig. 2.7. Corallomycetella repens. A: Perithecia/synnemata on natural substrata; B: Perithecia on natural substrata; C: Synnemata on natural substrata; D: Perithecium in 3% KOH; E: Ascospore (surface view); F: Ascus; G: Conidial masses; H, I: Synnema in 3% KOH; J–K: Conidiophores/phialides of synnema; L: Conidia produced by synnema; M: Mononematous anamorph; N: Colony after 2 wks at 25℃ on PDA; O, P: Colony reverse; Q: Colony of sterile isolate after 2 wks at 25℃ on PDA; R: Colony reverse of sterile isolate. Scale bars: A, G: 500µm; B, D: 100µm; C, H, I: 200µm; E, L: 10µm; F, J, K: 20µm; N–R: 10mm. 98 99 Fig. 2.8. Corallonectria jatrophae. A: Perithecia on natural substrata; B: Median section of perithecium; C: Ascus; D: Perithecium in 3% KOH; E: Ascospores (optical section); F: Ascospore (surface view); G: Colony after 2 wks at 25℃ on PDA; H, I: Colony reverse; J: Synnemata after 2 wks at 25℃ on PDA; K: Synnema after 8 wks at 25℃ on PDA; L: Phialides of synnema; M: Conidia produced by synnema. Scale bars: A, D: 200µm; B: 100µm; C: 20µm; E, F, L, M: 10µm; G–I: 10mm; J: 700µm; K: 1,000µm. 100 Chapter 3: Phylogenetic and taxonomic revision of the Cosmospora viliuscula species complex: Cosmospora species that grow on xylariaceous fungi. ABSTRACT The genus Cosmospora includes nectroid fungi that grow on polypores and xylariaceous fungi. The species growing on xylariaceous fungi have been known previously as ‘Cosmospora vilior,’ although recently these fungi have been referred to as Cosmospora viliuscula. In this paper, the phylogenetic relationships and taxonomy of C. viliuscula are revised. A phylogeny was generated with maximum likelihood and Bayesian inference methods based on a three-partition dataset (ITS, LSU, and mcm7-rpb1-tub2). We demonstrate that Cosmospora viliuscula represents a species complex of which each well-supported clade is regarded as species. A few rare species were also described, namely C. fomiticola, C. novazelandica, and C. stilbohypoxyli. For the first time the sexual morphs of C. arxii and C. khandalensis are described. INTRODUCTION The generic concept of Cosmospora Rabenh. (Nectriaceae, Hypocreales, Sordariomycetes, Ascomycota) was based on sexual state morphology (Rossman et al. 1999; also referred to as Cosmospora sensu Rossman). It included nectria-like species with small, reddish, KOH+, smooth, thin-walled, laterally collapsing when 101 dry, non- or weakly stromatic perithecia (Samuels et al. 1991, Rossman et al. 1999). Asexual states of Cosmospora sensu Rossman included fusarium-like, acremonium- like, Chaetopsina Rambelli, Stilbella Lindau, and Volutella Fr. (Rossman et al. 1999). Cosmospora species exhibit greater diversity in warm temperate and tropical regions, and have a fungicolous, insecticolous, rarely lignicolous, corticolous, and herbicolous habits (Samuels et al. 1991, Rossman et al. 1999). Given its range of asexual states and ecological habits, the genus was considered polyphyletic, and has been split into monophyletic groups based on asexual characteristics: Chaetopsina Rambelli, Nectricladiella Crous & Schoch, Fusicolla Bonord., Macroconia (Wollenw.) Gräfenhan et al., Microcera Desm., Pseudocosmospora C. Herrera & P. Chaverri,, Stylonectria Höhn. and Volutella Fr. (Schoch et al. 2000, Gräfenhan et al. 2011, Luo and Zhang 2010, 2012, Herrera et al. 2013). Herrera et al. (2013) presented a detail history of Cosmospora sensu Rossman. The name Cosmospora vilior (Starbäck) Rossman & Samuels (≡ Nectria vilior), a common tropical species of nectria-like fungi, was applied to collections growing on stromata of Xylariaceae (Xylariales, Sordariomycetes, Ascomycota). However, it was recognized early on that the type specimen of that name did not occur on a xylariaceous fungus (e.g. Samuels et al. 1990). It has since been demonstrated that the name “C. vilior” was misapplied to Cosmospora occurring on xylariaceous fungi (see Herrera et al. 2013). Herrera et al. (2013) referred to the group of fungi occurring on xylariaceous fungi as the Cosmospora viliuscula (Samuels, Yoshim. Doi & Rogerson) Rossman & Samuels species complex because its type specimen grew on a xylariaceous fungus (Kretzschmaria cf. deusta (Hoffm.) P.M.D. Martin). In addition, 102 Samuels et al. (1990) diagnosed Cosmospora viliuscula (as N. viliuscula) as being morphologically similar to ‘Nectria’ vilior but having slightly smaller ascospores. Cosmospora vilior has been transferred to the newly described genus Pseudocosmospora C. Herrera & P. Chaverri, whose species occur mainly on Eutypa Tul. & C. Tul. and Eutypella (Nitschke) Sacc. species (Diatrypaceae, Xylariales, Sordariomycetes, Ascomycota; Herrera et al. 2013). The present taxonomic treatment deals with the Cosmospora viliuscula species complex, which up to now, had been thought to be a single species. We describe many seven new species in this complex based on multi locus phylogenetic analyses, and report an apparent host specificity by the recognized species within this complex. Results from this study show once again the prevalence of morphologically similar species complexes in the Hypocreales and the utility of molecular methods for defining cryptic species. MATERIALS AND METHODS Herbarium specimens and cultures Fresh specimens of Cosmospora spp. were collected on trips to Argentina, Brazil, Costa Rica, Peru, and USA. Pure cultures were obtained by isolating single ascospores from the freshly collected samples and grown in cornmeal dextrose agar (CMD; Difco™ cornmeal agar + 2% w/v dextrose + antibiotics). Dried specimens were deposited at the U.S. National Fungus Collections (BPI), Beltsville, Maryland, USA, and the cultures derived from the fresh specimens were deposited at 103 Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands. Accession numbers are listed in Table 3.1. Herbarium specimens of Cosmospora spp. were borrowed from the U.S. National Fungus Collections (BPI); Royal Botanic Gardens Kew (K); William and Lynda Steere Herbarium, New York Botanical Garden (NY); New Zealand National Fungal Herbarium (PDD); and Herbarium of the Botany Department, Swedish Museum of National History (S). Extant cultures corresponding to these herbarium specimens were obtained from the culture collection at USDA, ARS, Systematic Mycology and Microbiology Laboratory (SMML) and CABI Bioscience Fungus Collection (formerly International Mycological Institute, IMI). Additional cultures were obtained from CBS and Ministry of Agriculture, Forestry and Fisheries (MAFF) culture collection (see Table 3.1). Morphological characterization Sexual state morphology was observed using a stereoscope (Olympus SZX12 Olympus, Tokyo, Japan). The features of the perithecia were characterized (e.g. color, shape, size, ornamentation, and habit). To observe their internal structures, the perithecia were rehydrated in 3% KOH, and the centrum isolated on a glass slide and covered with a coverslip. Microscopic characters such as asci and ascospores were observed under a compound microscope (Olympus BX50; Olympus, Tokyo, Japan). Colony morphology was characterized in two different growing media: cornmeal dextrose agar (CMD; Difco™ cornmeal agar + 2% w/v dextrose) and Difco™ potato dextrose agar (PDA). Two replicates with two pseudoreplicates were grown for each 104 isolate in each growing medium. Cultures were grown in an incubator that alternates between fluorescent light and darkness (12h/12h) at 25°C. Colony growth rate was measured weekly for two weeks. Colony color is described using the terminology in Rayner (1970). Asexual state morphology (in culture) was characterized in synthetic nutrient-poor agar (SNA; Nirenberg 1976). Isolates were grown under the conditions described above. A block of agar was cut, placed on a microscope slide, covered with a coverslip, and examined by light microscopy (Olympus BX50; Olympus, Tokyo, Japan). Measurements of continuous characters, such as length and width, were made with Scion Image software beta 4.0.2 (Scion Corp., Frederick, Maryland), and were summarized by descriptive statistics, e.g., minimum, maximum, mean and standard deviation. DNA extraction, PCR, and sequencing DNA extraction was performed as described in Hirooka et al. (2010). Briefly, Cosmospora isolates were grown in Difco™ potato dextrose broth, and the mycelial mat was harvested after one week of growth for DNA extraction. DNA was extracted with PowerPlant® DNA Isolation Kit (MO BIO Laboratories Inc., Solana Beach, California). Six partial loci were amplified and sequenced. These loci comprised two ribosomal DNA regions and four protein-coding regions. These loci and the primers used to amplify them are listed in Table 3.2. PCR reaction mixtures were prepared as described in Herrera et al. (2013). PCR amplifications were carried out in an 105 Eppendorf Mastercycler thermocycler (Eppendorf, Westbury, New York) under the cycle conditions listed in Table 3.2. PCR products were cleaned with ExoSAP-IT® (USB Corp., Cleveland, Ohio) and the cleaned products were sequenced at the DNA Sequencing Facility (Center for Agricultural Biotechnology, University of Maryland, College Park, Maryland) or McLAB DNA sequencing services (San Francisco, California). Sequences were assembled and edited with Sequencher 4.9 (Gene Codes, Madison, Wisconsin). Sequences were deposited in GenBank (accession numbers are listed in Table 3.1). Phylogenetic analyses Multiple sequence alignment for each locus was performed via the MAFFT v.6 web service (http://mafft.cbrc.jp/alignment/server/; Katoh et al. 2002, 2013) with the E- INS-i alignment strategy and the 1PAM / κ=2 scoring matrix for nucleotide sequences. Alignments were manually edited in in Mesquite 2.75 (Maddison and Maddison 2011). Alignments were deposited in TreeBase under accession number XXXXX. PartitionFinder (Lanfear et al. 2012) was used with default settings to find the best-fit partitioning scheme among the sequenced loci. CONCATEPILLAR 1.4 (Leigh et al. 2008) was used to determine which partitions could be concatenated and analyzed to estimate a phylogeny. Partitions were concatenated if the p-value was greater than the estimated α-level correction of 0.025, which indicated that the null hypothesis, i.e., congruence of loci, could not be rejected. 106 JModeltest (Guindon and Gascuel 2003; Darriba et al. 2012) was used to infer the model of nucleotide substitution for each locus with default settings (11 substitution schemes, +F, +I, +G). The base tree for likelihood calculations was ML optimized. Once likelihood scores were calculated, the best model for each partition was selected according to the Akaike Information Criterion (AIC). The phylogeny for Cosmospora was estimated with maximum likelihood and Bayesian methods. Maximum likelihood (ML) analyses were performed with GARLI v2.0 (Genetic Algorithm for Rapid Likelihood Inference; Zwickl 2006) via the GARLI web service (http://www.molecularevolution.org; Bazinet and Cummings 2011), which uses a grid computing system associated with The Lattice Project (Cummings and Huskamp 2005; Bazinet and Cummings 2008). Fifty independent search replicates were performed to find the best tree with a fast ML stepwise- addition algorithm. Two thousand bootstrap replicates were used in the bootstrap analysis. Bayesian analyses were performed in MrBayes v3.2.2 (Ronquist et al. 2012). A majority rule consensus tree was generated by running four chains for 10,000,000 Markov Chain Montecarlo generations sampling trees every 100th generation, and discarding the first 25% of the sampled trees as burn-in. Tracer version 1.5 (Rambaut and Drummond 2007) was used to confirm whether the negative log likelihoods had reached convergence. Species Recognition Species were recognized in Cosmospora based on the modified criterion used by Pringle et al. (2005) of the genealogical concordance phylogenetic species 107 recognition (GCPSR; Taylor et al. 2000). According to the criterion used by Pringle et al. (2005) of the GCPSR, a putative species is recognized if a clade is well supported in the majority of single gene trees. Single gene trees were generated with MrBayes v3.2.2 as described above, except that only 5,000,000 Markov Chain Montecarlo generations were run. Morphological species recognition and ecological species recognition were used to support the species inferences made when applying GCPSR. Thus, a clade, or putative species, may be associated with unique morphological and/or ecological traits that set it apart from other closely related species. RESULTS Phylogenetic analyses PartitionFinder determined four partitions: ITS, LSU, mcm7+rpb1+tub2, and tef1. The null hypothesis that these partitions were congruent was tested in CONCATEPILLAR, which rejected this null hypothesis (P < 0.025). In a separate analysis, the partitions ITS, LSU, and mcm7+rpb1+tub2 were found to be congruent (P = 0.043; i.e., we failed to reject the null hypothesis that these loci were congruent), and were concatenated. Tef1 was analyzed separately (Supplemental Figure 3.5). The ITS, LSU, and mcm7+rpb1+tub2 concatenated matrix comprised of 3327 total characters; 681 characters were parsimony informative; 254 characters were parsimony uninformative; and 1991 characters were invariable. Table 3.2 lists information for each individual locus. 108 The topologies of the generated phylogenetic trees for ML and BI were not congruent (Figures 3.1, 3.2). The negative log likelihoods for the phylogenetic trees were −18580.293 and −18297.8348, respectively. The topologies differed primarily in deep node relationships of the genus Cosmospora (node labeled “A” in Figures 3.1 and 3.2; 74% ML BP; 100% BI PP). Basal relationships were unresolved in the Bayesian tree with a polytomy that includes the following lineages: Cosmospora fomiticola (described below), C. viridescens, and the Cosmospora viliuscula species complex (node labeled “B” in Figures 3.1 and 3.2). In addition, basal nodes of the Bayesian tree were unsupported (<70% BI PP). In the ML tree, the topology was resolved, although basal nodes were poorly supported (<70% ML BP). Basal species such as C. coccinea and C. fomiticola occur on polypores (Polyporaceae, Basidiomycota), while C. viridescens occurs on various ascomycetous and other substrates. All lineages within the Cosmospora viliuscula species complex corresponding to recognized species received high ML BP and BI PP support (with one exception; Figures 3.1 and 3.2; Table 3.3). At least two genes supported the recognized species (Supplemental Figures S3.1–S3.6; Table 3.3). Clades within this complex correlate with specific fungal hosts belonging to the Xylariaceae (Ascomycota). Morphological studies Little morphological variation exists among species of the Cosmospora viliuscula complex. The confidence intervals of character means (e.g. ascospore length, width, ratio of length and width; conidia length, width, ratio of length and width; colony growth) generally overlapped among species in the complex with some exceptions. 109 The ascospores of Cosmospora novazelandica (described below) were longer and wider compared to other species in this complex. The conidia length of Cosmospora arxii and C. stilbohypoxyli (described below) were not different, but had longer conidia compared to other species in the complex. Cosmospora stilbohypoxyli had wider conidia. Full pairwise comparisons are presented in Supplemental Table 3.1. DISCUSSION Cosmospora Rabenh. The generic concept of Cosmospora is based on its type Cosmospora coccinea Rabenh., which was lectotypified by Rossman et al. (1999). Its asexual state was described as Verticillium olivaceum W. Gams, although it is unrelated to Verticillium Nees in the strict sense. Morphologically the asexual state of C. coccinea varies from acremonium-like (single phialide, unbranched) to verticillium-like (branching into 1– 3 phialides). The asexual state of some species of Cosmospora produce secondary branches, ultimately terminating in 1–3 phialides. The asexual state is the character that unites species in Cosmospora sensu stricto (Gräfenhan et al. 2011). Herrera et al. (2013) described Pseudocosmospora for a cosmospora-like clade that has an acremonium-like asexual state, but is unrelated to Cosmospora species. Pseudocosmospora species have different hosts, which belong to the Diatrypaceae (Xylariales, Ascomycota). The reported fungal hosts of Cosmospora species belong to two unrelated fungal families: Xylariaceae (Ascomycota) and Polyporaceae (Basidiomycota). The genus Cosmospora sensu stricto is monophyletic and well supported (node 110 labeled A in Figs. 3.1 and 3.2; 74% ML BP; 100% BI PP). However, basal relationships are poorly resolved. In the ML tree, basal nodes have low support values (<70% ML BP), and in the Bayesian tree, a basal polytomy is present. Cosmospora is likely to have undergone a putative early fast radiation given this basal phylogenetic structure. Similar deep node relationships have been observed in other organisms that have undergone a fast radiation (e.g., birds, Pons et al. 2005; fishes, Chen et al. 2003; plants, Fishben and Soltis 2004; snakes, Kelly et al. 2009; among others). Reconstructing basal relationships in systems undergoing early radiations is difficult because phylogenetic signal is often masked by multiple hits, which increase with time (reviewed in Donoghue and Sanderson 1992). Phylogenetic resolution can be accomplished by increasing the number of characters and/or by doing a more exhaustive sampling of taxa (reviewed in Hillis and Wiens 2000). Species recognition Species were recognized as described in Herrera et al. (2013), except that we follow the GCPSR criterion used by Pringle et al. (2005). All clades with high support (>95% BI PP) in the majority of single gene trees were recognized as species (Supplemental Figures S3.1–S3.6). Further support for recognizing these clades as species is their apparent host specificity, and in some instances, these recognized species exhibit unique morphological characters that differentiate them. Species such as Cosmospora viliuscula are well supported (100% ML BP; 100% BI PP), produce a penicillate asexual state, and have Kretzschmaria cf. deusta as its host. This host differs from Kretzschmaria deusta in that it occurs in tropical regions, while the native range of K. deusta is in temperate regions (Rogers & Ju 1998). One species of 111 Cosmospora, C. ustilinae, occurs on K. deusta in temperate regions, and is highly supported as well (100% ML BP; 100% BI PP). Cosmospora scruposae produces long phialides, occurs on Xylaria scruposa, and is highly supported (100% ML BP; 100% BI PP). Similarly, C. arxii, C. clavi, and C. micropedis occur on diverse hosts (Hypoxylon fragiforme/H. howeanum, Kretzschmaria clavus/K. sp., and K. micropus/H. cyclopicum, respectively), and have maximum support. Species on each host set are probably closely related, but have not been treated phylogenetically. Identification of the hosts for C. khandalensis could not be determined to species rank. A more holistic approach, i.e. studying both parasite and host together, is needed as suggested by Herrera et al. (2013). We also recognize some singletons as species based on the rule of rarity (reviewed in Lim et al. 2012) given the apparent high fidelity of Cosmospora species to their hosts. Cosmospora fomiticola, C. novazelandica, and C. stilbohypoxyli occur on Fomes fomentarius (Polyporaceae, Basidiomycota), Annulohypoxylon bovei (Xylariaceae, Ascomycota), and Stilbohypoxylon quisquiliarum (Xylariaceae, Ascomycota), respectively. With extensive searching in herbaria, we were able to identify additional specimens with the same hosts and similar morphology as these Cosmospora species. However, no extant cultures are associated with these specimens, and thus, DNA could not be extracted. These species have been poorly sampled due to lack of experts in their regions of origin. Apart from the unique ecological roles of these fungi, these species bear morphological differences that set them apart from other recognized species. For example, C. novazelandica has cells that protrude from the apex of the perithecia, which results in a roughened appearance, and C. stilbohypoxyli has 112 extremely slow growing colonies in PDA. Additionally, in the phylogenetic tree these species have relatively long branches, which suggest that there have been multiple substitutions per site since their speciation. TAXONOMY Cosmospora Rabenh., Hedwigia 2: 59. 1862. Generic type: Cosmospora coccinea Rabenh., Fungi europ. exsicc.: no. 459. 1862. Habitat: On polypores (e.g., Fomes (Fr.) Fr., Fomitopsis P. Karst., Inonotus P. Karst., and Stereum Hill ex Pers.), xylariaceous fungi (e.g., Annulohypoxylon Y.M. Ju, J.D. Rogers & H.M. Hsieh, Hypoxylon Bull., Kretzschmaria Fr., Stilbohypoxylon Henn., and Xylaria Hill ex Schrank); often isolated from soil. Distribution: Cosmopolitan. Descriptions and illustrations: Gräfenhan et al. (2011); Rossman et al. (1999) Notes: The present paper follows the generic concept of Cosmospora by Gräfenhan et al. (2011). In Gräfenhan et al. (2011), the genus was restricted to cosmospora-like fungi tending to occur on other fungi, particularly on polypores and xylariaceous fungi, and have an acremonium-like to verticillium-like asexual state. The perithecia are nonstromatic, collapsing laterally, scarlet, and generally less than 300 μm. The ascospores are ellipsoid, minutely verrucose, verrucose, or tuberculate, yellow-brown at maturity, and <10 μm. On PDA, colonies are velvety, slightly flocculose, olivaceous, and on CMD, colonies are dark-green, flat. Key to species of Cosmospora sensu stricto 1. On bone and Ruzenia spermoides (Lasiosphaeriaceae)………......…..C. viridescens 113 1. On Polyporaceae and Xylariaceae (excluding Biscogniauxia spp.) ……………….2 2. On Polyporaceae……………………………………………………………………3 2. On Xylariaceae……………………………………………………………………...4 3. On Inonotus spp.; ascospores tuberculate……………………………….C. coccinea 3. On Fomes fomentarius; ascospores verrucose…………………………C. fomiticola 4. On Hypoxylon fragiforme and H. howeanum………………………………..C. arxii 4. On other xylariaceous fungi………………………………………………………...5 5. On Annulohypoxylon spp…………………………………………………………...6 5. On other xylariaceous fungi………………………………………………………...8 6. On Annulohypoxylon bovei; ascospore length mean =10 µm……………………………………………………………………C. novazelandica 6. On Annulohypoxylon spp.; ascospore length mean <10 µm………………………..7 7. On Annulohypoxylon cohaerens and A. multiforme; ascospore length mean =7.5 µm; on CMD, colony amber………………..……………………….C. annulohypoxyli 7. On an Annulohypoxylon sp.; ascospore length mean = 6.8 µm; on CMD, colony dark-green……………………………………………………………...C. khandalensis 8. On Kretzschmaria spp……………………………………………………………...9 8. On other xylariaceous fungi……………………………………………………….13 9. On ustulinoid fungi………………………………………………………………..10 9. On kretzschmariod fungi…………………………………………………………..11 114 10. On Kretzschmaria deusta (temperate forest); asexual state simple; ascospore length mean =7.0 µm; conidia length mean =4.7 µm……………………...C. ustulinae 10. On Kretzschmaria cf. deusta (tropical forest); asexual state penicillate; ascospore length mean =7.8 µm; conidia length mean =4.0 µm.…………………….C. viliuscula 11. On Kretzschmaria cetrarioides; ascospore length mean >10 µm…………….………………………………………………………………..C. rickii 11. On other kretzschmariod fungi with ascospore length mean < 10 µm………………………….……………………………………………………...…12 12. On Kretzschmaria clavus and K. cf. pavimentosa; ascospores 7.5 × 3.9 µm (mean); conidia 4.4 × 2.1 µm (mean).………………………………………….C. clavi 12. On Kretzschmaria micropus and Hypoxylon cyclopicum; ascospores 6.9 × 3.7 µm (mean); conidia 3.7 × 1.8 µm (mean).…………………………………...C. micropedis 13. On Stilbohypoxylon quisquiliarum; conidia 5.9 × 2.8 µm (mean); on CMD, 9.5 mm diam (mean)……………………………………………..……….C. stilbohypoxyli 13. On Xylaria scruposa; conidia 4.8 × 2.0 µm (mean); on CMD, 49.5 mm diam (mean)…………………………………………………………………….C. scruposae Cosmospora annulohypoxyli C. Herrera & P. Chaverri, sp. nov. Fig. 3.3. Mycobank MB XXXXXX Holotype: USA, Louisiana, East Baton Rouge Parish, Port Hudson, Port Hudson State Commemorative Area, on Annulohypoxylon cf. cohaerens, on bark of unidentified tree, 19 Aug. 1996, G.J. Samuels, M. Blackwell & M. Camara, BPI 744521, ex- 115 holotype culture G.J.S. 96-186 = CBS XXXXXX. Etymology: In reference to the fungal host, Annulohypoxylon species. Sexual state: Perithecia solitary, sometimes in groups of a few (< five), superficial, subglobose with a blunt apex, collapsing laterally, scarlet, smooth, 207–274 × 181– 232 μm (mean = 232.6 × 198.4; SD 21.2, 15.2; n = 10). Asci cylindrical, eight- spored, uniseriately arranged, 50–68 × 4.0–5.7 μm (mean = 59.0 × 5.0; SD 4.9, 0.6; n = 20). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, minutely verrucose, yellow-brown, 6.5–8.9 × 3.2–4.2 µm (mean = 7.5 × 3.6; SD 0.5, 0.2; n = 60). Culture and asexual state: On PDA colonies 30–39 mm diam (mean = 35.1; SD 2.8; n = 7) after 14 d at 25°C, velvety, slightly floccose, olivaceous, reverse amber. On CMD colonies 40–46 mm diam (mean = 42.7; SD 2.4; n = 7) after 14 d at 25°C, flat, amber, with aerial mycelium sparsely spread, reverse concolorous. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like; conidiophores simple, unbranched, or dichotomously branched. Phialides monophialidic, cylindrical, collarette flared, hyaline, length (26–)36–68 μm (mean = 50.4; SD 8.5; n = 20), width at base 1.8–2.6 μm (mean = 2.2; SD 0.3; n = 20), width at tip 0.8–1.4 μm (mean = 1.1; SD 0.2; n = 20). Conidia ovoid to ellipsoidal, unicellular, smooth, hyaline, 3.0–5.4 × 1.6–2.7 μm (mean = 4.0 × 2.0; SD 0.5, 0.2; n = 60). Habitat: Fungicolous on Annulohypoxylon cohaerens and A. multiforme 116 (Xylariaceae), on unidentified bark. Distribution: United States (Indiana, Louisiana). Additional specimens and isolates examined: USA, Indiana, Yellow Wood State Forest, Brown Co., alt. 200 m., 39°09’ N, 86°06’ W, Jackson Creek Management Trail, on Annulohypoxylon cf. multiforme, on bark of unidentified tree, 30 Sep. 1995, G.J. Samuels, BPI 737773, culture G.J.S. 95-199 = CBS XXXXXX. Notes: This species is only known to grow on Annulohypoxylon cohaerens and A. multiforme. Cosmospora novazelandica and C. khandalensis also occur on Annulohypoxylon species, but have different ascospore lengths. Cosmospora novazelandica has longer ascospores (mean = 10 μm) than C. annulohypoxyli (mean = 7.5 μm), and C. khandalensis have slightly shorter ascospores (mean = 6.8 μm). Cosmospora arxii (W. Gams) Gräfenhan & Schroers, Studies in Mycology 68: 95. 2011. Fig. 3.4. Basionym: Acremonium arxii W. Gams, Cephalosporium-artige Schimmelpilze (Stuttgart) p. 123. 1971. Asexual state: acremonium-like. Habitat: Fungicolous on Hypoxylon fragiforme (Pers.) J. Kickx f. and H. howeanum Peck (Xylariaceae). Distribution: France, Germany, and USA (KY, NY). Sexual state: Perithecia solitary, superficial, globose with a blunt apex, collapsing 117 laterally, scarlet, smooth, 172–226 × 162–209 μm (mean = 206.2 × 186.8; SD 19.4, 18.7; n = 8). Asci cylindrical, eight-spored, uniseriately arranged 55.7–68.0 × 4.5–5.9 μm (mean = 63.1 × 5.2; SD 3.7, 0.4; n = 17). Ascospores ellipsoid, equally two- celled, one-septate, slightly constricted at septum, minutely verrucose, yellow-brown, 6.4–8.6(–9.9) × 3.0–4.2 µm (mean = 7.6 × 3.5; SD 0.7, 0.3; n = 40). Culture and asexual state: On PDA colonies 16–23 mm diam (mean = 20.1; SD 2.1; n = 8) after 14 d at 25°C, velvety, slightly floccose, pale salmon-pink, reverse concolorous. On CMD colonies 23–31 mm diam (mean = 27.1; SD 3.0; n = 8) after 14 d at 25°C, flat, hyaline, with no aerial mycelium, reverse concolorous. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like; conidiophores simple, unbranched, or dichotomously branched. Phialides monophialidic, cylindrical, collarette flared, hyaline, length 42–68 μm (mean = 52.1; SD 7.7; n = 20), width at base 1.6–2.3 μm (mean = 2.0; SD 0.2; n = 20), width at tip 0.9–1.3 μm (mean = 1.2; SD 0.1; n = 20). Conidia ellipsoidal, unicellular, smooth, hyaline, 4.0–8.4 × 1.5–2.4 μm (mean = 5.4 × 2.0; SD 1.0, 0.2; n = 60). Specimens and isolates examined: France, Foret de I’Hermitage, on Hypoxylon howeanum (originally identified as H. fuscum), on Prunus spinosa, 17 Apr. 2008, A. Gminder, BPI 879925, culture A.R. 4521 = CBS XXXXXX. USA, Kentucky, Clermont, Bernhein Arbor & Research Forest, Jackson-Yoe Loop, 37º54.3225’N, 85º38.4079’W, on Hypoxylon fragiforme, on Fagus grandifolia, 27 Jun. 2010, C. Herrera (C.H. 10-05), BPI XXXXXX, culture G.J.S. 10-247 = CBS XXXXXX. Additional descriptions and illustrations: Gams (1971), Gräfenhan et al. (2011). 118 Notes: Identity of these isolates was confirmed by sequencing RNA polymerase II 2nd largest subunit (RPB2; A.R. 4521: JQ014128; G.J.S. 10-247: JQ014118) and comparing the sequences against the sequence of the ex-type culture, CBS 748.69 (HQ897725 retrieved from GenBank). CBS 748.69 was 99% similar to G.J.S. 10-247 and 97% similar to A.R. 4521. Unique to this species is its fungal hosts (Hypoxylon fragiforme or H. howeanum) and its salmon-pink colony on PDA. Cosmospora clavi C. Herrera & P. Chaverri, sp. nov. Fig. 3.5. Mycobank MB XXXXXX Holotype: Costa Rica, Heredia, Puerto Viejo de Sarapiquí, La Selva Biological Station , Sendero Tres Rios and Camino Experimental Norte, N 10°26’7.3”, W 84°00’31.4”, elev. 600 m, on Kretzschmaria clavus, 17 Mar. 2010, P. Chaverri (P.C. 1167), G.J. Samuels, A.Y. Rossman, C. Salgado & C. Herrera, holotype BPI XXXXXX, ex-holotype culture G.J.S. 10-112 = CBS XXXXXX. Etymology: Referring to the fungal host of the type specimen, Kretzschmaria clavus. Sexual state: Perithecia solitary, superficial, globose with blunt apex to obpyriform, collapsing laterally, scarlet, smooth 181– 258 × 135–245µm (mean = 214.3 × 179.1; SD 27.8, 30.5; n = 15). Asci cylindrical, eight-spored, uniseriately arranged, 49–67 × 5–6 µm (mean = 56.3 × 5.2; SD 3.9, 0.4; n = 30). Ascospores ellipsoid, equally two- celled, one-septate, slightly constricted at septum, verrucose, yellow-brown, 6.1–9.1 × 3.2–4.7 µm (mean = 7.5 × 3.9; SD 0.6, 0.3; n = 90). Culture and asexual state: On PDA colonies (32–)37–53 mm diam (mean = 43.7; SD 6.5; n = 15) after 14 d at 25°C, velvety, floccose, greenish-olivaceous or pale-yellow, 119 with a sienna pigment diffusing in medium, reverse umber. On CMD colonies 50–61 mm diam (mean = 54.4; SD 3.4; n = 16) after 14 d at 25°C, flat, dark-green, becoming greenish-yellow at edge, reverse concolorous. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like; conidiophores simple, unbranched, or dichotomously branched, rarely with three phialides. Phialides monophialidic, cylindrical, collarette flared, hyaline, length 21– 68 μm (mean = 47; SD 11.2; n = 49), width at base 1.5–2.7 μm (mean = 2.1; SD 0.3; n = 49), width at tip 0.9–1.4 μm (mean = 1.1; SD 0.1; n = 39). Conidia rarely ovoid, ellipsoidal to reniform, unicellular, smooth, hyaline, 3.5–6.6 × 1.5–2.6 μm (mean = 4.4 × 2.1; SD 0.5, 0.2; n = 150). Habitat: Fungicolous on Kretzschmaria clavus and K. cf. pavimentosa (Xylariaceae), on unidentified bark. Distribution: Possibly pan Neotropical, known from Brazil, Costa Rica, Puerto Rico and Venezuela. Additional specimens and isolates examined: Brazil, State of Amazonas, Pico Rondon, Perimetral Norte Road, km 211, vine forest ca. 3 hr. walk from FUNAI, 01º31'N, 62º48'W, on Kretzschmaria clavus, on bark of unidentified tree, 24–26 Mar. 1984, G.J. Samuels (1076), J. Pipoly & W. Rodrigues, NY, culture G.J.S. 84-290 = CBS XXXXXX. Puerto Rico, Caribbean Nat. Forest, Luquillo Mts., El Verde Research Area, along La Prieta Creek, elev. 350–400 m., on Kretzschmaria cf. pavimentosa, 20 Feb. 1996, G.J. Samuels (8047) & H.-J. Schroers, BPI 744678, culture G.J.S. 96-7 = CBS XXXXXX. Cordillera Central, Chario Azul, off Rte.184, 120 elev. 550 m, on Kretzschmaria cf. pavimentosa, 25 Feb. 1996, G.J. Samuels (8106), H.-J. Schroers & D.J. Lodge, BPI 745249, culture G.J.S. 96-48 = CBS 448.96. Venezuela, Edo. Bolivar, on road between El Dorado and Sta. Elena, ca 118 km S of El Dorado, trail up N-facing slope of Uei-Tepui, from old military camp ‘Ciento Veinticinco’, on Kretzschmaria clavus, on bark of unidentified tree, 5 Aug 1972, K.P. Dumont, R.F. Cain, G.J.Samuels & C. Blanco, Dumont-VE 6918 (NY). Distrito Federal, on the road between El Portachuelo and Chichiriviche, ca 7 km south of Chichiriviche, on Kretzschmaria clavus, on bark of unidentified tree, 30 July 1972, K.P. Dumont, R.F. Cain, G.J. Samuels, B. Manara, Dumont-VE 6706 (NY). Notes: This Cosmospora species grows on Kretzschmaria clavus and on a Kretzschmaria species with an ustulinoid shape. Cosmospora coccinea Rabenh., Fungi europ. exsicc.: no. 459. 1862. Fig. 3.6. = Nectria cosmariospora Ces. & De Not., Comm. Soc. Crittog. Ital. 1(4): 195. 1863. ≡ Dialonectria cosmariospora (Ces. & De Not.) Cooke, Grevillea 12(no. 64): 110. 1884. = Verticillium olivaceum W. Gams, Cephalosporium-artige Schimmelpilze (Stuttgart) p. 123. 1971. Asexual state: Acremonium-like. Habitat: Fungicolous on Inonotus spp. on Fagus and Alnus. Distribution: Europe. Specimens and isolates examined: Germany, Bavaria, Burghausen-Unterhadermark, on dead crusts of Inonotus nodulosus, on fallen branch of Fagus sylvatica, 02 Oct. 121 1993, R. Boesmiller, BPI 802729, culture A.R. 2741 = CBS 114050, also A.R. 2743. Additional descriptions and illustrations: Gams (1971; as V. olivaceum), Samuels et al. (1991; as N. cosmariospora), Rossman et. al (1999), Gräfenhan et al. (2011). Notes: Cosmospora coccinea was lectotypified by Rossman et al. (1999), and reported to grow on pores of Inonotus spp. Its ascospores are tuberculate. Cosmospora cymosa is also known to occur on Inonotus spp., and is presumed to be sister species with C. coccinea (Gräfenhan et al. 2011). However, the sexual state of C. cymosa is not known. It is possible that C. cymosa is a morphological variation of C. coccinea. Cosmospora fomiticola C. Herrera & P. Chaverri, sp. nov. Fig. 3.7. Mycobank MB XXXXXX Holotype: New Zealand, Westland, Mt Aspiring National Park, Haast Pass, on Fomes fomentarius, 12 Apr. 1983, G.J. Samuels, R.E. Beever & R.H. Petersen, PDD 46398, ex-holotype culture G.J.S. 83-194 = CBS XXXXXX. Etymology: From fomitis (latin) = tinder and -cola (latin) = growing on; referring to the host of this species, the tinder fungus, a common name for Fomes fomentarius. Sexual state: Perithecia solitary, rarely in groups of a few, superficial, globose to ovoid with an acute apex, collapsing laterally, orange, smooth, 212–304 × 176–272 μm (mean = 240.4 × 201.3; SD 33.2, 35.4; n = 6). Asci cylindrical, eight-spored, uniseriately arranged, 59–85 × 5–8 μm (mean = 73.9 × 6.0; SD 7.7, 1.1; n = 10). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, 122 minutely verrucose, yellow-brown, 6.9–9.7 × 2.8–4.1 µm (mean = 7.9 × 3.6; SD 0.6, 0.3; n = 50). Culture and asexual state: On PDA colonies 24–27.5 mm diam (mean = 25.8; n = 2) after 14 d at 25°C, velvety, slightly floccose, white, with a sienna pigment diffusing in medium, reverse umber. On CMD colonies 12–17 mm diam (mean = 14.5; n = 2) after 14 d at 25°C, flat, olivaceous, with white aerial mycelium towards center, with an olivaceous pigment diffusing into medium, reverse concolorous. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like; conidiophores branched with 3–4 phialides. Phialides monophialidic, cylindrical, collarette flared, hyaline, length 19–42 μm (mean = 35; SD 6.3; n = 10), width at base 1.3–2.8 μm (mean = 1.9; SD 0.4; n = 10), width at tip 0.9–1.3 μm (mean = 1.1; SD 0.1; n = 10). Conidia ovoid to ellipsoidal, unicellular, smooth, hyaline, 2.8–4.8 × 1.6– 2.4 μm (mean = 3.7 × 2.0; SD 0.5, 0.2; n = 30). Habitat: Fungicolous on Fomes fomentarius (Polyporaceae, Basidiomycota). Distribution: New Zealand. Additional specimens examined: New Zealand, North Island, Gisborne District, Track to Lake Ruapani (Site 74), on Fomes sp., 31 May 1983, A.Y. Rossman, P. Matsushima, P. Johnston & G.J. Samuels, BPI 745118. Notes: This monotypic species is the only species known to occur on Fomes fomentarius. The shape of the perithecia resembles the shape a lemon with a minute acute apex. Also, the colonies produced by C. fomiticola on PDA and CMD are very 123 distinct. On PDA, the colony is floccose and white, and on CMD, the colony is olivaceous with sporulation occurring towards the center. Cosmospora khandalensis (Thirum. & Sukapure) Gräfenhan & Seifert, Stud. Mycol. 68: 96. 2011. Fig. 3.8. Basionym: Cephalosporium khandalense Thirum. & Sukapure, Mycologia 58: 359. 1966. Habitat: Fungicolous on Annulohypoxyon sp. (Xylariaceae); asexual state on decaying stem and stump of Bambusa. Distribution: Argentina, Brazil, India, and Japan. Sexual state: Perithecia solitary, superficial, globose with a blunt apex, collapsing laterally, scarlet, smooth, 163–228 × 143–208 µm (mean = 188.9 × 164.9; SD 20.3, 19.5; n = 11). Asci cylindrical, eight-spored, uniseriately arranged, 44–61 × 4–7 µm (mean = 52.7 × 5.2; SD 4.5, 0.7; n = 15). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, verrucose, yellow-brown, 5.5–8.0 × 2.6– 4.2 µm (mean = 6.8 × 3.3; SD 0.5, 0.3; n = 80). Culture and asexual state: On PDA colonies (23–)31.5–43 mm diam (mean = 36.5; SD 5.8; n = 12) after 14 d at 25°C, velvety, floccose, citrine, with a sienna pigment diffusing in medium, reverse sienna. On CMD colonies 40–51 mm diam (mean = 45.6; SD 3.4; n = 11) after 14 d at 25°C, flat, dark-green, becoming pale-luteous at edge, with white aerial mycelium sparsely spread, reverse concolorous. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like; 124 conidiophores simple, unbranched, or dichotomously branched. Phialides monophialidic, cylindrical, collarette flared, hyaline, length 34–64 μm (mean = 50.5; SD 9.2; n = 30), width at base 1.5–2.6 μm (mean = 2.0; SD 0.2; n = 30), width at tip 0.8–1.3 μm (mean = 1.0; SD 0.1; n = 30). Conidia ovoid to ellipsoidal, unicellular, smooth, hyaline, 3.0–5.2 × 1.5–2.4 μm (mean = 3.8 × 2.0; SD 0.4, 0.2; n = 90). Holotype of Cosmospora khandalensis: India, Maharashtra, Khandala, on decaying stem and stump of Bambusa, Aug. 1964., M. J. Thirumalachar, holotype HACC 148. (not seen), isotypes CBS H-15076 (not seen) = K(M) 169348, ex-type cultures ATCC 16091 (not seen) = CBS 356.65 (not seen) = IMI 112790 = MUCL 7974 (not seen). Specimens and isolates examined: Argentina, Tucuman Province, Tucuman, San Javier trail just past Yerba Buena, on Annulohypoxylon sp., 21 Apr. 2011, C. Salgado, BPI XXXXXX, Culture A.R. 4798 = CBS XXXXX; Tucuman Province, Tucuman, San Javier trail just past Yerba Buena, on Annulohypoxylon sp., 21 Apr. 2011, A. Romero, BPI XXXXXX, Culture A.R. 4799 = CBS XXXXX. Brazil, Estado do Pará, Belterra, Maguari, Floresta Nacional do Tapajós, S2°46'59.6'', W55°01'38.9'', elev. 28 m., on Annulohypoxylon sp., on decaying tree, 7 May 2011, O. Liparini Pereira & P. Chaverri (P.C. 1306), BPI XXXXXX, culture CBS XXXXXX. Japan, Chinen-son, Shimajiri-gun, Okinawa Pref., Okinawa Islands, on unidentified pyrenomycete, 19 Jan. 2003, Y. Hirooka (h81), TFM FPH-7823 (not seen), culture MAFF 251500. Additioinal descriptions and illustrations: Sukapure & Thirumalachar (1966), Hirooka et al. (2008; as Cosmospora triqua), Gräfenhan et al. (2011). 125 Notes: The sexual state of Cosmospora khandalensis is reported for the first time. Isolates derived from single ascospores (e.g. A.R. 4798, A.R. 4799, and P.C. 1306) formed a well-supported clade with the ex-type culture of C. khandalensis (IMI 112790). This result resolved the identity of these isolates, and suggested a wide distribution of this species. Cosmospora micropedis C. Herrera & P. Chaverri, sp. nov. Fig. 3.9. Mycobank MB XXXXXX Holotype: French Guiana, Saul, ca. 20 km SW of Saul (03º60'N, 53º20'W) toward Mt. Galbao (03º50'N, 53º20'W), elev. 650 m, on Kretzschmaria micropus, 22 Jan. 1986, G.J. Samuels (3182) & J.R. Boise, NY, ex-holotype culture G.J.S. 86-108 = CBS XXXXXX. Etymology: Referring to the fungal host of the type specimen, Kretzschmaria micropus. Sexual state: Perithecia solitary, superficial, subglobose with a blunt apex, collapsing laterally, scarlet, smooth, 186–227 × 167–233 μm (mean = 208.3 × 196.8; SD 14.7, 23.1; n = 6). Asci cylindrical, eight-spored, uniseriately arranged, 50–61 × 4–7 μm (mean = 55.5 × 5.3; SD 3.5, 0.6; n = 9). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, verrucose, yellow-brown, 5.2–8.7 × 2.8– 4.4 µm (mean = 6.9 × 3.7; SD 0.6, 0.3; n = 84). Culture and asexual state: On PDA colonies 41–54 mm diam (mean = 47.6; SD 4.3; n = 12) after 14 d at 25°C, velvety, slightly floccose, olivaceous, with an amber 126 pigment diffusing into medium, reverse amber. On CMD colonies 48–63 mm diam (mean = 57.6; SD 5.2; n = 12) after 14 d at 25°C, flat, dark-green, becoming pale- luteous towards edge, with white aerial mycelium sparsely spread, reverse concolorous. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like; conidiophores simple, unbranched. Phialides monophialidic, cylindrical, collarette flared, hyaline, length 22–53 μm (mean = 41.2; SD 7.9; n = 29), width at base 1.6–2.4 μm (mean = 2.1; SD 0.2; n = 29), width at tip 0.9–1.4 μm (mean = 1.1; SD 0.1; n = 29). Conidia ovoid to ellipsoidal, unicellular, smooth, hyaline, 2.9–5.8 × 1.2–2.4 μm (mean = 3.7 × 1.8; SD 0.5, 0.2; n = 90). Habitat: Fungicolous on Kretzschmaria micropus and Hypoxylon cyclopicum (Xylariaceae). Distribution: Possibly pan Neotropical, known from Brazil, Costa Rica, French Guiana and Guadeloupe. Additional specimens and isolates examined: Brazil, Estado do Pará, Município de Rurópolis, Comunidade Novo Horizonte, S4°06'49.7”, W55°00'24.9'', elev. 69 m., on Hypoxylon cyclopicum, on rotten wood, 02 May 2011, G. Barata, O. Liparini Pereira, D.N. Skaltsas & P. Chaverri (P.C. 1285), BPI XXXXXX, culture CBS XXXXXX; 0–3 km S. of Central Portion of Serra Araca & 0–8 km E. of Rio Javari, elev. 60 m, 00º49’N, 63º19’W, on Hypoxylon cyclopicum, 1–5 & 12–13 Mar. 1984, G.J. Samuels (661), NY; Serra Araca, elev. 60 m, on Hypoxylon cyclopicum, 10–13 Mar. 1984, G.J. Samuels (805), NY. Costa Rica, Heredia, Puerto Viejo de Sarapiquí, La Selva Biological Station , Sendero Tres Rios and Camino Experimental Norte, N 127 10°26’7.3”, W 84°00’31.4”, elev. 800 m, on Hypoxylon cyclopicum, 17 Mar. 2010, P. Chaverri (P.C. 1164), G.J. Samuels, A.Y. Rossman, C. Salgado & C. Herrera, BPI XXXXXX, culture G.J.S. 10-113 = CBS XXXXXX; Puerto Viejo de Sarapiquí, La Selva Biological Station , Sendero Tres Rios and Camino Experimental Norte, N 10°26’7.3”, W 84°00’31.4”, elev. 800 m, on Hypoxylon cyclopicum, 17 Mar. 2010, P. Chaverri (P.C. 1158), G.J. Samuels, A.Y. Rossman, C. Salgado & C. Herrera, BPI XXXXXX, culture G.J.S. 10-121 = CBS XXXXXX. Guadeloupe, Trace Delgrès, Basse Terre, on Kretzschmaria micropus, Jan. 1997, J. Vivant, BPI 744480. Notes: This Cosmospora species grows on Kretzschmaria micropus and Hypoxylon cyclopicum. Experts in the Xylariaceae consider these fungal hosts to be morphological variants of the same species (Rogers & Ju 1998). The conidia of C. micropedis are generally smaller compared to those of other Cosmospora species growing on Kretzschmaria species. Cosmospora novazelandica C. Herrera & P. Chaverri, sp. nov. Fig. 3.10. Mycobank MB XXXXXX Holotype: New Zealand, Buller, 21 Km S. of Murchison, on Annulohypoxylon bovei, 17 Apr. 1983, G.J. Samuels, P.R. Johnson, R.E. Beever & R.H. Petersen, PDD 46401, ex-holotype culture G.J.S. 83-197 = CBS XXXXXX. Etymology: In reference to the geographical origin of this species, New Zealand. Sexual state: Perithecia solitary, superficial, obpyriform, collapsing laterally, scarlet, with cells protruding around the apex (appearing roughened), 292– 330 × 211– 330 128 μm (mean = 312.1 × 289.9; SD 19.1, 54.1; n = 4). Asci cylindrical to clavate, eight- spored, uniseriately arranged, 70–90 × (5–)6–8 μm (mean = 80.5 × 7.0; SD 5.8, 0.8; n = 18). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, minutely verrucose, yellow-brown, 8.0–11.39 × 4.0–5.8 µm (mean = 10.0 × 4.8; SD 0.6, 0.4; n = 60). Culture and asexual state: On PDA colonies 16–20 mm diam (mean = 18.3; SD 1.7; n = 4) after 14 d at 25°C, velvety, slightly floccose, citrine to olivaceous, with a citrine pigment slightly diffusing into medium, reverse olivaceous. On CMD colonies 18–19 mm diam (mean = 18.5; SD 0.6; n = 4) after 14 d at 25°C, flat, citrine, with an citrine pigment diffusing in medium, reverse greyish yellow-green to citrine green. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like; conidiophores simple, unbranched. Phialides monophialidic, cylindrical, collarette flared, hyaline, length 38–51 μm (mean = 42.7; SD 4.2; n = 10), width at base 1.9–3.0 μm (mean = 2.2; SD 0.4; n = 10), width at tip 1.0–1.2 μm (mean = 1.1; SD 0.1; n = 10). Conidia ovoid to ellipsoidal, unicellular, smooth, hyaline, 2.6–5.3 × 1.4–2.4 μm (mean = 3.8 × 1.8; SD 0.5, 0.2; n = 50). Habitat: Fungicolous on Annulohypoxylon bovei (Xylariaceae) on unidentified bark. Distribution: New Zealand. Additional specimens examined: New Zealand, South Island, Boyle River Lodge, 172º23’E, 42º31’S, on Annulohypoxylon bovei, on unidentified bark, 13 Sep. 1981, L. Brako (No. 3991A), NY. 129 Notes: Cosmospora novazelandica is host specific to Annulohypoxylon bovei. Also, this species is unusual in respect to the ornamentation at the apex in which cells are protruding, thus giving the apex the appearance of being roughened. Perithecial ornamentation is generally absent in Cosmospora. Additionally, this species have the largest ascospores among species of the Cosmospora viliuscula species complex. Cosmospora rickii (Rehm) Rossman & Samuels, Stud. Mycol. 42: 124. 1999. Basionym: Nectria rickii Rehm, Hedwigia 44: 2. 1905. = Nectria episphaeria var. kretzschmariae Henn., Bot. Jb. 14(4): 364. 1891. ≡ Nectria kretzschmariae (Henn.) Weese, Akad. Wiss. Wien Sitzungsber., Math.-Naturwiss. Kl., Abt. 1, 125: 506. 1916. = Nectria stigme Rehm, Hedwigia 44: 2. 1905. Anamorph: not known. Habitat: Fungicolous on Kretzschmaria cetrarioides. Distribution: Brazil and Republic of the Congo. Holotype of Nectria rickii: Brazil, Sao Leopoldo, on disintegrated stroma of Kretzschmaria cetrarioides (as K. lichenoides), 1903, S.J. Rick, F10125. (holotype of N. rickii). Additional specimens examined: Brazil, S. Sao Leopoldo, on Kretzschmaria cetrarioides (as K. lichenoides), 1903, S.J. Rick, F10189 ( S - holotype of N. stigme). Republic of the Congo (as Loango), on Kretzschmaria cetrarioides (as K. pechuelii), 130 1876, Pechuel-Lösche, B (holotype of Nectria episphaeria var. kretzschmariae not seen; destroyed by the war-dependent fire of 1943). Notes: Weese (1916) was the last researcher to have examined all three holotype specimens. He concluded that N. episphaeria var. kretzschmariae, N. rickii, and N. stigme represented the same species. He raised N. episphaeria var. kretzschmariae to species rank in 1916. The principle of priority applies only to names within the same rank, thus N. rickii or N. stigme, published in the same article in 1905 and thus having equal priority, provide older names at the species rank. Rossman et al. (1999) already selected N. rickii to represent this species and transferred the name to Cosmospora. The holotype of N. rickii consists of the mature form (ascospores ellipsoid, minutely verrucose, yellow-brown, 14.1–18.3 × 6.1–8.9 μm, mean = 16.0 × 7.5 μm) while the holotype of N. stigme represents an immature form of the same species (ascospores ellipsoid, minutely verrucose, yellow-brown, 7.1–9.6(–13.2) × 3.8–4.8(–7.4) μm, mean = 8.0 × 4.1 μm). The holotype of Nectria episphaeria var. kretzschmariae has been destroyed. All three type specimens are reported to occur on the same host, Kretzschmaria cetrarioides, which supports the idea that these specimens each represent the same single species. A living culture from a specimen is needed to determine the phylogenetic placement of this species. Cosmospora scruposae C. Herrera & P. Chaverri, sp. nov. Fig. 3.11. Mycobank MB XXXXXX Holotype: French Guiana, Montagne de Kaw, Route de l'est, km 50, on Xylaria scruposa, 25 Mar. 1986, G.J. Samuels & C. Feuillet, GJS 4487 (NY), ex-holotype 131 culture G.J.S. 86-331 = CBS XXXXXX. Etymology: In reference to its fungal host, Xylaria scruposa. Sexual state: Perithecia solitary, superficial, subglobose with an acute apex, collapsing laterally, orange, or scarlet, smooth, 177–239 × 159–224 µm (mean = 212.6 × 189.2; SD 17.0, 18.0; n = 18). Asci cylindrical, eight-spored, uniseriately arranged, 45–71 × 4–6 µm (mean = 56.4 × 4.8; SD 6.4, 0.5; n = 20). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, verrucose, yellow-brown, 6.0–9.0 × 2.9–4.8 µm (mean = 7.3 × 3.7; SD 0.6, 0.3; n = 130). Culture and asexual state: On PDA colonies 28–41.5 mm diam (mean = 33.3; SD 3.8; n = 16) after 14 d at 25°C, velvety, floccose, olivaceous buff, with a sienna to umber pigment diffusing into medium, sometimes with white aerial mycelium densely spread, reverse sienna to umber. On CMD colonies 33–55 mm diam (mean = 49.5; SD 6.6; n = 17) after 14 d at 25°C, flat, white, sometimes with an amber pigment, reverse concolorous. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like; conidiophores simple, unbranched, or dichotomously branched, rarely terminating in three phialides. Phialides monophialidic, cylindrical, collarette flared, hyaline, length 36–66 μm (mean = 59.6; SD 9.0; n = 38), width at base 1.4–2.8 μm (mean = 2.1; SD 0.4; n = 38), width at tip 0.8–1.3 μm (mean = 1.1; SD 0.2; n = 38). Conidia ovoid to ellipsoidal, unicellular, smooth, hyaline, 3.5–6.7 × 1.4–2.8 μm (mean = 4.8 × 2.0; SD 0.8, 0.3; n = 90). Habitat: Fungicolous on Xylaria cf. scruposa (Xylariaceae). 132 Distribution: Possibly pan Neotopical, known from French Guiana, Guyana, Puerto Rico and Venezuela. Additional specimens and isolates examined: French Guiana, Piste de Saint-Elie, km 16 on road between Sinnamary and St. Elie, ECEREX, ORSTOM research area, 05°20' N, 53°W, on stromata of Xylaria cf. scruposa Feb–Mar 1986, G.J. Samuels, G.J.S. 3899 (NY); Paul Isnard Area, ca. 150 km S of St. Laurent du Moroni, Citron, Mt. Lucifer, 04º70'N, 53º90'W, on stroma of Xylaria cf. scruposa, Mar. 1986, G.J. Samuels & P. Searwar, GJS 4129 (NY), culture G.J.S. 86-278 = CBS XXXXXX; Paul Isnard Area, ca. 150 km S of St. Laurent du Moroni, Citron, small ridge at SW edge of the village, 04º70'N, 53º90'W, on Xylaria cf. scruposa, 16–17 Mar. 1986, G.J. Samuels & P. Searwar, G.J.S. 4326 (NY), culture G.J.S. 86-315 = CBS XXXXXX; Montagne de Kaw, Route de l'est, km 27, 04º60'N, 52º40'W, on Xylaria cf. scruposa, 21 Mar. 1986, G.J. Samuels & C. Feuillet, G.J.S. 4393 (NY), culture G.J.S. 86-320 = CBS XXXXXX. Guyana, Upper Demerara-Berbice Region, Upper Demerara subregion, Mabura Hill, along Waraputa Creek, elev. 0–50 m, 05º14'N, 58º47'W, on stroma of Xylaria cf. scruposa, 28 Mar. 1987, G.J. Samuels & L. Wong Kam, G.J.S. 5303 (NY); Demerara-Mahaica Region, Mahaica subregion, Linden Highway between Georgetown & Yarowcabra, Yarowcabra Forestry Station, elev. 50–100 m, 06º30'N, 58º15'W, on stromata of Xylaria cf. scruposa, 26–27 Apr. 1987, G.J. Samuels & J. Pipoly, G.J.S. 5563 (NY); Cuyuni-Mazarun Region, Mazarun Subregion, along Koatze River, ca. 2 km E of Pong River, ca. 5 hr walk of Chinoweing Village, elev. 600–650 m, 05º28'N, 60º04'W, on stroma of Xylaria cf. scruposa, Feb–Mar 1990, G.J. Samuels, J. Pipoly, G. Gharbarran, J. Chin & R. 133 Edwards, G.J.S. 5002 (NY). Puerto Rico, Caribbean National Forest, Luquillo Mts., La Prieta Creek, elev. 350–400 m, on stroma of Xylaria cf. scruposa, 20 Feb. 1996, G.J. Samuels (8039) & H.-J. Schroers, BPI 744671, culture G.J.S. 96-6 = CBS 455.96. Venezuela, Edo. Aragua, Henry Pittier National Park, Rancho Grande Biological Station, Toma Trail to water source, elev. 1200–1300 m, 10°21’N, 67°41’W, on stroma of Xylaria cf. scruposa, 03 Dec. 1990, G.J. Samuels (7853), B. Hein & S.M. Huhndorf, BPI 745150, culture G.J.S. 90-224 = CBS XXXXXX; Edo. Aragua, Henry Pittier National Park, Rancho Grande Biological Station, Trail to Guacamayo, elev. 1250–1400 m, 10°21’N, 67°41’W, on stroma of Xylaria cf. scruposa, 04 Dec. 1990, G.J. Samuels (7891), B. Hein & S.M. Huhndorf, BPI 744778, culture G.J.S. 90-217= CBS XXXXXX. Notes: Cosmospora species growing on Xylaria tend to have perithecia with an acute apex. Although fresh cultures were apparently dark-green as indicated by the dried culture specimens in herbarium packets, regrown isolates of C. scruposae produced a white colony on CMD, thus the white colony cannot be considered to an unique character of this species. It suggests that the stored isolates have degraded in storage such that they no longer produce the pigmentation. Cosmospora stilbohypoxyli C. Herrera & P. Chaverri, sp. nov. Fig. 3.12. Mycobank MB XXXXXX Holotype: Argentina, Tucuman Province, San Javier, on Stilbohypoxylon quisquiliarum, on decorticated wood, 20 Apr. 2011, C. Salgado, BPI XXXXXX, ex- holotype culture A.R. 4783 = CBS XXXXXX. 134 Etymology: In reference to its fungal host, Stilbohypoxylon quisquiliarum. Sexual state: Perithecia solitary, superficial, subglobose with blunt apex, sometimes appearing acute, collapsing laterally, scarlet, smooth, 152–188 × 145–185 μm (mean = 169 × 158; SD 11.2, 12.8; n = 8). Asci narrowly clavate, eight-spored, uniseriately arranged, 45–61 × 4– 6 μm (mean = 51.6 × 4.9; SD 4.5, 0.5; n = 10). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, minutely verrucose, yellow-brown, 6.5–7.9(–10.1) × 3.0–4.0 µm (mean = 6.9 × 3.5; SD 0.4, 0.2; n = 40). Culture and asexual state: On PDA colonies 5–6 mm diam (mean = 5.3; SD 0.6; n = 4) after 14 d at 25°C, velvety, slightly floccose, herbage-green, with a slight sulphur- yellow pigment diffusing into medium, reverse concolorous. On CMD colonies 8– 11.5 mm diam (mean = 9.5; SD 1.5; n = 4) after 14 d at 25°C, flat, greenish-yellow at center, becoming white towards edge, reverse concolorous. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like; conidiophores simple, unbranched. Phialides monophialidic, cylindrical, collarette flared, hyaline, length 34–44 μm (mean = 40.1; SD 3.2; n = 10), width at base 2.0–3.0 μm (mean = 2.3; SD 0.3; n = 10), width at tip 1.1–1.6 μm (mean = 1.3; SD 0.2; n = 10). Conidia ovoid to ellipsoidal, unicellular, smooth, hyaline, 4.6–7.5 × 2.2–3.7 μm (mean = 5.9 × 2.8; SD 0.6, 0.3; n = 30). Habitat: Fungicolous on Stilbohypoxylon quisquiliarum (Xylariaceae) on unidentified bark. 135 Distribution: Argentina and Venezuela. Additional specimens and isolates examined: Venezuela, Distrito Federal, South of Los Caracas, along Rio Los Caracas, on Stilbohypoxylon quisquiliarum, on bark of unidentified tree, 22 July 72, K.P. Dumont (Dumont-VE 5665), R.F. Cain, G.J. Samuels & B. Manara, NY. Notes: Cosmospora stilbohypoxyli is only known to grow on Stilbohypoxylon quisquiliarum. This species is extremely slow growing on PDA and CMD compared to other species. Cosmospora ustulinae (Teng) C. Herrera & P. Chaverri, comb. nov. Fig. 3.13. Mycobank MB XXXXXX Basionym: Nectria ustulinae Teng, Sinensia, Shanghai 4: 275 (1934). Habitat: Fungicolous on Kretzschmaria deusta (Xylariaceae). Distribution: China, Japan, Portugal, USA (AL, FL, MI, NY, TN). Holotype of Nectria ustulinae: China, Kiangsu, Pao-hua Shan, on stroma of Kretzschmaria deusta (as Hypoxylon ustulatum), S.C. Teng (2027), holotype BPI 553261. Sexual state: Perithecia solitary, in clusters (<10), rarely densely aggregated, superficial, subglobose with a blunt apex, collapsing laterally, scarlet, smooth, 185– 225 × 146–195 μm (mean = 205.6 × 169.9; SD 15.9, 17.7; n = 6). Asci narrowly clavate, eight-spored, uniseriately arranged, 41–61 × 4–5 μm (mean = 50.3 × 4.8; SD 136 6.5, 0.4; n = 12). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, verrucose, yellow-brown, 5.8–8.3 × 2.7–4.8 µm (mean = 7.0 × 3.6; SD 0.6, 0.4; n = 62). Culture and asexual state: On PDA colonies 35–51 mm diam (mean = 42.9; SD 5.3; n = 14) after 14 d at 25°C, velvety, slightly floccose, olivaceous to citrine, sometimes becoming greenish-yellow at edge, with a slight greenish-yellow or sienna pigment diffusing in medium, sometimes black droplets forming at center of colony, reverse concolorous. On CMD colonies 44–52 mm diam (mean = 48.4; SD 2.8; n = 14) after 14 d at 25°C, flat, dark-green, or greenish-yellow at the center, becoming hyaline towards edge of colony, reverse concolorous. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like; conidiophores simple, unbranched, or dichotomously branched. Phialides monophialidic, cylindrical, collarette flared, hyaline, length 35–57 μm (mean = 45.9; SD 5.8; n = 28), width at base 1.7–3.1 μm (mean = 2.3; SD 0.3; n = 28), width at tip 0.9–1.3 μm (mean = 1.1; SD 0.1; n = 28). Conidia ovoid to ellipsoidal, unicellular, smooth, hyaline, 3.3–6.5 × 1.6–3.1 μm (mean = 4.7 × 2.2; SD 0.7, 0.3; n = 120). Epitype designated herein: USA, Tennessee, Blount Co., Great Smoky Mts. National Park, 25 mi W Gatlinburg, Cades Cove, Gum Swamp, elev. 1800 ft, 35°35’13.7”N, 83°50’19.2”W, on stroma of Kretzschmaria deusta, 06 Sep. 2005, S. Huhndorf, BPI 871089, ex-epitype culture A.R. 4215 = CBS XXXXXX. Additional specimens and isolates examined: Japan, Kumamoto Prefecture, Kikushi City, Kikuchi valley, on remnants of old stroma of Kretzschmaria cf. deusta, 31 May 137 2004, Y. Hirooka (h278), BPI XXXXXX, culture MAFF 241532. USA, Alabama, Talledega Nat. Forest, Cheaha State Park, Clay Co., Creek Trail, on stroma of Kretzschmaria deusta, 23 Sep. 1992, G.J. Samuels, C.T. Rogerson, S.M. Huhndorf, BPI 802840, culture G.J.S. 92-95 = CBS XXXXXX. Portugal, isolated from cut treated stump of Clethra arborea, 2000, culture IMI 389101. Additional description: Teng (1934; no illustrations). Notes: This species grows on Kretzschmaria deusta in temperate forests. In contrast to the asexual state of C. viliuscula, which is highly branched, i.e. penicillate, the asexual state of C. ustulinae is simple, unbranched or branching into two phialides. Cosmospora viliuscula (Samuels) Rossman & Samuels, Stud. Mycol. 42: 126 (1999). Fig. 3.14. Basionym: Nectria viliuscula Samuels, Mem. New York Bot. Gard. 59: 44 (1990). Habitat: Fungicolous on Kretzschmaria cf. deusta (Xylariaceae). Distribution: Australia, Costa Rica, Indonesia and New Zealand. Holotype of Nectria viliuscula: Indonesia, North Sulawsi, Eastern Dumoga-Bone National Park, Gn. Muajat, Danau Alia, elev. 1400 m, 00°45’N, 124°25’E, on Kretzschmaria cf. deusta (as Hypoxylon cf. deustum), 26 Oct. 1985, G.J. Samuels 2385, Holotype BO (Not Seen), Isotype NY 01013285. Sexual state: Perithecia solitary, or in clusters (<10), superficial, subglobose with a blunt apex, collapsing laterally, scarlet, smooth, 198–255 × 159–236 μm (mean = 138 224.9 × 236; SD 20.7, 29.4; n = 10). Asci cylindrical, eight-spored, uniseriately arranged, 46–63 × 4–6(–7) μm (mean = 54.2 × 5.1; SD 4.9, 0.7; n = 12). Ascospores ellipsoid, equally two-celled, one-septate, slightly constricted at septum, verrucose, yellow-brown, 6.2–9.8 × 2.2–5.1 µm (mean = 7.8 × 3.8; SD 0.8, 0.4; n = 54). Culture and asexual state: On PDA colonies 39–56 mm diam (mean = 46.4; SD 6.7; n = 11) after 14 d at 25°C, velvety, slightly floccose, amber, or olivaceous, sometimes sienna pigment diffusing in medium, sometimes with white aerial mycelium sparsely or densely spread, reverse sienna. On CMD colonies 27–55 mm diam (mean = 45.8; SD 10.2; n = 12) after 14 d at 25°C, flat, white, reverse concolorous. Sporulation on SNA abundant, arising directly from agar surface. Asexual state acremonium-like to penicillate; conidiophores simple, unbranched, or dichotomously branched; forming primary, secondary, and rarely tertiary branches, each terminal branch producing 2–4 phialides. Phialides monophialidic, cylindrical, collarette flared, hyaline, length 28– 57 μm (mean = 43.6; SD 6.3; n = 30), width at base 1.4–2.5 μm (mean = 1.8; SD 0.3; n = 30), width at tip 0.8–1.2 μm (mean = 1.0; SD 0.1; n = 30). Conidia ovoid to ellipsoidal, unicellular, smooth, hyaline, 2.2–5.7 × 1.3–2.9 μm (mean = 4.0 × 2.1; SD 0.6, 0.3; n = 90). Epitype designated herein: Australia, Queensland, Lake Barrine, elev. 750 m, 17.0°14.0’43.0”S, 145°38.0’, 21.0”E, on stroma of Kretzschmaria cf. deusta (as Ustulina deusta), 20 Feb. 2009, P. Chaverri (P.C. 858) & A.Y. Rossman, BPI 878994, ex-epitype culture G.J.S. 09-411 = CBS XXXXXX. Additional specimens and isolates examined: Costa Rica, Heredia, Puerto Viejo de 139 Sarapiquí, La Selva Biological Station , Sendero Tres Rios and Camino Experimental Norte, N 10°26’7.3”, W 84°00’31.4”, elev. 100–300 m, on stroma of Kretzschmaria cf. deusta, 17 Mar. 2010, P. Chaverri (P.C. 1150), G.J. Samuels, A.Y. Rossman, C. Salgado & C. Herrera, BPI XXXXXX, culture G.J.S. 10-114 = CBS XXXXXX. New Zealand, Auckland Prov. Bay of Islands Co., Puketi Forest, on stroma of Kretzschmaria cf. deusta (as Ustulina sp.), 02 Mar. 1973, J.M. Dingley, PDD 30869, culture G.J.S. 73-2 = CBS XXXXXX. Additional descriptions and illustrations: Samuels, Doi, & Rogerson (1990), Samuels et al. (1991), Rossman et al. (1999). Notes: This species grows on Kretzschmaria cf. deusta in tropical forests. In addition this species is unique in producing a penicillate asexual state. Cosmospora viridescens (C. Booth) Gräfenhan & Seifert, Stud. Mycol. 68: 96. 2011. Basionym: Nectria viridescens C. Booth, Mycol. Pap. 73: 89. 1959. Habitat: On bone and fungicolous on Ruzenia spermoides (Lasiosphaeriaceae). Distribution: Czech Republic, Denmark and United Kingdom. Holotype of Nectria viridescens: UK, England, Yorkshire, Sawley Woods, on black pyrenomycete on branches of Salix, 22 Apr. 1954, C. Booth, holotype IMI 56376 (not seen), isotype BPI 553304 = DAOM 83074 (not seen); Wales, Llanrwst, Gwydyr Forest, on Ruzenia spermoides, on Betula, May 1958, C. Booth, paratype K(M) 169349, ex-paratype culture IMI 73377a. Additional isolates examined: Czech Republic, Sumava Mts. National Park, vicinity 140 of Breznik, Mt. Studna hora, on bark of dead standing trunk, 11 Aug. 1999, M. Reblova, culture CBS 102430; Sumava Mts. National Park, vicinity of Breznik, Pytlacky, on dead tree, 14 Aug. 1999, M. Reblova, culture CBS 102433. Denmark, on bone, 24 Jul. 1998, T. Laessoe, culture A.R. 2783 = CBS XXXXXX. Descriptions and illustrations: Booth (1959), Gräfenhan et al. (2011). Notes: The isolate IMI 73377a represents the only living culture derived from specimens cited by Booth (1959) in his original description of Nectria viridescens. By definition, K(M) 169349 represents a paratype of C. viridescens, and phylogenetic placement of this species in the strict sense was possible with this isolate. It clustered with other isolates previously considered to be C. viridescens (see Gräfenhan et al. 2011), but the clade may consist of a species complex. The ascospores are ellipsoid, slightly constricted at septum, minutely verrucose, yellow-brown, 7.6–12.3 × 3.7–4.7 μm (mean = 9.4 × 4.3 μm). Additional accepted species not treated in this paper Cosmospora berkeleyana (P. Karst.) Gräfenhan, Seifert & Schroers, Stud. Mycol. 68: 95. 2011. Basionym: Verticillium berkeleyanum P. Karst., Meddeland. Soc. Fauna Fl. Fenn. 18: 64. 1891. ≡ Acremonium berkeleyanum (P. Karst.) W. Gams, Netherlands J. Pl. Pathol. 88: 76. 1982. Asexual state: Acremonium-like. 141 Habitat: On Inonotus radiatus and Stereum hirsutum. Distribution: Canada, Finland, Germany, and Netherlands. Descriptions and illustrations: Karsten (1891), Gams (1971), and Gams & Zaayen (1982). Notes: The sexual state of this species is unknown. The identity of the isolate CBS 258.70 reported to be growing on Inonotus radiatus is questionable. Given the host, the isolate could be a morphological variant of Cosmospora coccinea. This isolate has not been sequenced. Cosmospora butyri (J.F.H. Beyma) Gräfenhan, Seifert & Schroers, Stud. Mycol. 68: 96. 2011. Basionym: Tilachlidium butyri J.F.H. Beyma, Zentralbl. Bakteriol., 2 Abt. 99: 388. 1938. ≡ Acremonium butyri (J.F.H. Beyma) W. Gams, Cephalosporium-artige Schimmelpilze (Stuttgart) p. 126. 1971. Asexual state: Acremonium-like. Habitat: Isolated from butter. Distribution: Denmark (only known from the type). Descriptions and illustrations: Beyma (1938) and Gams (1971). Notes: The sexual state of this species is unknown. Summerbell et al. (2011) noted 142 that the ex-type culture might comprise a mixed culture. We suspect that C. butyri has a fungal host, but it has not been found. The reported substrate suggests that this fungus might have a secondary nutrition mode (e.g., saprophytic). Cosmospora cymosa (W. Gams) Gräfenhan, Seifert & Schroers, Stud. Mycol. 68: 96. 2011. Basionym: Acremonium cymosum W. Gams, Cephalosporium-artige Schimmelpilze (Stuttgart) p. 131. 1971. Asexual state: Acremonium-like. Habitat: On Inonotus radiatus. Distribution: Germany. Descriptions and illustrations: Gams (1971). Notes: The sexual state of this species is unknown. The phylogeny in Gräfenhan et al. (2011) includes C. coccinea and C. cymosa as sister taxa. It is possible that C. cymosa is a morphological variant of C. coccinea given that they are 97% similar based on ITS sequences. However, protein-coding loci showed them to be more divergent. Differences are found primarily at third codon position sites of the protein-coding genes. Cosmospora lavitskiae (Zhdanova) Gräfenhan, Seifert & Schroers, Stud. Mycol. 68: 96. 2011. Basionym: Gliomastix lavitskiae Zhdanova, Mikrobiol. Zhurn. 28: 37. 1966. 143 Asexual state: Acremonium-like. Habitat: Isolated from soil rhizosphere of Zea mays. Distribution: Ukraine. Descriptions and illustrations: Zhdanova (1966) and Gams (1971). Notes: The sexual state of this species is unknown. We suspect that C. lavitskiae has a fungal host, but it has not been found. This fungus could have a secondary nutrition mode (e.g., saprophytic). 144 Ta bl e 3. 1. Is o la te s a n d a cc es sio n n u m be rs u se d in th e ph yl o ge n et ic a n a ly se s. G en eB a n k A cc es sio n N o . tu b2 JF 83 28 38 K J6 76 28 4 K J6 76 28 5 K J6 76 25 5 K C2 91 90 8 K J6 76 26 3 K J6 76 28 8 K J6 76 29 3 - K J6 76 26 2 K J6 76 26 4 K J6 76 26 5 te f1 JF 83 25 17 K J6 76 36 5 K J6 76 36 6 K J6 76 33 8 K C2 91 84 3 K J6 76 34 5 K J6 76 36 9 - - K J6 76 34 4 K J6 76 34 6 K J6 76 34 7 rp b1 JF 83 27 63 JQ 03 10 66 JQ 03 10 65 JQ 03 10 86 K C2 91 86 9 K J6 76 22 4 K J6 76 24 1 K J6 76 24 6 - K J6 76 22 3 K J6 76 22 5 JQ 03 10 81 m cm 7 K C2 91 79 5 JN 99 33 07 JN 99 33 08 JN 99 33 36 JN 99 33 26 K J6 76 30 6 K J6 76 32 5 K J6 76 33 0 - - K J6 76 30 7 JN 99 33 19 LS U JF 83 26 79 JN 93 98 17 JN 93 98 16 JN 93 98 39 JN 93 98 24 K J6 76 18 7 K J6 76 20 4 K J6 76 20 9 X X X X X X K J6 76 18 6 K J6 76 18 8 JN 93 98 34 IT S JF 83 25 94 JN 99 56 34 JN 99 56 35 JN 99 56 21 JN 99 56 29 K J6 76 15 0 K J6 76 16 7 K J6 76 17 2 X X X X X X K J6 76 14 9 K J6 76 15 1 JN 99 56 25 G eo gr a ph ic O ri gi n Fr en ch G u ia n a U SA U SA Fr an ce U SA U SA U kr ai n e Ca n ad a Ca n ad a B ra zi l B ra zi l Pu er to Ri co Su bs tr a te /H o st B ar k An n u lo hy po xy lo n cf . m u lti for m e An n u lo hy po xy lo n cf . co ha er en s H yp o xy lo n cf. ho w ea n u m H yp o xy lo n fra gi for m e u n id en tif ie d bl ac k py re n o m yc et e o n u n de sc rib ed pl an t su bs tr at e o n w o o dp ile H yp o xy lo n sp . K re tz ch m a ri a sp . K re tz ch m a ri a sp . K re tz ch m a ri a sp . H er ba ri u m N o . B PI 88 10 71 B PI 73 77 73 B PI 74 45 21 B PI 87 99 25 B PI X X X X X X - - - B PI X X X X X X - - B PI 74 52 49 Is o la te N o . A . R . 45 47 , CB S 12 38 26 G . J.S . 95 - 19 9, CB S X X X X X X G . J.S . 96 - 18 6, CB S X X X X X X A . R . 45 21 , CB S X X X X X X G . J.S . 10 - 24 7, CB S X X X X X X G . J.S . 96 - 25 1, CB S 12 39 61 IM I 1 33 98 4 K A S 11 05 K A S 50 72 G . J.S . 87 - 4, CB S 12 39 41 CB S 25 1. 78 G . J.S . 96 - 48 , CB S 44 8. 96 Sp ec ie s Co ra llo m yc et el l a el eg a n s Co sm o sp o ra a n n u lo hy po x yli Co sm o sp o ra a n n u lo hy po x yli Co sm o sp o ra a rx ii Co sm o sp o ra a rx ii Co sm o sp o ra sp . Co sm o sp o ra sp . Co sm o sp o ra sp . Co sm o sp o ra sp . Co sm o sp o ra cl a vi Co sm o sp o ra cl a vi Co sm o sp o ra cl a vi 145 Ta bl e 3. 1. C o n tin u ed . G en eB a n k A cc es sio n N o . tu b2 K J6 76 27 5 K J6 76 28 6 H M 48 45 89 K J6 76 25 2 K J6 76 27 4 K J6 76 25 6 K J6 76 25 8 K J6 76 25 9 K J6 76 28 7 K J6 76 29 5 te f1 K J6 76 35 6 K J6 76 36 7 H M 48 45 15 K J6 76 33 5 K J6 76 35 5 K J6 76 33 9 K J6 76 34 0 K J6 76 34 1 K J6 76 36 8 K J6 76 37 5 rp b1 K J6 76 23 3 K J6 76 23 9 G Q5 06 02 0 K J6 76 21 5 K J6 76 23 2 K J6 76 21 7 K J6 76 21 9 K J6 76 22 0 K J6 76 24 0 K J6 76 24 8 m cm 7 K J6 76 31 6 - - - K J6 76 31 4 K J6 76 30 0 K J6 76 30 2 K J6 76 30 3 K J6 76 32 4 K J6 76 33 1 LS U K J6 76 19 6 K J6 76 20 2 G Q5 05 99 0 K J6 76 17 8 K J6 76 19 5 K J6 76 18 0 K J6 76 18 2 K J6 76 18 3 K J6 76 20 3 K J6 76 21 1 IT S K J6 76 15 9 K J6 76 16 5 H M 48 45 37 K J6 76 14 1 K J6 76 15 8 K J6 76 14 3 K J6 76 14 5 K J6 76 14 6 K J6 76 16 6 K J6 76 17 4 G eo gr a ph ic O ri gi n B ra zi l Pu er to Ri co G er m an y G er m an y N ew Ze al an d A rg en tin a A rg en tin a A rg en tin a In di a Ja pa n Su bs tr a te /H o st K re tz sc hm a ri a cl a vu s K re tz ch m a ri a sp . In o n o tu s n o du lo su s In o n o tu s n o du lo su s Fo m es fom en ta ri u s An n u lo hy po xy lo n sp . An n u lo hy po xy lo n sp . An n u lo hy po xy lo n sp . o n de ca yi n g st em an d st u m p o f B a m bu sa o n u n id en tif ie d py ro n o m yc et e H er ba ri u m N o . G . J.S . 10 76 (N Y ) B PI 74 46 78 B PI 80 27 29 - PD D 46 39 8 B PI X X X X X X B PI X X X X X X B PI X X X X X X IM I 1 12 79 0 - Is o la te N o . G . J.S . 84 - 29 0, CB S X X X X X X G . J.S . 96 - 7, CB S X X X X X X A . R . 27 41 , CB S 11 40 50 A . R . 27 43 , CB S X X X X X X G . J.S . 83 - 19 4 , CB S X X X X X X A . R . 47 70 , CB S X X X X X A . R . 47 98 , CB S X X X X X A . R . 47 99 , CB S X X X X X IM I 1 12 79 0 M A FF 24 15 00 Sp ec ie s Co sm o sp o ra cl a vi Co sm o sp o ra cl a vi Co sm o sp o ra co cc in ea Co sm o sp o ra co cc in ea Co sm o sp o ra fom iti co la Co sm o sp o ra kh a n da le n sis Co sm o sp o ra kh a n da le n sis Co sm o sp o ra kh a n da le n sis Co sm o sp o ra kh a n da le n sis Co sm o sp o ra kh a n da le n sis 146 Ta bl e 3. 1. C o n tin u ed . G en eB a n k A cc es sio n N o . tu b2 K J6 76 26 9 K J6 76 27 1 K J6 76 27 7 K J6 76 29 7 K C2 91 90 7 K C2 91 90 6 JF 83 28 40 H M 48 48 76 K J6 76 26 6 K J6 76 27 8 K J6 76 27 9 te f1 K J6 76 35 1 K J6 76 35 2 K J6 76 35 8 K J6 76 37 7 K C2 91 84 9 K C2 91 85 1 JF 83 25 18 H M 48 48 51 K J6 76 34 8 K J6 76 35 9 K J6 76 36 0 rp b1 K J6 76 22 8 K J6 76 23 0 K J6 76 23 5 K J6 76 25 0 K C2 91 86 8 K C2 91 86 7 JF 83 27 65 G Q5 06 03 2 K J6 76 22 6 K J6 76 23 6 K J6 76 23 7 m cm 7 K J6 76 31 0 K J6 76 31 2 K J6 76 31 8 K J6 76 33 3 K J6 76 31 5 K J6 76 32 0 - K J6 76 32 3 K J6 76 30 8 K J6 76 31 9 K J6 76 32 0 LS U K J6 76 19 1 K J6 76 19 3 K J6 76 19 8 K J6 76 21 3 K C2 91 77 7 K C2 91 77 9 JF 83 26 81 G Q5 06 00 3 K J6 76 18 9 K J6 76 19 9 K J6 76 20 0 IT S K J6 76 15 4 K J6 76 15 6 K J6 76 16 1 K J6 76 17 6 K C2 91 73 2 K C2 91 74 8 JF 83 25 96 H M 48 48 55 K J6 76 15 2 K J6 76 16 2 K J6 76 16 3 G eo gr a ph ic O ri gi n Co st a R ic a Co st a R ic a Fr en ch G u ia n a B ra zi l N ew Ze al an d Fr en ch G u ia n a V en ez u el a Pu er to Ri co Ja m ai ca Fr en ch G u ia n a Fr en ch G u ia n a Su bs tr a te /H o st H yp o xy lo n cy cl o pi cu m H yp o xy lo n cy cl o pi cu m K re tz sc hm a ri a m ic ro pu s H yp o xy lo n cf . cy cl o pi cu m An n u lo hy po xy lo n bo ve i Xy la ri a sc ru po sa Xy la ri a sc ru po sa Xy la ri a sc ru po sa Xy la ri a sc ru po sa Xy la ri a sc ru po sa Xy la ri a sc ru po sa H er ba ri u m N o . B PI X X X X X X B PI X X X X X X G JS 31 82 (N Y ) B PI X X X X X X PD D 46 40 1 G JS 43 26 (N Y ) B PI 74 47 78 B PI 74 46 71 CU P- M J 8 58 (N Y ) G JS 41 29 (N Y ) G JS 43 93 (N Y ) Is o la te N o . G . J.S . 10 - 11 3, CB S X X X X X X G . J.S . 10 - 12 1, CB S X X X X X X G . J.S . 86 - 10 8, CB S X X X X X X P. C. 12 85 , CB S X X X X X X G . J.S . 83 - 19 7, CB S 12 40 32 G . J.S . 86 - 31 5, CB S X X X X X X G . J.S . 90 - 21 7, CB S X X X X X X G . J.S . 96 - 6, CB S 45 5. 96 C. T. R . 71 - 62 , CB S X X X X X X G . J.S . 86 - 27 8, CB S X X X X X X G . J.S , 86 - 32 0, CB S X X X X X X Sp ec ie s Co sm o sp o ra m ic ro pe di s Co sm o sp o ra m ic ro pe di s Co sm o sp o ra m ic ro pe di s Co sm o sp o ra m ic ro pe di s Co sm o sp o ra n o va ze la n di ca Co sm o sp o ra sc ru po sa e Co sm o sp o ra sc ru po sa e Co sm o sp o ra sc ru po sa e Co sm o sp o ra sc ru po sa e Co sm o sp o ra sc ru po sa e Co sm o sp o ra sc ru po sa e 147 Ta bl e 3. 1. C o n tin u ed . G en eB a n k A cc es sio n N o . tu b2 K J6 76 28 0 K J6 76 28 1 K J6 76 26 7 K J6 76 27 3 K J6 76 27 6 K J6 76 28 3 K J6 76 28 9 K J6 76 29 0 K J6 76 29 4 K J6 76 25 7 K J6 76 25 4 K J6 76 28 2 te f1 K J6 76 36 1 K J6 76 36 2 K J6 76 34 9 K J6 76 35 4 K J6 76 35 7 K J6 76 36 4 K J6 76 37 0 K J6 76 37 1 K J6 76 37 4 - K J6 76 33 7 K J6 76 36 3 rp b1 JQ 03 10 70 JQ 03 10 68 K J6 76 22 7 K J6 76 23 1 K J6 76 23 4 K J6 76 23 8 K J6 76 24 2 K J6 76 24 3 K J6 76 24 7 K J6 76 21 8 JQ 03 10 88 JQ 03 10 67 m cm 7 - - K J6 76 30 9 K J6 76 31 3 K J6 76 31 7 K J6 76 32 2 K J6 76 32 6 K J6 76 32 7 - K J6 76 30 1 - - LS U JN 93 98 22 JN 93 98 20 K J6 76 19 0 K J6 76 19 4 K J6 76 19 7 K J6 76 20 1 K J6 76 20 5 K J6 76 20 6 K J6 76 21 0 K J6 76 18 1 JN 93 98 41 JN 93 98 18 IT S JN 99 56 31 JN 99 56 32 K J6 76 15 3 K J6 76 15 7 K J6 76 16 0 K J6 76 16 4 K J6 76 16 8 K J6 76 16 9 K J6 76 17 3 K J6 76 14 4 JN 99 56 19 JN 99 56 33 G eo gr a ph ic O ri gi n Fr en ch G u ia n a V en ez u el a Th ai la n d N ew Ze al an d In do n es ia U ga n da En gl an d - Ca n ad a A rg en tin a U SA U SA Su bs tr a te /H o st Xy la ri a sc ru po sa Xy la ri a sc ru po sa Xy la ri a sp . An n u lo hy po xy lo n sp . Xy la ri a sp . Xy la ri a sp . Xy la ri a cf . po ly m o rp ha st em tis su e o f V iti s Xy la ri a cf . po ly m o rp ha St ilb o hy po xy lo n qu is qu ili a ru m K re tz sc hm a ri a de u st a K re tz sc hm ar ia de u st a H er ba ri u m N o . G JS 44 87 (N Y ) B PI 74 51 50 B PI 87 15 86 PD D 44 44 0 G JS 23 84 (N Y ) B PI 73 77 07 K (S ) 1 69 35 0 - - B PI X X X X X X B PI 87 10 89 B PI 80 28 40 Is o la te N o . G . J.S . 86 - 33 1, CB S X X X X X X G . J.S . 90 - 22 4, CB S X X X X X X G . J.S . 01 - 30 1, CB S X X X X X X G . J.S . 82 - 27 5, CB S X X X X X X G . J.S . 85 - 20 0, CB S X X X X X X G . J.S . 95 - 14 2, CB S X X X X X X IM I 3 18 02 5 IM I 3 62 24 0 K A S 37 51 A . R . 47 83 , CB S X X X X X X A . R . 42 15 , CB S X X X X X X G . J.S . 92 - 95 , CB S X X X X X X Sp ec ie s Co sm o sp o ra sc ru po sa e Co sm o sp o ra sc ru po sa e Co sm o sp o ra sp . Co sm o sp o ra sp . Co sm o sp o ra sp . Co sm o sp o ra sp . Co sm o sp o ra sp . Co sm o sp o ra sp . Co sm o sp o ra sp . Co sm o sp o ra st ilb o hy po x yl i Co sm o sp o ra u st u lin a e Co sm o sp o ra u st u lin a e 148 Ta bl e 3. 1. C o n tin u ed . G en eB a n k A cc es sio n N o . tu b2 K J6 76 29 1 K J6 76 29 6 K C2 91 90 5 K J6 76 27 0 K J6 76 27 2 K J6 76 25 3 K J6 76 26 0 K J6 76 26 1 K J6 76 29 2 K C2 91 93 2 K C2 91 93 5 K C2 91 91 2 te f1 K J6 76 37 2 K J6 76 37 6 K C2 91 84 1 - K J6 76 35 3 K J6 76 33 6 K J6 76 34 2 K J6 76 34 3 K J6 76 37 3 K C2 91 84 2 K C2 91 83 2 K C2 91 83 0 rp b1 K J6 76 24 5 K J6 76 24 9 K C2 91 86 6 K J6 76 22 9 JQ 03 10 93 K J6 76 21 6 K J6 76 22 1 K J6 76 22 2 K J6 76 24 4 K C2 91 89 2 K C2 91 89 4 K C2 91 87 1 m cm 7 K J6 76 32 9 K J6 76 33 2 - K J6 76 31 1 - K J6 76 29 9 K J6 76 30 4 K J6 76 30 5 K J6 76 32 8 K C4 79 77 3 K C2 91 79 8 K C2 91 79 6 LS U K J6 76 20 7 K J6 76 21 2 JN 93 98 26 K J6 76 19 2 JN 93 98 23 K J6 76 17 9 K J6 76 18 4 K J6 76 18 5 K J6 76 20 8 K C2 91 77 1 K C2 91 75 9 K C2 91 75 7 IT S K J6 76 17 0 K J6 76 17 5 JN 99 56 27 K J6 76 15 5 JN 99 56 30 K J6 76 14 2 K J6 76 14 7 K J6 76 14 8 K J6 76 17 1 K C2 91 74 4 K C2 91 75 1 K C2 91 72 1 G eo gr a ph ic O ri gi n Po rt u ga l Ja pa n A u st ra lia Co st a R ic a N ew Ze al an d D en m ar k Cz ec h R ep u bl ic Cz ec h R ep u bl ic W al es U SA N ew Ze al an d U SA Su bs tr a te /H o st st u m p o f C le th ra a rb o re a K re tz sc hm a ri a cf . de u st a K re tz sc hm a ri a cf . de u st a K re tz sc hm a ri a cf . de u st a K re tz sc hm ar ia cf . de u st a B o n e st an di n g tr u n k de ad tr ee Ru ze n ia sp er m o id es D ia tr yp e st ig m a sc al e in se ct Eu ty pe lla sp . H er ba ri u m N o . - B PI X X X X X X B PI 87 89 94 B PI X X X X X X PD D 30 86 9 ? - - K (S ) 1 69 34 9 - - B PI 88 41 65 Is o la te N o . IM I 3 89 10 1 M A FF 24 15 32 G . J.S . 09 - 41 1, CB S X X X X X X G . J.S . 10 - 11 4, CB S X X X X X X G . J.S . 73 - 2, CB S X X X X X X A . R . 27 83 CB S 10 24 30 CB S 10 24 33 IM I 7 33 77 a G . J.S . 10 - 19 3, C. H . 10 - 01 A . R . 45 80 A . R . 45 62 Sp ec ie s Co sm o sp o ra u st u lin a e Co sm o sp o ra u st u lin a e Co sm o sp o ra vi liu sc u la Co sm o sp o ra vi liu sc u la Co sm o sp o ra vi liu sc u la Co sm o sp o ra vi rid es ce n s Co sm o sp o ra vi rid es ce n s Co sm o sp o ra vi rid es ce n s Co sm o sp o ra vi rid es ce n s D ia lo n ec tr ia ep isp ha er ia M ic ro ce ra la rv a ru m Ps eu do co sm o sp o r a eu ty pe lla e 149 Ta bl e 3. 2. Lo ci u se d in th e ph yl o ge n et ic a n a ly se s. In fo rm a tio n o n th e pr im er s, in cl u di n g ba se pa ir s, PC R pr o to co ls, a n d m o de ls o f n u cl eo tid e su bs tit u tio n a re in di ca te d. Te f1 H K Y + G 35 2 20 1 80 0 te f1 - 72 8, te f1 - 98 6 (C ar bo n e & K o hn 19 99 ) 66 ° C, 55 s, 9x 56 ° C, 55 s, 35 × C o m bi n ed — 33 27 68 1 25 4 19 91 — — Tu b2 G TR + I+ G 19 70 62 4 18 8 91 6 B tu b- TI , B tu b- T2 (O ’ D o n n el l & Ci ge ln ik 19 97 ) 55 ° C, 30 s, 35 × R pb 1 cr pb 1a , rp b1 c (C as tle bu ry et al . 20 04 ) 50 ° C, 2 m in , 40 × M cm 7 m cm 7- 70 9f o r, m cm 7- 13 48 re v (S ch m itt et al . 20 09 ) 56 ° C, 50 s, 38 × LS U H K Y + I 78 3 29 20 72 3 LR 5, LR O R (V ilg al ys & H es te r 19 90 ) 53 ° C, 1 m in , 40 × IT S SY M + I+ G 57 4 28 46 35 2 IT S5 , IT S4 (W hi te et al . 19 90 ) Lo cu s N u cl eo tid e su bs tit u tio n m o de ls In cl u de d sit es Ph yl o ge n et ic a lly in fo rm a tiv e sit es U n in fo rm a tiv e po ly m o rp hi c sit es In v a ri a bl e sit es Pr im er s u se d (re fe re n ce ) PC R pr o to co l: A n n ea lin g te m p. & cy cl es 150 Ta bl e 3. 3. Su pp o rt re ce iv ed by ea ch re co gn iz ed sp ec ie s. C o m bi n ed (B I P P/ M L BP ) 10 0% /1 00 % 10 0% /1 00 % 10 0% /7 3% 10 0% /9 7% 10 0% /1 00 % 10 0% /1 00 % 10 0% /1 00 % 10 0% /1 00 % 10 0% /1 00 % 10 0% /1 00 % 10 0% /9 9% tu b2 (B I P P) 10 0% 10 0% 10 0% - 10 0% 10 0% 10 0% 10 0% 10 0% 10 0% 10 0% te f1 (B I P P) 94 % 98 % - - 99 % 94 % - - 97 % 93 % - rp b1 (B I P P) 10 0% 10 0% 10 0% 10 0% 10 0% 97 % 10 0% 10 0% 10 0% 10 0% 94 % m cm 7 (B I P P) 10 0% 10 0% 10 0% 98 % N /A 10 0% 10 0% - 99 % 10 0% 10 0% LS U (B I P P) 94 % 10 0% - - 10 0% - - - - - - IT S (B I P P) - 10 0% - - 10 0% 98 % - - - 10 0% 91 % Co sm o sp o ra a n n u lo hy po x ili Co sm o sp o ra a rx ii Co sm o sp o ra sp . Co sm o sp o ra cl a vi Co sm o sp o ra co cc in ea Co sm o sp o ra kh a n da le n sis Co sm o sp o ra m ic ro po di s Co sm o sp o ra sc ru po sa e Co sm o sp o ra u st u lin a e Co sm o sp o ra vi liu sc u la Co sm o sp o ra vi rid es ce n s 151 Su pp le m en ta l T ab le 3 .1 . M or ph ol og ica l d iff er en ce s i n th e Co sm os po ra vi liu sc ul a sp ec ie s c om pl ex (K -W te st , P < 0. 05 ). A) A sc os po re Le ng th . B ) A sc os po re W id th . C ) A sc os po re Q ra tio . D ) C on id ia Le ng th . E ) C on id ia W id th . F ) Co ni di a Qr at io . G ) G ro w ith in C M D. H ) G ro w ith in P DA . C. annulohypoxili ** = C . f om iti co la e xc lu de d in G & H co m pa ris on s C. arxii DF C. clavi BC DG BC DF GH C. fomiticola** DF BC DF C. khandalensis AB AD F AB DF G AB C C. micropodis AC EG H AC DF GH AB DE AC BC EG C. novaezelandica AB AB DF AB CD EG H AB AB AB CG H C. scruposae DF CG BE FH AC DF AB DF AC DF H AB DF G C. stilbohypoxili AD E AE F AB DE GH AC DE DE DE GH AB DE DE FG C. ustulinae AD EF AC DE FH AB AC DF BD EF DE AB DE H EF DE H C. viliuscula DF H CD F AB AC E AB EH AD FH AB DE H AD EF C. a nn ul oh yp ox ili C. a rx ii C. cl av i C. fo m iti co la ** C. kh an da le ns is C. m icr op od is C. n ov ae ze la nd ica C. sc ru po sa e C. st ilb oh yp ox ili C. u st ul in ae C. vi liu sc ul a 152 Fig. 3.1 ML phylogeny (best tree; LnL = −18580.293) of C. viliuscula species complex based on three partition (ITS, LSU, mcm7-rpb1- tub2) dataset. Label A, Cosmospora node; label B, Cosmospora viliuscula species complex. Black bold branches indicate >70% ML BP. 153 Fig. 3.2 BI phylogeny (consensus tree; LnL = −18297.835) of C. viliuscula species complex based on three partition (ITS, LSU, mcm7-rpb1- tub2) dataset. Label A, Cosmospora node; label B, Cosmospora viliuscula species complex. Gray bold branches indicate >90% BI PP; black bold branches indicate >95% BI PP. 0.03 GJS83197 AR4799 GJS90217 KAS1105 GJS967 GJS10113 CBS123941 IMI389101 KAS3751 GJS86278 CH1001 - Dialonectria episphaeria GJS86320 MAFF241500 GJS83194 AR2783 CBS102430 GJS86108 GJS86331 IMI362240 AR4783 GJS01301 AR4798 AR4215 IMI733779 AR4580 - Microcera larvarum MAFF241532 GJS95199 GJS966 GJS96186 AR2743 GJS10247 GJS95142 GJS82275 GJS9295 IMI318025 IMI112790 GJS10114 GJS10121 AR4562 - Pseudocosmospora eutypellae AR4770 GJS90224 CBS102433 AR2741 GJS96251 AR4521 KAS5072 CBS44896 GJS09411 IMI133984 PC1285 GJS86315 GJS732 PC1306 GJS85200 GJS10112 AR4547 - Corallomycetella elegans CBS25178 GJS84290 CTR7162 // // A B C. fomiticola C. coccinea C. viridescens C. arxii C. novazelandica C. annulohypoxyli C. khandalensis C. scruposae C. stilbohypoxyli C. clavi C. ustulinae C. viliuscula C. micropedis Fig. 3.3 Cosmospora annulohypoxyli bar = 4 mm. (C) Perithecia on natural substrata. in 3% KOH. Scale bar = 100 bar = 10 µm. (G) Phialide. Cultures after 3 wks at 25 25 C on CMD. Scale bar = 154 . (A) Habit. Scale bar = 3 mm. (B) Habit. Scale bar = 200 µm. (D) Perithecium µm. (E) Asci. Scale bar = 10 µm. (F) Ascospore. Scale Scale bar = 10 µm. (H) Conidia. Scale bar = 10 C on PDA. Scale bar = 10 mm. (K) Cultures aft 10 mm Scale µm. (I−J) er 3 wks at Fig. 3.4 Cosmospora arxii substrata. Scale bar = 200 Asci. Scale bar = 10 µm. 10 µm. (G) Phialide. Scale bar = Scale bar = 10 mm. (J) Cultures after 3 wks at 25 155 . (A) Habit. Scale bar = 4 mm. (B) Perithecia on natural µm. (C) Perithecium in 3% KOH. Scale bar = 100 (E) Ascospore. Scale bar = 10 µm. (F) Conidia. 10 µm. (H−I) Cultures after 3 wks at 25 C on CMD. Scale bar = µm. (D) Scale bar = C on PDA. 10 mm Fig. 3.5 Cosmospora clavi substrata. Scale bar = 200 Asci. Scale bar = 10 µm. bar = 10 µm. (I) Conidia. PDA. Scale bar = 10 mm. mm 156 . (A−B) Habit. Scale bar = 4 mm. (C) Perithecia on natural µm. (D) Perithecium in 3% KOH. Scale bar = (F) Ascospore. Scale bar = 10 µm. (G−H) Phialide. Scale bar = 10 µm. (J−K) Cultures after 3 wks at 25 (L) Cultures after 3 wks at 25 C on CMD. Scale bar = 100 µm. (E) Scale C on 10 Fig. 3.6 Cosmospora coccinea natural substrata. Scale bar = µm. (D) Ascospore. Scale bar = Conidia. Scale bar = 10 µm. 10 mm. (J) Cultures after 3 wks at 25 157 . (A) Habit. Scale bar = 4 mm. (B) Perithecia on 200 µm. (C) Perithecium in 3% KOH. Scale bar = 10 µm. (E−F) Phialide. Scale bar = 10 µm. ( (H−I) Cultures after 3 wks at 25 C on PDA. C on CMD. Scale bar = 10 mm 100 G) Scale bar = Fig. 3.7 Cosmospora fomiticola natural substrata. Scale bar = µm. (D) Asci. Scale bar = Scale bar = 10 µm. (G) Conidia. on PDA. Scale bar = 10 mm. mm 158 . (A) Habit. Scale bar = 3 mm. (B) Perithecia on 200 µm. (C) Perithecium in 3% KOH. Scale bar = 10 µm. (E) Ascospore. Scale bar = 10 µm. (F) Scale bar = 10 µm. (H) Cultures after 3 wks at 25 (I) Cultures after 3 wks at 25 C on CMD. Scale bar = 100 Phialide. C 10 Fig. 3.8 Cosmospora khandalensis natural substrata. Scale bar = µm. (D) Asci. Scale bar = Phialide. Scale bar = 10 µ wks at 25 C on PDA. Scale bar = Scale bar = 10 mm 159 . (A) Habit. Scale bar = 5 mm. (B) Perithecia on 200 µm. (C) Perithecium in 3% KOH. Scale bar = 10 µm. (E) Ascospores. Scale bar = 10 µm. (F m. (H) Conidia. Scale bar = 10 µm. (I−J) Cultures 10 mm. (K) Cultures after 3 wks at 25 C on CMD. 100 −G) after 3 Fig. 3.9 Cosmospora micropedis natural substrata. Scale bar = µm. (D) Asci. Scale bar = and conidia. Scale bar = 10 bar = 10 mm. (I) Cultures 160 . (A) Habit. Scale bar = 1 mm. (B) Perithecia on 200 µm. (C) Perithecium in 3% KOH. Scale bar = 10 µm. (E) Ascospores. Scale bar = 10 µm. (F µm. (G−H) Cultures after 3 wks at 25 C on PDA. after 3 wks at 25 C on CMD. Scale bar = 10 mm 100 ) Phialides Scale Fig. 3.10 Cosmospora novazelandica 200 µm. (B) Perithecium in 3% KOH. Scale bar = around the perithecial apex. Ascospores. Scale bar = 10 Cultures after 3 wks at 25 25 C on CMD. Scale bar = 161 . (A) Perithecia on natural substrata. 100 µm. (C) Cells protruding Scale bar = 10 µm. (D) Asci. Scale bar = 10 µ µm. (F) Phialides and conidia. Scale bar = 10 µ C on PDA. Scale bar = 10 mm. (H) Cultures after 3 wks at 10 mm Scale bar = m. (E) m. (G) Fig. 3.11 Cosmospora scruposae natural substrata. Scale bar = µm. (D) Asci. Scale bar = Scale bar = 10 µm. (G) Conidia C on PDA. Scale bar = 10 mm. 10 mm 162 . (A) Habit. Scale bar = 2 mm. (B) Perithecia on 200 µm. (C) Perithecium in 3% KOH. Scale bar = 10 µm. (E) Ascospores. Scale bar = 10 µm. (F . Scale bar = 10 µm. (H−I) Cultures after 3 wks at 25 (J) Cultures after 3 wks at 25 C on CMD. 100 ) Phialides. Scale bar = Fig. 3.12 Cosmospora stilbohypoxyli natural substrata. Scale bar = µm. (D) Asci. Scale bar = Scale bar = 10 µm. (G) Conidia on PDA. Scale bar = 10 mm mm 163 . (A) Habit. Scale bar = 1 mm. (B) Perithecia on 200 µm. (C) Perithecium in 3% KOH. Scale bar = 10 µm. (E) Ascospores. Scale bar = 10 µm. (F . Scale bar = 10 µm. (H) Cultures after 3 wks at 25 . (I) Cultures after 3 wks at 25 C on CMD. Scale bar = 100 ) Phialides. C 10 Fig. 3.13 Cosmospora ustulinae natural substrata. Scale bar = µm. (D) Asci. Scale bar = Scale bar = 10 µm. (G) Conidia C on PDA. Scale bar = 10 mm. 10 mm 164 . (A) Habit. Scale bar = 4 mm. (B) Perithecia on 200 µm. (C) Perithecium in 3% KOH. Scale bar = 10 µm. (E) Ascospores. Scale bar = 10 µm. (F . Scale bar = 10 µm. (H−I) Cultures after 3 wks at 25 (J) Cultures after 3 wks at 25 C on CMD. 100 ) Phialides. Scale bar = Fig. 3.14 Cosmospora viliuscul natural substrata. Scale bar = µm. (D) Asci. Scale bar = Scale bar = 10 µm. (G) Conidia C on PDA. Scale bar = 10 mm. 10 mm 165 a. (A) Habit. Scale bar = 4 mm. (B) Perithecia on 200 µm. (C) Perithecium in 3% KOH. Scale bar = 10 µm. (E) Ascospores. Scale bar = 10 µm. (F . Scale bar = 10 µm. (H−I) Cultures after 3 wks at 25 (J) Cultures after 3 wks at 25 C on CMD. 100 ) Phialides. Scale bar = 166 Supplemental Fig. S3.1 C. viliuscula species complex inferred from ITS. Gray bold branches indicate >90% BI PP; black bold branches indicate >95% BI PP. 167 Supplemental Fig. S3.2 C. viliuscula species complex inferred from LSU. Gray bold branches indicate >90% BI PP; black bold branches indicate >95% BI PP. 168 Supplemental Fig. S3.3 C. viliuscula species complex inferred from mcm7. Gray bold branches indicate >90% BI PP; black bold branches indicate >95% BI PP. 169 Supplemental Fig. S3.4 C. viliuscula species complex inferred from rpb1. Gray bold branches indicate >90% BI PP; black bold branches indicate >95% BI PP. 170 Supplemental Fig. S3.5 C. viliuscula species complex inferred from tef1. Gray bold branches indicate >90% BI PP; black bold branches indicate >95% BI PP. 171 Supplemental Fig. S3.6 C. viliuscula species complex inferred from tub2. Gray bold branches indicate >90% BI PP; black bold branches indicate >95% BI PP. 172 Chapter 4: Pseudocospeciation of the mycoparasite Cosmospora with their associated fungal hosts. ABSTRACT Species of Cosmospora are parasites of other fungi (mycoparasites), including species belonging to the Xylariales. Based on prior taxonomic work, these fungi were determined to be highly host specific. We suspected that the association of Cosmospora and their hosts could not be a result of random chance, and tested the cospeciation of Cosmospora and the their hosts with contemporary methods (e.g. Parafit, PACo, and Jane). The cophylogeny of Cosmospora and their hosts was found to be congruent, but only host-parasite links in more recent evolutionary lineages of the host were determined as coevolutionary. Reconciliation reconstructions determined at least five host switch events early in the evolution of Cosmospora. This pattern is more likely to be explained by pseudocospeciation (i.e., host switches followed by cospeciation), which also produce congruent cophylogenies. INTRODUCTION Evolutionary relationships of fungus-fungus systems have been rarely studied. Millanes et al. (2014) studied the Biatoropsis Räsänen-Usnea Dill. ex Adans. system (a fungal parasite-fungal host association), and demonstrated that host-switch events played a more prevalent role than cospeciation events in their reconciliation 173 reconstructions of Biatoropsis and Usnea phylogenies. Also, the fungal cultivars of the fungus-growing ants (fungi belonging to the Agaricaceae and Tricholomataceae) and their associated fungal parasites, Escovopsis J.J. Muchovej & Della Lucia, have been shown to have highly congruent phylogenies (Currie et al. 2003). In other non- fungal systems, host-parasite relationships have also produced congruent cophylogenies (e.g. Clayton and Johnson 2003; Banks et al. 2006; Hosokawa et al. 2006; Marussich and Machado 2007; Hughes et al. 2007; Noda et al. 2007; Jackson et al. 2008; Lanterbecq et al. 2010; Göker et al. 2011), which have been taken as evidence of cospeciation between hosts and parasites. However, congruent cophylogenies can also result from other evolutionary mechanisms besides cospeciation such as coevolution and sequential evolution. Coevolution is the evolution in two or more species that leads to reciprocal evolutionary changes, and in sequential evolution, changes in one taxon leads to changes in the other taxon, but the change is not reciprocal (reviewed in Ridley 2004). Cospeciation involves the joint speciation of two or more species that are ecologically associated (e.g. host-parasites; Page 2003). There are also evolutionary events that would lead to incongruent cophylogenies: 1) duplication (independent speciation), 2) host-switching, and 3) lineage sorting (e.g. extinction and “missing the boat”; reviewed in Page 2003; reviewed in Paterson and Banks 2001). In the present study, we studied the association between species of Cosmospora Rabenh. (sensu lato; a mycoparasite—a fungus that parasitizes other fungi) and their associated fungal hosts. Cosmospora (Ascomycota, Hypocreales, Nectriaceae) is a fungal genus that was determined to be artificial, and segregated into many 174 monophyletic genera (Schoch and Crous 2000; Lou and Zhuang 2010, 2012; Gräfenhan et al. 2011; Herrera et al. 2013). The sexual fruiting bodies (perithecia) in Cosmospora sensu lato are highly conserved to the degree of being indistinguishable. Briefly, the perithecia are reddish, small-sized (<300 microns), and pear-shaped (Fig. 4.1A). The sexual spores (ascospores) are ellipsoid to ellipsoid-fusiform, one-septate, yellow-brown and warted at maturity (Samuels et al. 1991; Rossman et al. 1999). The perithecia usually grow in-clusters on other fungi, scale insects, rarely on wood and herbaceous substrata (Rossman 1983; Samuels et al. 1991; Rossman et al. 1999). Cosmospora-like fungi are reported to be most common in recently disturbed forest stands (Chaverri and Vilchez 2006), and to have much greater diversity in warm temperate and tropical regions (Rossman et al. 1999). However, they are not infrequent outside those regions. Most cosmospora-like fungi are mycoparasites of fungi in the families Xylariaceae and Diatrypaceae (Xylariales, Ascomycota; Gräfenhan et al. 2011). Tsuneda (1982) first described the attack by these mycoparasites. Briefly, the fruiting bodies of the fungal host are penetrated by the Cosmospora species, and the fleshy insides of the fungal host are slowly attacked and consumed by the Cosmospora’s vegetative hyphae. It is thought that the slow attack ensures an extended period of nutrient uptake. The fungal host is able to mature but not to release ascospores. Ultimately, the host’s fleshy insides are replaced by vegetative hyphae of the Cosmospora. The mycoparasitic attack ends with the formation of its own perithecia directly on the surface of the host’s fruiting bodies (Fig. 4.1B), while simultaneously consuming its own vegetative hyphae for the production of perithecia (Tsuneda 1982). 175 Cosmospora sensu stricto include species that grow on xylariaceous fungi (fungi belonging to the Xylariaceae, Xylariales, Ascomycota). During the taxonomic revision of these fungi (see Herrera et al. 2014), it was observed that these species demonstrated a high degree of host-specificity (i.e., their association was not random). Host-specificity is a trait that often characterizes the intimate relationship of a host and its associated parasite. Given this host-specific trait, we hypothesized that species of Cosmospora have cospeciated with their xylariaceous fungal hosts following Fahrenholz’ rule (i.e., host and associated parasites form cophylogenies; reviewed in Ridley 2004). In this paper, we investigated associations between Cosmospora species and their associated xylariaceous hosts. METHODS Cosmospora phylogeny Thirteen species were selected based on the availability of host data (see below). Sequences were generated in prior taxonomic work (Herrera et al. 2013; Herrera et al. 2014). Briefly, DNA was extracted from mycelium grown for one week in Difco™ potato dextrose broth with PowerPlant® DNA Isolation Kit (MO BIO Laboratories Inc., Solana Beach, California). Internal transcribed spacer (ITS), large subunit nuclear ribosomal DNA (LSU), DNA replication licensing factor (mcm7), RNA polymerase II Subunit one (rpb1), and β-tubulin (tub2) were amplified in an Eppendorf Mastercycler thermocycler (Eppendorf, Westbury, New York) and sequenced at the DNA Sequencing Facility (Center for Agricultural Biotechnology, 176 University of Maryland, College Park, Maryland). The selected species and the associated sequences are listed in Table 4.1. Sequences were aligned via the MAFFT v.6 web service (http://mafft.cbrc.jp/alignment/server/; Katoh et al. 2002, 2013) implementing the E- INS-i alignment strategy and the 1PAM / κ=2 scoring matrix for nucleotide sequences. Alignments were manually edited in Mesquite 2.75 (Maddison and Maddison 2011). Ambiguous regions were excluded. The best-fit partitioning scheme among the sequenced loci and the model of nucleotide substitution for each partition were determined with PartitionFinder v1.1.1 (Lanfear et al. 2012) using the default settings. Phylogenetic analysis was performed using GARLI v2.01 (Genetic Algorithm for Rapid Likelihood Inference; Zwickl 2006) via the GARLI web service (http://www.molecularevolution.org; Bazinet and Cummings 2011), which uses a grid computing system associated with The Lattice Project (Cummings and Huskamp 2005; Bazinet and Cummings 2008). Fifty independent search replicates were performed to find the best tree with a fast ML stepwise-addition algorithm. One thousand bootstrap replicates were used in the bootstrap analysis. Host phylogeny Effort was made to extract DNA directly from the fruiting bodies of the host. However, in most cases, we obtained sequences of the associated Cosmospora species suggesting that the mycoparasite had already attacked the host. We were able to obtain ITS sequences from the hosts of Cosmospora khandalensis and 177 Pseudocosmospora joca by amplifying DNA with Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare Bio-Sciences Corp., Piscataway, New Jersey) following the manufacturer’s instructions. The identity of the host for P. joca was determined to be Biscogniauxia capnodes (Xylariaceae), while the host for C. khandalensis could only be identified to genus rank as Annulohypoxylon (Xylariaceae). In our previous taxonomic revision of the Cosmospora viliuscula species complex (Herrera et al., Chapter 3), we conservatively identified the hosts in the complex based on morphological characters. We extracted sequences (ITS, actA, rpb2, and tub2) from GenBank for these species, and these sequences are listed in Table 4.2. Phylogenetic analysis was performed as described for the mycoparasite (above). Cophylogenetic analyses Thirteen host species and thirteen Cosmospora species were included in the cophylogenetic analyses. We performed two distance-based methods: PACo (Balbuena et al. 2013) and ParaFit (Legendre et al. 2002). Additionally, two tree- reconciliation methods were performed: Jane v.4 (Conow et al. 2010) and CoRe-PA v0.5.1 (Merkle et al. 2010). A tanglegram between Cosmospora species and their associated host was generated with TreeMap v3.0β (Charleston 2011). Distance-based methods were implemented in R (R Core Team 2013) with the APE package (Paradis et al. 2004). Host and parasite phylogenies were transformed into matrices of patristic distances, and transformed again into principle coordinates to describe the phylogenies. The host principle coordinates, parasite principle 178 coordinates, and host-parasite association matrices were used to test the degree of congruence between the host and parasite phylogenies with a global host-parasite statistic, and the significance of the statistic was determined using a permutation test. 100000 permutations were run for PACo, whereas 999 permutations were run for ParaFit. PACo and ParaFit algorithms test the null hypothesis that the host and parasite phylogenies are independent (or randomly associated). In an evaluation of tree reconciliation methods (CoRe-PA, Jane and TreeMap), CoRe- PA was determined to be the most precise tool available in predicting the associations between hosts and parasites, although it does not produce an optimal estimate of the number of cospeciation and switching events. Jane was determined to yield the correct estimate of cospeciation events (Keller-Schmidt et al. 2011). Because they are based on the optimality criterion of Maximum Parsimony, these methods seek to find the cophylogeny with the minimum cost. CoRe-PA and Jane assign costs to four evolutionary events: cospeciation, duplication, host switch and sorting. Additionally, Jane assigns a cost to failure to diverge. We used the default cost settings in CoRe-PA and Jane. RESULTS Phylogenetic analyses PartitionFinder determined three partitions in the Cosmospora supermatrix, which included 3217 total characters (ITS: 570; LSU: 782; mcm7: 615; rpb1: 690; and tub2: 560). These partitions were ITS, LSU, and mcm7+rpb1+tub2. The best model of nucleotide substitution was TIMef+I, K80+I, and TrNef+G, for each partition 179 respectively. The negative log likelihood for the best tree was −10885.2025. Cosmospora lineages were well supported with some exceptions (Fig. 4.2). Three partitions were determined for the host supermatrix that comprised 3159 total characters (ITS: 466; actA: 301; rpb2: 1199; and tub2: 1193). These partitions were ITS, actA+rpb2, and tub2. TIMef+G, K80+I, and TrN+G were selected as the best models for each partition, respectively. The negative log likelihood for the best tree was −12636.6736. Lineages of xylariaceous fungi were well supported with some exceptions (Fig. 4.2). Distance-based analyses A procrustean superimposition plot of axes one and two, corresponding to patristic distances of Cosmospora and their fungal hosts, suggested three groups of host- parasite associations (Fig. 4.3). One group is composed of Cosmospora species associated with Annulohypoxylon and Hypoxylon. Another group is composed of Cosmospora species associated with Kretzschmaria, Stilbohypoxylon and Xylaria. A third group is composed of Dialonectria episphaeria, Pseudocosmospora and their associated hosts. Distance-based methods supported an overall congruence between the phylogenies of Cosmospora and their associated hosts. The PACo analysis produced a residual sum of squares (m2XY) of 0.4193 with an associated permutational P = 0.00001. Similarly, the ParaFit global fit statistic was 0.0275 (P = 0.005). The contribution of each host- parasite to the global fit was assessed with a jackknife procedure applied in PACo, which estimated the squared residual and its 95% confidence interval of each 180 individual link (Fig. 4.4). Most links associated with Kretzschmaria, Stilbohypoxylon and Xylaria hosts contributed relatively little to the residual sum of squares. The Eutypa lata-Pseudocosmospora eutypae and Eutypella scoparia-Pseudocosmospora eutypellae links were also determined to contribute relatively little to the residual sum of squares. ParaFitLink1 analysis also considered these links + Kretzschmaria deusta- Cosmospora ustulinae as coevolutionary at 0.05 significance level. Tree reconciliation analyses The Tanglegram between Cosmospora and host phylogenies showed some internal congruence (Fig. 4.2). The reconciliation of the Cosmospora tree with the host tree revealed a maximum of seven cospeciation events might have happened in their evolution (Fig. 4.5). This reconciliation also contained five host-switches and three sorting events. The total cost for this reconciliation was 18 in CoRe-Pa and 13 in Jane. Jane generated another equally parsimonious reconciliation between Cosmospora and host trees (Fig. 4.6). This reconciliation had six cospeciations, six host-switches, and one sorting event. In only one instance out of 100 did a better random sample solution produced a reconciliation cost below 13 (P = 0.01). DISCUSSION Distance based methods confirmed that the phylogenies of Cosmospora and the fungal host were more congruent than expected by chance (PACo, P = 0.00001; ParaFit, P = 0.005). The global congruence between host and parasite phylogenies have been interpreted as a result of cospeciation in many studies prior to this one (e.g. ants and plants, Itino et al. 2001; fungi and plants, Jackson 2004; penguins and their 181 lice, Banks et al. 2006; mycoviruses and their fungal hosts, Göker et al. 2011; among others). However, not all individual host-parasite links were found to be coevolutionary (Fig. 4.4). Most host-parasite links considered coevolutionary included Cosmospora associated with Kretzschmaria, Stilbohypoxylon, and Xylaria hosts. These host genera represent recent evolutionary lineages of the Xylariaceae (Tang et al. 2009; Hsieh et al. 2010). Charleston and Robertson (2002) observed a similar global congruency of host-parasite cophylogenies and codivergences occurring at the tip of the host phylogeny. Given that there was a large difference in evolutionary rates between host and parasites, Charleston and Robertson (2002) determined that the observed evolutionary pattern could not be explained by cospeciation events alone, and suggested that this pattern was a result of host- switches followed by cospeciation events. Cospeciation is expected to have congruent phylogenies but also to have similar divergence times (reviewed in Page 2003). Similar congruent topologies as seen in cospeciation could arise as a result of host- switches followed by cospeciation events (or pseudocospeciation) but not have similar divergence times (Hafner and Nadler 1988; reviewed in Page 2003; de Vienne et al. 2007, 2013). Tree reconciliation based methods also supported the idea that the cophylogeny between Cosmospora and their fungal hosts could not be interpreted from strict cospeciation events (Fig. 4.5 and 4.6). The reconciled trees contained five–six host switch events (Fig. 4.5 and 4.6), which occurred early in the host phylogeny. Cospeciation events were more prevalent towards the tip of the host phylogeny. Divergence time estimates could not be determined in the current study due the lack 182 of fossil records for fungi in general (Taylor and Berbee 2006). Calibration points are needed within the in-group of study to obtain more accurate estimates of divergence times. Therefore, we cannot determine whether or not Cosmospora and the host have similar divergent times or not. Strict speciation cannot be ruled out, but strict cospeciation is probably unlikely given the relatively high number of suspected host switch events in the reconciliation reconstructions. Pseudocospeciation represents a better hypothesis to explain the apparent congruency between Cosmospora and the host phylogenies. Pseudocospeciation is often confused in the literature as cospeciation given the significant global congruency between host and parasite phylogenies, even though the parasites have been shown to diverge more recently than the host (e.g. Reed et al. 2007; Light and Hafner 2008). The lack of congruency in divergence times (or temporal congruency) between host and parasites should have refuted the hypothesis of cospeciation (e.g. Charleston and Robertson 2002; Sorenson et al. 2004; Huyse and Volckaert 2005). De Vienne et al. (2013) reviewed cospeciation literature, and determined than only seven percent of the literature represented convincing cases of cospeciation. These cases involved symbionts that were transmitted vertically, which does seem to be the case for Cosmospora. In contrast, Hafner and Nadler (1988) posited that pseudocospeciation resulted from host switches by the symbiont onto closely related hosts of the original host (horizontal transmission) followed by speciation on the new host. The resulting phylogenies of the host and the symbiont resemble the phylogenetic signature of cospeciation (i.e. cophylogenies) as result of the conserved host switching of the symbionts (Hafner and Nadler 1988; Charleston 183 and Robertson 2002; Sorenson et al. 2004; Huyse and Volckaert 2005). Host switching consists of a two-step process (reviewed in Norton and Carpenter 1998). Firstly, the acquisition of a new host by the parasite requires that the new host is found within the parasite’s range and is related to the old host (i.e. phylogenetically similar; e.g. Davies and Pedersen 2008), or has a similar ecological habitat to the old host (i.e. ecologically similar; e.g. Nikoh and Fukatsu 2000). Secondly, the parasite has to adapt to the new host in a way that diminishes gene flow between populations on the old host and populations on the new host. Ultimately, the parasite on the new host will speciate as a result of limited gene flow over time. Host switching involves an initial decrease in host specificity during the colonization of a new host, and an increase in host specificity as speciation on the new host occurs (Norton and Carpenter 1998). This study represents a preliminary account of the evolutionary relationships between cosmospora-like fungi and their associated hosts, and further study of this group of fungi is likely to yield intriguing and complex results. Some species of Cosmospora sensu stricto are associated with basidiomycetes (Basidiomycota, Mycota; Herrera et al. 2014; Gräfenhan et al. 2011), which could represent a putative inter-phylum host- switch early in the evolution of Cosmospora. In other fungi, rapid speciation was observed after host switches, particularly those exploiting new adaptive zones (Zaffarano et al. 2008; Chaverri and Samuels 2013). Additionally, species of Microcera Desm. (Nectriaceae, Hypocreales, Ascomycota), a former group of fungi of Cosmospora sensu lato, are parasites of scale insects (Coccoidea, Hemiptera, 184 Insecta; Gräfenhan et al. 2011) and lichens (unpublished data). This lineage of cosmospora-like fungi could represent a putative interkingdom host switch. 185 Ta bl e 4. 1. Is ol at es an d ac ce ss io n nu m be rs u se d in th e p hy lo ge ne tic an al ys es Ge ne Ba nk A cc es sio n No . tu b2 KJ 67 62 85 KC 29 19 08 KJ 67 62 75 KJ 67 62 59 KC 29 19 07 KJ 67 62 80 KJ 67 62 94 KJ 67 62 57 KJ 67 62 54 rp b1 JQ 03 10 65 KC 29 18 69 KJ 67 62 33 KJ 67 62 20 KC 29 18 68 JQ 03 10 70 KJ 67 62 47 KJ 67 62 18 JQ 03 10 88 m cm 7 JN 99 33 08 JN 99 33 26 KJ 67 63 16 KJ 67 63 03 KJ 67 63 15 - - KJ 67 63 01 - LS U JN 93 98 16 JN 93 98 24 KJ 67 61 96 KJ 67 61 83 KC 29 17 77 JN 93 98 22 KJ 67 62 10 KJ 67 61 81 JN 93 98 41 IT S JN 99 56 35 JN 99 56 29 KJ 67 61 59 KJ 67 61 46 KC 29 17 32 JN 99 56 31 KJ 67 61 73 KJ 67 61 44 JN 99 56 19 Ge og ra ph ic or igi n US A US A Br az il Ar ge nt in a Ne w Z ea la nd Fr en ch G ui an a Ca na da Ar ge nt in a US A Ho st An nu lo hy po xy lo n cf . c oh ae re ns Hy po xy lo n fra gi fo rm e Kr et zs ch m ar ia cla vu s An nu lo hy po xy lo n sp . An nu lo hy po xy lo n bo ve i Xy la ria sc ru po sa Xy la ria cf . po lym or ph a St ilb oh yp ox ylo n qu isq ui lia ru m Kr et zs ch m ar ia de us ta He rb ar iu m No . BP I 7 44 52 1 BP I X XX XX X G. J.S . 1 07 6 (N Y) BP I X XX XX X PD D 46 40 1 GJ S 44 87 (N Y) - BP I X XX XX X BP I 8 71 08 9 Iso la te N o. G. J.S . 9 6- 18 6, CB S X XX XX X G. J.S . 1 0- 24 7, CB S X XX XX X G. J.S . 8 4- 29 0, CB S X XX XX X A. R. 4 79 9, C BS XX XX X G. J.S . 8 3- 19 7, CB S 1 24 03 2 G. J.S . 8 6- 33 1, CB S X XX XX X KA S 3 75 1 A. R. 4 78 3, CB S X XX XX X A. R. 4 21 5, C BS XX XX XX ID co de Ca n Ca r Cc l Ck h Cn o Cs c Cs p Cs t Cu s Sp ec ie s Co sm os po ra an nu lo hy po xil i Co sm os po ra a rx ii Co sm os po ra cl av i Co sm os po ra kh an da le ns is Co sm os po ra no va ez el an di ca Co sm os po ra sc ru po sa e Co sm os po ra sp . Co sm os po ra st ilb oh yp ox ili Co sm os po ra us tu lin ae 186 Ta bl e 4. 1. C on tin ue d Ge ne Ba nk A cc es sio n No . tu b2 KC 29 19 32 KC 29 19 25 KC 29 19 12 KC 29 19 24 rp b1 KC 29 18 92 KC 29 18 84 KC 29 18 71 KC 29 18 87 m cm 7 KC 47 97 73 KC 29 18 05 KC 29 17 96 KC 29 18 01 LS U KC 29 17 71 KC 29 17 66 KC 29 17 57 KC 29 17 62 IT S KC 29 17 44 KC 29 17 35 KC 29 17 21 KC 29 17 46 Ge og ra ph ic or igi n US A Fr an ce US A Ar ge nt in a Ho st Di at ry pe st ig m a Eu ty pa sp . Eu ty pe lla sp . Bi sc og ni au xia sp . He rb ar iu m No . - BP I 8 84 16 4 BP I 8 84 16 5 BP I 8 84 17 5 Iso la te N o. G. J.S . 1 0- 19 3, C. H. 1 0- 01 C. H. 1 1- 01 A. R. 4 56 2 A. R. 4 77 9 ID co de De p Pe 1 Pe 2 Pj o Sp ec ie s Di al on ec tri a ep isp ha er ia Ps eu do co sm os po ra eu ty pa e Ps eu do co sm os po ra eu ty pe lla e Ps eu do co sm os po ra jo ca 187 Ta bl e 4. 2. Is ol at es an d ac ce ss io n nu m be rs u se d in th e p hy lo ge ne tic an al ys es Ge ne Ba nk A cc es sio n No . rp b2 GQ 85 30 20 GQ 84 47 89 - - - JX 50 77 79 GQ 84 83 43 GQ 84 83 41 GQ 84 47 66 - - - - tu b2 EF 02 56 05 EF 02 56 11 DQ 84 00 92 AY 95 16 54 AY 95 17 19 AY 95 16 75 GQ 49 59 54 GQ 49 59 52 AY 95 16 55 - DQ 00 70 03 DQ 00 69 73 GQ 29 40 29 ac t EF 02 55 90 EF 02 55 96 - AY 95 17 65 AY 95 18 31 AY 95 17 87 GQ 45 23 64 GQ 45 23 62 AY 95 17 66 - - - - IT S EF 02 61 19 EF 02 61 26 - EF 02 61 41 JN 97 94 19 EF 02 61 31 GU 32 24 60 GU 32 24 58 EF 02 61 40 XX XX XX X DQ 00 69 45 DQ 00 69 28 GQ 29 39 62 As so cia te d w ith : Co sm os po ra st ilb oh yp ox ili Co sm os po ra cl av i Co sm os po ra u st ul in ae Co sm os po ra no va ez el an di ca Co sm os po ra a rx ii Ps eu do co sm os po ra jo ca Co sm os po ra sp . Co sm os po ra sc ru po sa e Co sm os po ra an nu lo hy po xil i Co sm os po ra kh an da le ns is Di al on ec tri a ep isp ha er ia Ps eu do co sm os po ra eu ty pa e Ps eu do co sm os po ra eu ty pe lla e Ge og ra ph ic or igi n Fr en ch G ui an a Fr en ch G ui an a Fr an ce Ta iw an Fr an ce Ta iw an US A M ar tin iq ue Fr an ce Ar ge nt in a US A Fr an ce US A? Iso la te / sp ec im en no . 17 2 (JD R) 11 4 (JD R) JF 05 15 4 YM J 90 08 19 14 YM J 3 87 YM J 1 38 10 12 (J DR ) CL L5 02 5 YM J 3 10 AR 47 99 b UC DD Ca sh 20 0 CB S 2 89 .8 7 DF M AL 10 0 ID co de Sq u Kc l Kd e Ab o Hf r Bc a Xp o Xs c Ac o As p Ds t El a Es c Sp ec ie s St ilb oh yp ox ylo n qu isq ui lia ru m Kr et zs ch m ar ia cl av us Kr et zs ch m ar ia d eu st a An nu lo hy po xy lo n bo ve i Hy po xy lo n fra gi fo rm e Bi sc og ni au xia ca pn od es Xy la ria p ol ym or ph a Xy la ria sc ru po sa An nu lo hy po xy lo n co ha er en s An nu lo hy po xy lo n sp . Di at ry pe st ig m a Eu ty pa la ta Eu ty pe lla sc op ar ia 188 Fig. 4.1. Cosmospora species. A. Cosmospora perithecia (reddish). B. Median section of Cosmospora perithecia (stained in Lactic Acid: yellow) and fruiting body of xylariaceous host (dark). Cosmospora perithecia growing directly above the host perithecia (empty spaces). A B 189 10 0 10 0 10 0 72 66 95 62 10 0 60 95 10 0 10 0 75 10 0 10 0 10 0 94 92 < 50 81 52 72 190 Fig. 4.2. Tanglegram between Cosmospora (red) and host (black) phylogenies. Solid lines between Cosmospora species and the associated host indicate host-parasite associations. ML bootstraps are provided for each node. See Tables 1 and 2 for abbreviations of taxa. Fig. 4.3. Procrustean superimposition plot of ordinations of Cosmospora Coordinates of patristic distances. The rotated and scaled to fit the fungal hosts for abbreviations of taxa. 191 Cosmospora and fungal hosts. and their fungal hosts are Principal Correspondence Cosmospora configuration (dots) has been ordination (arrow tips). See Tables 1 and 2 The 192 Figure 4.4. Contributions of individual host-parasite links to the Procrustean fit. Jacknifed squared residuals (bars) and upper 95% confidence intervals (error bars) resulting from applying PACo to patristic distances. Asterisks identify links significantly supported (α = 0.05) by ParaFitLink1. The median squared residual value is shown as a dashed line. See Tables 1 and 2 for abbreviations of taxa. Fig. 4.5. Reconciliation between isomorphic solutions with seven cospeciations, five duplica three losses (total cost = 13). The reconciliation of generated with Jane v.4. Blue and black lines represent hosts, respectively. Empty circles represent cospeciation switches; dash lines represent sorting taxa. 193 Cosmospora and host phylogenies. One of 263 tions & host-switches, and Cosmospora and host trees was Cosmospora and their fungal s; Arrows represent host events. See Tables 1 and 2 for abbreviations of - Fig. 4.6. Equally parsimonious reconciliation between phylogenies. One of 68 isomorphic solutions with six cospeciations, six duplications & host-switches, and one loss (total cost = 13). The reconciliation of host trees was generated with Jane v.4. Blue and black lines represent and their fungal hosts, respectively. Empty circles represent cospeciations; Arrows represent host-switches; dash lines represent sorting events. See Tables 1 and 2 for abbreviations of taxa. 194 Cosmospora and host Cosmospora Cosmospora and 195 Conclusions This dissertation represents one of the most comprehensive systematic works performed on the genus Cosmospora (in the broad sense) so far. It included more taxa than any other previous study. Many of which have been recognized as new species. Additionally, a few names (e.g. Cosmospora vilior) were stabilized with the selection of an epitype, which enabled determining correct classification of the fungus based on phylogeny. However, a high proportion of singleton lineages were also observed in the phylogenies. Singleton lineages do not conform to the species recognition concept (i.e., Genealogical Concordance Phylogenetic Species Recognition, GCPSR) used in this dissertation. By definition, a singleton lineage cannot be called a clade; a clade is composed by two or more specimens/isolates (reviewed in Vinuesa 2010). It was decided to recognize selected singleton lineages as species if the lineages were morphologically and/or ecologically distinct that sets them apart from other species (reviewed in Lim et al. 2012). The host of Cosmospora species, in particular, has been recognized in this dissertation to be a very informative character to diagnose Cosmospora species. Because Cosmospora species appear to have a high fidelity to their associated hosts, the recognized singleton species are likely to represent species. By recognizing singleton species, it was thought that it would make it easier to recognize the species again in nature, and fresh samples can be used to expand the morphological ranges of the species. 196 Two new genera, nine new combinations, and eleven new species were described in the taxonomic work included in this dissertation. Although progress has been made in increasing fungal diversity knowledge, diversity of cosmospora-like fungi is likely to be much higher that our current knowledge. The unrecognized singleton lineages, the many herbarium specimens, and fresh collections with new hosts are evidence that more work is needed. Unfortunately, extracting DNA from the fleshy insides of fruiting bodies stored in herbaria has been unsuccessful, and their phylogenetic position could not be determined. Either, the DNA of herbarium specimens has been degraded, or a better DNA extraction method needs to be found for this material. Additionally, DNA should be extracted from the fungal host for future collections of cosmospora-like fungi. It would provide a more accurate identity of the fungal hosts, and a better dataset for coevolutionary studies between Cosmospora species and their associated fungal hosts. Chapter 4 represents one of the first fungal studies studying evolutionary relationships between a mycoparasite and the associated fungal host (e.g., Currie et al. 2003; Millanes et al. 2014). The results suggested that host switch events were common early in the evolution of Cosmospora species, while cospeciation events more prevalent late in the evolution of Cosmospora species. This phylogenetic pattern is consistent with pseudocospeciation reported in other systems (e.g., Hafner and Nadler 1988; Charleston and Robertson 2002; Sorenson et al. 2004; Huyse and volckaert 2005). Given that cosmospora-like fungi represent the only group of fungi in the family Nectriaceae that have fungicolous, insecticolous, and lichenicolous life- 197 style habits, future work will probably focus on evolution of life of lifestyle habits at the molecular level. The data generated in this dissertation can be use to identify Cosmospora species. The dichotomous keys inside the dissertation are useful in the identification of Cosmospora species based on based on morphological and host characters. DNA sequences were deposited to GenBank, and unknown samples can be blasted against the deposited sequences. Sequence alignments were also deposited to a public online database, TreeBASE. Additionally, isolates were deposited to The Centraalbureau voor Schimmelcultures (CBS), Utrecht, The Netherlands, for long-term storage. CBS is a public fungal repository of strain cultures, and the strain cultures are available to all scientists for a relatively small fee that is attributed to the costs of handling and maintenance of cultures. New specimen collections were deposited to the U.S. National Fungus Collections (BPI), USDA-ARS, Beltsville, Maryland, and the specimens can be borrowed from BPI herbarium for study. 198 Bibliography Balbuena Díaz-Pinés JA, Míguez Lozano R, Blasco Costa MI. 2013. PACo: A Novel Procrustes Application to Cophylogenetic Analysis. Plos One 8(4): e61048. Banks JC, Palma RL, Paterson AM. 2006. Cophylogenetic relationships between penguins and their chewing lice. Journal of Evolutionary Biology 19(1): 156–166. Bazinet AL, Cummings MP. 2008. The Lattice Project: a grid research and production environment combining multiple grid computing models. In: Weber MHW, ed. Distributed & Grid Computing - Science Made Transparent for Everyone. Principles, Applications and Supporting Communities. Marburg.: Rechenkraft.net. p. 2–13. Bazinet AL, Cummings MP. Computing the Tree of Life: Leveraging the Power of Desktop and Service Grids. Parallel and Distributed Processing Workshops and Phd Forum (IPDPSW), 2011 IEEE International Symposium on; 16-20 May 2011 2011. p. 1896–1902. Bills GF, Platas G, Overy DP, Collado J, Fillola A, Jiménez MR, Martín J, del Val AG, Vicente F, Tormo JR, Peláez F, Calati K, Harris G, Parish C, Xu D, Roemer T. 2009. Discovery of the parnafungins, antifungal metabolites that inhibit mRNA polyadenylation, from the Fusarium larvarum complex and other Hypocrealean fungi. Mycologia 101 (4): 449–472. doi:10.3852/08-163. 199 Blackwell M. 2011. The Fungi: 1, 2, 3… 5.1 million species? American Journal of Botany 98 (3): 426–438. Booth C, Holliday P. 1973. Sphaerostilbe repens. Commonwealth Mycological Institute Descriptions Pathogenic Fungi and Bacteria 391: 1–2. Booth C. 1959. Studies of Pyrenomycetes: IV. Nectria (Part I). Mycol Pap 73: 1–115. Carbone I, Kohn LM. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553–556. Castlebury LA, Rossman AY, Sung GH, Hyten AS, Spatafora JW. 2004. Multigene phylogeny reveals new lineage for Stachybotrys chartarum, the indoor air fungus. Mycol Res 108: 864–872. Charleston MA, Robertson DL. 2002. Preferential Host Switching by Primate Lentiviruses Can Account for Phylogenetic Similarity with the Primate Phylogeny. Systematic Biology 51(3): 528–535. Charleston MA. 2011. TreeMap version 3.0β. Available from: http://sites.google.com/site/cophylogeny. Chaverri P, Liu M, Hodge KT. 2008. A monograph of the entomopathogenic genera Hypocrella, Moelleriella, and Samuelsia gen. nov. (Ascomycota, Hypocreales, Clavicipitaceae), and their aschersonia-like anamorphs in the Neotropics. Studies in Mycology 60: 1–66. 200 Chaverri P, Salgado C, Hirooka Y, Rossman AY, Samuels GJ. 2011. Delimitation of Neonectria and Cylindrocarpon (Nectriaceae, Hypocreales, Ascomycota) and related genera with Cylindrocarpon-like anamorphs. Studies in Mycology, 68: 57–78. Chaverri P, Samuels GJ. 2013. Evolution Of Habitat Preference And Nutrition Mode In A Cosmopolitan Fungal Genus With Evidence Of Interkingdom Host Jumps And Major Shifts In Ecology. Evolution 67(10): 2823–2837. Chaverri P, Vilchez B. 2006. Hypocrealean (Hypocreales, Ascomycota) Fungal Diversity in Different Stages of Tropical Forest Succession in Costa Rica. Biotropica. 38(4): 531–543. Chen W-J, Bonillo C, Lecointre G. 2003. Repeatability of clades as a criterion of reliability: a case study for molecular phylogeny of Acanthomorpha (Teleostei) with larger number of taxa. Molecular Phylogenetics and Evolution 26 (2):262–288. doi:http://dx.doi.org/10.1016/S1055-7903(02)00371-8. Clayton DH, Johnson KP. 2003. Linking coevolutionary history to ecological process: doves and lice. Evolution 57(10): 2335–2341. Clement M, Posada D, Crandall KA. 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology, 9: 1657–1659. Conow C, Fielder D, Ovadia Y, Libeskind-Hadas R. 2010. Jane: a new tool for the cophylogeny reconstruction problem. Algorithms for Molecular Biology 5(1): 16. Cooke MC. 1884. Notes on Hypocreaceae. Grevillea 12: 77–83. 201 Cozzi G, Stornelli C, Moretti A, Logrieco A, Porcelli F. 2002. Field evaluation of Fusarium larvatum formulations in the biocontrol of Saissetia oleae on olive in Apulia. Acta Horticulturae 586: 811–814. Cummings MP, Huskamp JC. 2005. Grid computing. Educause Review 40: 116–117. Cummings MP, Neel MC, Shaw KL. 2008. A genealogical approach to quantifying lineage divergence. Evolution, 62: 2411–2422. Darriba D, Taboada GL, Doallo R, Posada D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9 (8): 772–772. Davies TJ, Pedersen AB. 2008. Phylogeny and geography predict pathogen community similarity in wild primates and humans. Proceedings of the Royal Society B 275 (1643): 1695–1701. De Queiroz K. 2007. Species concepts and species delimitation. Syst Biol 56: 879– 886. De Vienne DM, Giraud T, Shykoff JA. 2007. When can host shifts produce congruent host and parasite phylogenies? A simulation approach. Journal of Evolutionary Biology 20(4): 1428–1438. De Vienne DM, Refrégier G, López-Villavicencio M, Tellier A, Hood ME, Giraud T. 2013. Cospeciation vs host-shift speciation: methods for testing, evidence from natural associations and relation to coevolution. New Phytologist 198(2): 347–385. 202 Donoghue MJ, Sanderson MJ. 1992. The suitability of molecular and morphological evidence in reconstructing plant phylogeny. In: Soltis PS, Soltis DE, Doyle JJ, eds. Molecular systematics of plants. Springer. p. 340–368. Fishbein M, Soltis DE. 2004. Further Resolution of the Rapid Radiation of Saxifragales (Angiosperms, Eudicots) Supported by Mixed-Model Bayesian Analysis. Systematic Botany 29 (4):883–891. doi:10.1600/0363644042450982. Ganassi S, Moretti A, Stornelli C, Fratello B, Pagliai AMB, Logrieco A, Sabatini MA. 2001. Effect of Fusarium, Paecilomyces and Trichoderma formulations against aphid Schizaphis graminum. Mycopathologia 151 (3): 131–138. Göker M, Scheuner C, Klenk H-P, Stielow JB, Menzel W. 2011. Codivergence of Mycoviruses with Their Hosts. PLoS ONE 6(7): e22252. Gräfenhan T, Schroers HJ, Nirenberg HI, Seifert KA. 2011. An overview of the taxonomy, phylogeny, and typification of nectriaceous fungi in Cosmospora, Acremonium, Fusarium, Stilbella, and Volutella. Studies in Mycology 68(1): 79–113. Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696–704. Hafner MS, Nadler SA. 1988. Phylogenetic trees support the coevolution of parasites and their hosts. Nature 332: 258–259. Hart MW, Sunday J. 2007. Things fall apart: biological species form unconnected parsimony networks. Biology Letters, 3: 509–512. 203 Hawksworth DL. 1991. The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycological research 95 (6): 641–655. Hawksworth DL. 2001. The magnitude of fungal diversity: the 1.5 million species estimate revisited. Mycological research 105 (12): 1422–1432. Herrera CS, Rossman AY, Samuels GJ, Chaverri P. 2013a. Pseudocosmospora, a new genus to accommodate Cosmospora vilior and related species. Mycologia 105(5): 1287–1305. Herrera CS, Rossman AY, Samuels GJ, Lechat C, Chaverri P. 2013b. Revision of the genus Corallomycetella with Corallonectria gen. nov. for C. jatrophae (Nectriaceae, Hypocreales). Mycosystema 32 (3): 518–544. Herrera CS, Rossman AY, Samuels GJ, Liparini Pereira O, Chaverri P. 2014. Phylogenetic and taxonomic revision of the Cosmospora viliuscula species complex: Cosmospora species that grow on xylariaceous fungi. In: Herrera CS, Systematics of the genus Cosmospora (Nectriaceae, Hypocreales), and cospeciation of Cosmospora species with their associated fungal hosts. [Ph.D. dissertation]. Universtiy of Maryland–College Park. Hillis DM, Wiens JJ. 2000. Molecules versus morphology in systematics: conflicts, artifacts, and misconceptions. In: Wiens JJ, ed. Phylogenetic analysis of morphological data. Smithsonian Institution Press, Washington, DC. p. 1–19. 204 Hirooka Y, Kobayashi T, Ono T, Rossman AY, Chaverri P. 2010. Verrucostoma, a new genus in the Bionectriaceae from the Bonin Islands, Japan. Mycologia 102: 418– 429. Hirooka Y, Rossman AY, Chaverri P. 2011. A morphological and phylogenetic revision of the Nectria cinnabarina species complex. Studies in Mycology 68 (1): 35–56. Hirooka Y, Rossman AY, Samuels GJ, Lechat C, Chaverri P. 2012. A monograph of Allantonectria, Nectria, and Pleonectria (Nectriaceae, Hypocreales, Ascomycota) and their pycnidial, sporodochial, and synnematous anamorphs. Studies in Mycology, 71: 1–210. Hosokawa T, Kikuchi Y, Nikoh N, Shimada M, Fukatsu T. 2006. Strict host- symbiont cospeciation and reductive genome evolution in insect gut bacteria. PLoS Biol 4(10): e337. Hsieh H-M, Lin C-R, Fang M-J, Rogers JD, Fournier J, Lechat C, Ju Y-M. 2010. Phylogenetic status of Xylaria subgenus Pseudoxylaria among taxa of the subfamily Xylarioideae (Xylariaceae) and phylogeny of the taxa involved in the subfamily. Molecular Phylogenetics and Evolution 54(3): 957–969. Hughes J, Kennedy M, Johnson KP, Palma RL, Page RDM. 2007. Multiple Cophylogenetic Analyses Reveal Frequent Cospeciation between Pelecaniform Birds and Pectinopygus Lice. Systematic Biology 56(2): 232–251. 205 Huyse T, Volckaert FAM. 2005. Comparing host and parasite phylogenies: Gyrodactylus flatworms jumping from goby to goby. Systematic Biology 54 (5): 710–718. Itino T, Davies SJ, Tada H, Hieda Y, Inoguchi M, Itioka T, Yamane S, Inoue T. 2001. Cospeciation of ants and plants. Ecological Research 16(4): 787–793. Jackson AP, Machado CA, Robbins N, Herre EA. 2008. Multi-locus phylogenetic analysis of neotropical figs does not support co-speciation with the pollinators: The importance of systematic scale in fig/wasp cophylogenetic studies. Symbiosis 45(1- 3): 57–72. Jackson AP. 2004. A Reconciliation Analysis Of Host Switching In Plant-Fungal Symbioses. Evolution 58(9): 1909–1923. Jang J-H, Lee JH, Ki C-S, Lee NY. 2012. Identification of Clinical Mold Isolates by Sequence Analysis of the Internal Transcribed Spacer Region, Ribosomal Large- Subunit D1/D2, and β-Tubulin. Ann Lab Med, 32: 126–132. Katoh K, Misawa K, Kuma Ki, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30(14): 3059–3066. Katoh K, Standley DM. 2013. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30(4): 772–780. 206 Katoh K, Toh H. 2008. Recent developments in the MAFFT multiple sequence alignment program. Brief Bioinform 9: 286–298. Keller-Schmidt S, Wieseke N, Klemm K, Middendorf M. 2011. Evaluation of host parasite reconciliation methods using a new approach for cophylogeny generation. Working paper from Bioinformatics Leipzig. Available from http://www.bioinf.uni- leipzig.de/working/11-013. Kelly CMR, Barker NP, Villet MH, Broadley DG. 2009. Phylogeny, biogeography and classification of the snake superfamily Elapoidea: a rapid radiation in the late Eocene. Cladistics 25 (1):38–63. doi:10.1111/j.1096-0031.2008.00237.x. Lanfear R, Calcott B, Ho SYW, Guindon S. 2012. PartitionFinder: Combined Selection of Partitioning Schemes and Substitution Models for Phylogenetic Analyses. Molecular Biology and Evolution 29(6): 1695–1701. Lanterbecq D, Rouse GW, Eeckhaut I. 2010. Evidence for cospeciation events in the host-symbiont system involving crinoids (Echinodermata) and their obligate associates, the myzostomids (Myzostomida, Annelida). Molecular Phylogenetics and Evolution 54(2): 357–371. Legendre P, Desdevises Y, Bazin E. 2002. A Statistical Test for Host-Parasite Coevolution. Systematic Biology 51(2): 217–234. Leigh JW, Susko E, Baumgartner M, Roger AJ. 2008. Testing congruence in phylogenomic analysis. Syst Biol 57: 104–115. 207 Light JE, Hafner MS. 2008. Codivergence in heteromyid rodents (Rodentia: Heteromyidae) and their sucking lice of the genus Fahrenholzia (Phthiraptera: Anoplura). Systematic biology 57 (3): 449–465. Lim GS, Balke M, Meier R. 2011. Determining species boundaries in a world full of rarity: singletons, species delimitation methods. Syst Biol 61:165–169. Luo J, Zhuang W-Y. 2008. Two new species of Cosmospora (Nectriaceae, Hypocreales) from China. Fung Divers 31: 83–93. Luo J, Zhuang W-Y. 2010. Chaetopsinectria (Nectriaceae, Hypocreales), a new genus with Chaetopsina anamorphs. Mycologia 102(4): 976–984. Luo J, Zhuang W-Y. 2012. Volutellonectria (Ascomycota, Fungi), a new genus with Volutella anamorphs. Phytotaxa 44: 1–10. Maddison WP, Maddison DR. 2011. Mesquite: a modular system for evolutionary analysis. Version 2.75. Available from http://mesquiteproject.org. Marussich WA, Machado CA. 2007. Host-specificity and coevolution among pollinating and nonpollinating New World fig wasps. Molecular Ecology 16(9): 1925–1946. McNeill J, Barrie FR, Buck WR, Demoulin V, Greuter W, Hawksworth DL, Herendeen PS, Knapp S, Marhold K, Prado J. 2012. International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). Köeningstein, Germany: Koeltz Scientific Books. 208 Merkle D, Middendorf M, Wieseke N. 2010. A parameter-adaptive dynamic programming approach for inferring cophylogenies. BMC bioinformatics 11(Suppl 1): S60. Millanes AM, Truong C, Westberg M, Diederich P, Wedin M. 2014. Host switching promotes diversity in host-specialized mycoparasitic fungi: uncoupled evolution in the Biatoropsis-Usnea system. Evolution Forthcoming. Moravec Z. 1954. Dialonectria cosmariospora v Československu. Ceska Mykol 8: 92–95. Mostert L, Halleen F, Creaser M, Crous P. 2004. Cryptovalsa ampelina, a forgotten shoot and cane pathogen of grapevines. Australas Plant Pathol 33: 295–299. doi:10.1071/AP03095. Nikoh N, Fukatsu T. 2000. Interkingdom host jumping underground: phylogenetic analysis of entomoparasitic fungi of the genus Cordyceps. Molecular Biology and Evolution 17 (4): 629–638. Nirenberg HI. 1976. Untersuchungen über die morphologische und biologische Differenzierung in der Fusarium-Section Liseola. Mitt Biol Bund Land-Forstwirt Berlin-Dahlem 169: 1–117. Norton DA, Carpenter MA. 1998. Mistletoes as parasites: host specificity and speciation. Trends in Ecology & Evolution 13 (3): 101–105. Norvell, L. 2011. Fungal nomenclature. 1. Melbourne approves a new Code. Mycotaxon 116: 481–490. 209 Novicki TJ, LaFe K, Bui L, Bui U, Geise R, Marr K, Cookson BT. 2003. Genetic diversity among clinical isolates of Acremonium strictum determined during an investigation of a fatal mycosis. Journal of Clinical Microbiology, 41: 2623–2628. O'Donnell K, Cigelnik E. 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7: 103–116. Oda S, Kitade O, Inoue T, Kawai M, Kanuka M, Hiroshima K, Hongoh Y, Constantino R, Uys V, Zhong J, Kudo T, Ohkuma M. 2007. Cospeciation in the triplex symbiosis of termite gut protists (Pseudotrichonympha spp.), their hosts, and their bacterial endosymbionts. Molecular Ecology 16(6): 1257–1266. Page RDM. 2003. Tangled trees: Phylogeny, cospeciation, and coevolution. University of Chicago Press. Paradis E, Claude J, Strimmer K. 2004. APE: analyses of phylogenetics and evolution in R language. Bioinformatics 20(2): 289–290. Paterson AM, Banks J. 2001. Analytical approaches to measuring cospeciation of host and parasites: through a glass, darkly. International Journal for Parasitology 31(9): 1012–1022. Peterson KR, Pfister DH, Bell CD. 2010. Cophylogeny and biogeography of the fungal parasite Cyttaria and its host Nothofagus, southern beech. Mycologia, 102: 1417–1425. 210 Pons JM, Hassanin A, Crochet PA. 2005. Phylogenetic relationships within the Laridae (Charadriiformes: Aves) inferred from mitochondrial markers. Molecular Phylogenetics and Evolution 37 (3):686–699. doi:http://dx.doi.org/10.1016/j.ympev.2005.05.011. Porcelli F, Frisullo S. 1998. The fungus Nectria aurantiicola Berk. et Br. (Fusarium larvarum Fuckel) a biocontrol agent of the armored scale Suturaspis archangelskyae (Lindiger) in Apulia (Southern Italy). Entomologica 32: 109–119. Posada D, Crandall KA. 2001. Intraspecific gene genealogies: trees grafting into networks. Trends in Ecology & Evolution, 16: 37–45. Posada D. 2008. jModelTest: Phylogenetic model averaging. Mol Biol Evol 25: 1253–1256. Pringle A, Baker DM, Platt JL, Wares JP, Latgé JP, Taylor JW. 2005. Cryptic speciation in the cosmopolitan and clonal human pathogenic fungus Aspergillus fumigatus. Evolution, 59: 1886–1899. R Core Team. 2013. R: A language and environment for statistical computing. Vienna, Austria: R foundation for Statistical Computing. Available from: http://www.R-project.org. Rambaut A, Drummond AJ. 2013. Tracer v1.5. Available from: http://beast.bio.ed.ac.uk/Tracer. Rayner RW. 1970. A Mycological Colour Chart. Commonwealth Mycological Institute, Kew, Surrey. 211 Reed DL, Light JE, Allen JM, Kirchman JJ. 2007. Pair of lice lost or parasites regained: the evolutionary history of anthropoid primate lice. BMC Biol 5 (7). doi:10.1186/1741-7007-5-7. Rehner SA, Samuels GJ. 1995. Molecular systematics of the Hypocreales: a teleomorph gene phylogeny and the status of their anamorphs. Canadian Journal of Botany 73(S1): 816–823. Ridley ME. 2007. Evolution. New Delhi; New York: Oxford University Press. Rogers JD, Ju Y-M. 1998. The genus Kretzschmaria. Mycotaxon 68: 345–393. Rogerson CT. 1970. The Hypocrealean Fungi (Ascomycetes, Hypocreales). Mycologia 62 (5): 865–910. doi:10.2307/3757604. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 61: 539–542. Rossman AY. 1983. The phragmosporous species of Nectria and related genera. Kew, Surrey: Commonwealth Mycological Institute. Rossman AY. 1993. Holomorphic hypocrealean fungi: Nectria sensu stricto and teleomorphs of Fusarium. In: Reynolds DR, Taylor JW, eds. The fungal holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematic. CAB International, Wallingford, UK. p. 149–160. 212 Rossman AY. 1999. Genera of Bionectriaceae, Hypocreaceae and Nectriaceae (Hypocreales, Ascomycetes). Studies in Mycology 42: 1–248. Rossman AY. 2001. Molecular studies of the Bionectriaceae using large subunit rDNA sequences. Mycologia 93: 100–110. Samuels GJ, Dodd SL, Lu B-S, Petrini O, Schroers H-J, Druzhinina IS. 2006. The Trichoderma koningii aggregate species. Studies in Mycology, 56: 67–133. Samuels GJ, Doi Y, Rogerson CT. 1990. Hypocreales. Mem New York Bot Gard 59: 6–108. Samuels GJ, Dumont KP. 1982. The genus Nectria (Hypocreaceae) in Panama. Caldasia,13: 379–423. Samuels GJ, Lu B-S, Chaverri P, Candoussau F, Fournier J, Rossman A. 2009. Cyanonectria, a new genus for Nectria cyanostoma and its Fusarium anamorph. Mycol Prog 8: 49–58. doi:10.1007/s11557-008-0577-x. Samuels GJ, Rossman AY, Lowen RL, CT R. 1991. A Synopsis of Nectria subgen. Dialonectria. 164: 1–48. Samuels GJ, Seifert KA. 1987. Taxonomic implications of variation among hypocrealean anamorphs. In: Sugiyama J, ed. Pleomorphic Fungi: The diversity and its taxonomic implications. New York, NY: Elsevier. p. 29–56. Samuels GJ. 1973. The genus Macbridella with notes on Calostilbe, Herpotrichia, Phaeonectria, and Letendraea. Canadian Journal of Botany, 51: 1275–1283. 213 Schmitt I, Crespo A, Divakar PK, Fankhauser JD, Herman-Sackett E, Kalb K, Nelsen MP, Nelson NA, Rivas-Plata E, Shimp AD, Widhelm T, Lumbsch HT. 2009. New primers for promising single-copy genes in fungal phylogenetics and systematics. Persoonia 23: 35–40. doi:10.3767/003158509x470602. Schoch CL, Crous PW, Wingfield MJ, Wingfield BD. 2000. Phylogeny of Calonectria and selected hypocrealean genera with cylindrical macroconidia. Studies in Mycology 45: 45–62. Schuh RT. 2009. Biological Systematics: principles and applications (2nd ed.). Ithaca, NY: Cornell University Press. Seifert KA. 1985. A monograph of Stilbella and some allied Hyphomycetes. Studies in Mycology, 27: 1–235. Seo C, Sohn JH, Oh H, Kim BY, Ahn JS. 2009. Isolation of the protein tyrosine phosphatase 1B inhibitory metabolite from the marine-derived fungus Cosmospora sp. SF-5060. Bioorganic & Medicinal Chemistry Letters 19 (21): 6095–6097. doi:http://dx.doi.org/10.1016/j.bmcl.2009.09.025. Siebert JB. 2001. Eutypa: The economic toll on vineyards. Wines Vines (Apri): 50– 56. Sorenson MD, Balakrishnan CN, Payne RB. 2004. Clade-limited colonization in brood parasitic finches (Vidua spp.). Systematic Biology 53 (1): 140–153. 214 Summerbell RC, Gueidan C, Schroers HJ, de Hoog GS, Starink M, Rosete YA, Guarro J, Scott JA. 2011. Acremonium phylogenetic overview and revision of Gliomastix, Sarocladium, and Trichothecium. Studies in Mycology 68 (1):139–162. Sung G-H, Hywel-Jones NL, Sung J-M, Luangsa-ard JJ, Shrestha B, Spatafora JW. 2007. Phylogenetic classification of Cordyceps and the clavicipitaceous fungi. Stud Mycol 57: 5–59. Tang AMC, Jeewon R, Hyde KD. 2009. A re-evaluation of the evolutionary relationships within the Xylariaceae based on ribosomal and protein-coding gene sequences. Fungal Diversity 34: 127–155. Taylor JW, Berbee ML. 2006. Dating divergences in the Fungal Tree of Life: review and new analyses. Mycologia 98(6): 838–849. Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC. 2000. Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 31: 21–32. Templeton AR. 2001. Using phylogeographic analyses of gene trees to test species status and processes. Molecular Ecology,10: 779–791. Tsuneda A. 1982. Nectria episphaeria, a mycoparasite of Hypoxylon truncatum. Reports of the Tottori Mycological Institute 20: 42–46. Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172: 4238–4246. 215 Vinuesa P. 2010. Multilocus Sequence Analysis and Bacterial Species Phylogeny Estimation. In: Oren A, Papke RT, eds. Molecular Phylogeny of Microorganisms. Caister Academic Press, Norfolk, UK. p. 41–64. Weese J. 1916. Beiträge zur Kenntnis der Hypocreaceen. Sitzungsberichte der Kaiserlichen Akademie der Wissenschaften. Mathematisch-Naturwissenschaftliche Klasse 125: 465–575. White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Shinsky JJ, White TJ, eds. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, California, U.S.A. p. 315–322. Wollenweber HW. 1930. Fusaria autographica delineata. Berlin, published by the author. 658–1100. Zaffarano PL, McDonald BA, Linde CC. 2008. Rapid Speciation Following Recent Host Shifts In The Plant Pathogenic Fungus Rhynchosporium. Evolution 62(6): 1418–1436. Zhang XM, Zhuang WY. 2006. Phylogeny of some genera in the Nectriaceae (Hypocreales,Ascomycetes) inferred from 28S nrDNA partial sequences. Mycosystema 25: 15–22. Zwickl DJ. 2006. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. [Ph.D. dissertation]. The University of Texas at Austin.