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Persoonia 22, 2009: 129–138 www.persoonia.org RESEARCH ARTICLE doi:10.3767/003158509X461486

Geoglossomycetes cl. nov., Geoglossales ord. nov. and taxa above class rank in the Tree of Life

C.L. Schoch1, Z. Wang2, J.P. Townsend2, J.W. Spatafora3

Key words Abstract Featuring a high level of taxon sampling across Ascomycota, we evaluate a multi-gene phylogeny and propose a novel order and class in Ascomycota. We describe two new taxa, Geoglossomycetes and Geoglossales, Bayesian inference to host three earth tongue genera: Geoglossum, Trichoglossum and Sarcoleotia as a lineage of ‘Leotiomyceta’. hybrid classification Correspondingly, we confirm that these genera are not closely related to the genera Neolecta, Mitrula, Cudonia, maximum likelihood Microglossum, Thuemenidum, Spathularia and Bryoglossum, all of which have been previously placed within the . We also propose a non-hierarchical system for naming well-resolved nodes, such as ‘Saccharo- myceta’, ‘Dothideomyceta’, and ‘Sordariomyceta’ for supraordinal nodes, within the current phylogeny, acting as rankless taxa. As part of this revision, the continued use of ‘Leotiomyceta’, now as a rankless taxon, is proposed.

Article info Received: 16 April 2009; Accepted: 11 May 2009; Published: 5 June 2009.

INTRODUCTION The proposed informal, rankless names for well-supported clades above the class level in our phylogeny agrees with the The multi-gene sequence datasets generated by the research principles of the Phylocode (http://www.ohio.edu/phylocode/). consortium ‘Assembling the Fungal Tree of Life’ (AFTOL) It is our hope that such names should function as rankless have resulted in several multi-gene phylogenies incorporating taxa, facilitating the naming of additional nodes/clades as they comprehensive taxon sampling across Fungi (Lutzoni et al. become resolved. Eventual codification will follow the example 2004, Blackwell et al. 2006, James et al. 2006). AFTOL gener- of Hibbett et al. (2007) by applying principles of type names ated a data matrix spanning all currently accepted classes in and priority. A number of published manuscripts already provide the Ascomycota, the largest fungal phylum. The phylogenies background on other supraordinal relationships of Fungi; for produced by AFTOL prompted the proposal of a phylogenetic more complete treatments of the various classes, see Blackwell classification from phylum to ordinal level in fungi (Hibbett et et al. (2006). al. 2007). Although the Botanical Code does not require the During the AFTOL project a data matrix was generated span- principle of priority in ranks above family (McNeill et al. 2006), ning all currently accepted classes in the Ascomycota, the this principle was nevertheless followed for all taxa. The fol- largest fungal phylum. A multi-gene phylogeny was recently lowing ranked taxa were defined: subkingdom, phylum (suffix inferred from these data, demonstrating relevant patterns in -mycota, except for Microsporidia), subphylum (-mycotina), biological and morphological character development as well as class (-mycetes), subclass (-mycetidae) and order (-ales). As establishing several distinct lineages in Ascomycota (Schoch in Hibbett et al. (2007), several phylogenetically well-supported et al. 2009). Here we test whether the relationships reported in nodes above the rank of order could not be accommodated Schoch et al. (2009) remain valid by applying both maximum in the current hierarchical classification system based on the likelihood (ML) and Bayesian analyses on a more restricted International Code of Botanical Nomenclature. To remedy this but denser set of taxa, including expanded sampling in the deficiency, rankless (or unranked) taxa for unambiguously Geoglossaceae. resolved nodes with strong statistical support was proposed (Hibbett & Donoghue 1998). Hybrid classifications that include We will therefore address the taxonomic placement of a group both rankless and Linnaean taxa have since been discussed of fungi with earth tongue morphologies that are shown to be elsewhere (Jørgensen 2002, Kuntner & Agnarsson 2006), and unrelated to other known classes. This morphology is closely applied to diverse organisms from (Stenroos et al. 2002) associated with the family Geoglossaceae (Corda 1838). With and plants (Sennblad & Bremer 2002, Pfeil & Crisp 2005) to typical inoperculate asci and an exposed hymenium, Geoglos­ spiders (Kuntner 2006). These studies all attempt to create a saceae has long been thought to be a member of Leotiomy­ comprehensive code for phylogenetic nomenclature that retains cetes, though the content of the family itself has experienced the current Linnean hierarchical codes. many changes (Nannfeldt 1942, Korf 1973, Spooner 1987, Platt 2000, Wang et al. 2006a, b). It is currently listed with 48 In keeping with the practice of previous hybrid classifications, we and 6 genera in the Dictionary of the Fungi (Kirk et propose to use names corresponding to clades of higher taxa al. 2008). Several analyses using molecular data supported that were resolved in this phylogeny as well as preceding studies. a clade including three earth tongue genera, Geoglossum, Trichoglossum and Sarcoleotia (Fig. 1), and cast doubt upon 1 National Center for Biological Information (GenBank), National Library of Medicine, National Institute of Health, 45 Center Drive, MSC 6510, their positions in (Platt 2000, Gernandt et al. Bethesda, Maryland 20892-6510, USA; 2001, Lutzoni et al. 2004, Sandnes 2006, Spatafora et al. 2006, corresponding author e-mail: [email protected] Wang et al. 2006b). Here we present a comprehensive phylum- 2 Department of Ecology and Evolutionary Biology, Yale University, 165 wide phylogeny, including data from protein coding genes. We Prospect Street, New Haven, CT 06520, USA. 3 Department of Botany and Plant Pathology, Oregon State University, can confidently place the earth tongue family as separate from Corvallis, OR 97331, USA. currently accepted classes in Ascomycota.

© 2009 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 130 Persoonia – Volume 22, 2009

91 92 ‘Dothideo- myceta’ 6299 Eurotiomycetes 72 Lecanoromycetes Lichinomycetes Sordariomycetes Laboulbeniomycetes Monilinialaxa

5380 Botryotinia fuckeliana 7299 Lambertella subrenispora Mitrula elegans 99 Mitrula brevispora A Cudoniella clavus 35 100 78 98Lachnum virgineum 9092 ‘Sordario- Bryoglossum gracile Le

89Neofabrea malicorticis o myceta’ 85 tiom B Dermea acerina

Microglossum olivaceum y cetes lubrica

100 Thuemenidium atropurpureum 1 Thuemenidium atropurpureum 2 ‘Leotio- 52 Microglossum rufum 1 myceta’ 8492 A Microglossum rufum 2 61 C Cudonia circinans Spathularia velutipes 1 B Spathularia velutipes 2 Neobulgaria pura 58 99

Sarcoleotia globosa 1 Ge

Sarcoleotia globosa 2 C o gl

Sarcoleotia globosa 3 o Pezizo- Trichoglossum hirsutum 1 ss D o mycotina Trichoglossum hirsutum 2 m D y

Geoglossum umbratile ce ‘Saccharo- 10088 t myceta’ Geoglossum nigritum E es Asco- Geoglossum glabrum mycota Orbiliomycetes Saccharomycotina Saccharo- 74 72 mycetes E

0.1 Taphrinomycotina Taphrinomycetes substi- Neolectomycetes tutions Schizosaccharomycetes per site Basidiomycota

Fig. 1 A most likely tree obtained by RAxML for Ascomycota. Subphyla, class and rankless taxa are indicated. Classes containing fungi designated as earth tongues are indicated in black. The tree was rooted with outgroup Rhizopus oryzae (not shown). Bootstrap values are shown in orange and Bayesian poste- rior probabilities in blue. Orange, bold branches are supported by more than 80 % bootstrap and 95 % posterior probability, respectively. The full phylogeny, without collapsed clades, are shown in Fig. 2. The inset figures illustrate morphological ascomal diversity in the earth tongues. The species are as follows: A. Trichoglossum hirsutum; B. Geoglossum nigritum; C. Microglossum rufum; D. Spathularia velutipes; E. Geoglossum nigritum. Photo credits: A: Zhuliang Yang; B, D, E: Kentaro Hosaka; C: Dan Luoma.

MATERIALS AND METHODS deposited in GenBank from two previous studies (Wang et al. 2006a, b). To further minimise poorly aligned areas, 219 ad- Data were extracted from the complete data matrix obtained ditional columns, which proved variable when viewed in BioEdit from the WASABI database (www.aftol.org), incorporating with a 40 % shade threshold, were excluded from the original representatives for all currently accepted classes, and maxi- AFTOL inclusion set. The refined dataset consisted of 161 taxa mizing the number of orders and available data. Following the (including outgroups) and 4 429 characters for six different approach of James et al. (2006) we performed a combined loci: the nuclear small and large ribosomal subunits (nSSU, analysis, with both DNA and amino acid data, while allowing nLSU), the mitochondrial small ribosomal subunit (mSSU) and for missing data. This data was supplemented with additional fragments from three proteins: transcription elongation factor ribosomal sequences from earth tongue genera obtained and 1 alpha (TEF1) and the largest and second largest subunits of C.L. Schoch et al.: Geoglossomycetes 131

RNA polymerase II (RPB1, RPB2). A complete table with the is found in all ML bootstrap trees, suggesting that this taxon is published GenBank numbers is listed in Table 1. unstable in our analyses. No conflicts were detected in Neob­ The phylogenetic analysis was run in RAxML v7.0.0 (Stama- ulgaria genes under a previous study and missing data did not takis 2006), partitioning by gene (six partitions) and estimating affect important nodes (Schoch et al. 2009). A repeat run under unique model parameters for each gene, as in Schoch et al. maximum likelihood was done with Neobulgaria pura removed (2009). Models of evolution were evaluated as in Schoch et al. under the same settings but with only 100 bootstrap repetitions. (2009) with the same models selected. For DNA sequences, This trimmed dataset yielded a congruent phylogeny with in- this resulted in a general time reversible model (GTR) with a creased bootstrap for Leotiomycetes (78 %; data not shown). discrete gamma distribution composed of four rate classes The instability of the placement of Neobulgaria pura does not plus an estimation of the proportion of invariable sites. The compromise any of the conclusions we present here and may amino acid sequences were analysed with a RTREV model be due to various reasons. Improved taxon sampling will likely with similar accommodation of rate heterogeneity across sites help to resolve its placement in future analyses. and proportions of invariant sites. In addition, protein models for We find support for numerous backbone nodes in Ascomycota, TEF1 and RPB2 incorporated a parameter to estimate amino as did Schoch et al. (2009). Our phylum-wide sampling of acid frequencies. The tree shown in Fig. 1 was obtained by Ascomycota classes in this study, combined with the results using an option in RAxML running a rapid bootstrap analysis of a previous study (Schoch et al. 2009), facilitated addressing and search for the best-scoring ML tree in one single run. This the placement of the previously problematic and unsampled meant the GTRCAT model approximation was used, which does lineages such as the Geoglossaceae in relation to all currently not produce likelihood values comparable to other programs. accepted Ascomycota classes. The full tree is shown here as Fig. 2 and was deposited in TreeBASE (www.treebase.org). We also ran 100 repetitions of RAxML under a gamma rate distribution option. The best scoring tree was included in TreeBASE. Given their unique ascomatal development, ultrastructure of ascus apical apparatus, mossy habitat, and our multilocus gene A second analysis was run using Bayesian inference of maxi- phylogeny, Geoglossomycetes cl. & ord. nov. is justified here as mum likelihood in MrBayes v3.1.2 (Huelsenbeck & Ronquist in and ‘Leotiomyceta’. 2001, Altekar et al. 2004) using models and parameters that were comparable to the maximum likelihood run. Data were Geoglossomycetes, Geoglossales Zheng Wang, C.L. Schoch similarly partitioned and amino acids were analysed, so that & Spatafora, cl. & ord. nov. — MycoBank MB513351, a mixture of models with fixed rate matrices for amino acid MB513352 sequences could be evaluated. In all cases rate heterogeneity parameters were used by a discrete gamma distribution plus Ascomata solitaria vel gregaria, capitata, stipitata; stipe cylindricus, atrum, an estimation of the proportion of invariable sites. A metropolis glabrum vel furfuraceus. Regio hymeniali capitata, clavata vel pileata, in- coupled Markov Chain Monte Carlo analysis was run for 9 distinctum ex stipite; hymenium atrum, continuatcum stipite ad praematuro incrementi grado. Asci clavati, inoperculati, octospori, poro parvo in iodo million generations sampling every 200th cycle, starting from caerulescentes. Ascosporae elongatae, fuscae, pullae vel hyalinae, multi- a random tree and using 4 chains (three heated and one cold) septatae. Paraphyses filiformes, pullae vel hyalinae. Distributio generalis, under default settings. Two separate runs were confirmed to terrestris, habitaile locus fere uliginoso et muscoso. converge using Tracer v1.4.1 (http://tree.bio.ed.ac.uk/software/ tracer/). The first 10 000 sampled trees (2 million generations) Type . Geoglossum Pers., Neues Mag. Bot. 1: 116. 1794; Geoglos­ saceae. were removed as burn in each run. A 50 % majority rule con- sensus tree of 70 000 Bayesian likelihood trees from the two Ascomata scattered to gregarious, capitate, stipitate; stipe cylin- combined runs was subsequently constructed, and average drical, black, smooth to furfuraceous. Ascigerous portion capitate, branch lengths and posterior probabilities determined. The club-shaped to pileate, indistinguishable from stipe. Hymenium numbers of nodes shared with the most likely tree in Fig. 1 was surface black, continues with stipe at early development stage. determined and plotted on the branches. This tree was depos- Asci clavate, inoperculate, thin-walled, J+, usually 8-spored. ited in TreeBASE, along with the inclusive character set. Ascospores elongate, dark-brown, blackish to hyaline, septate when mature. Paraphyses filiform, blackish to hyaline. Global RESULTS distribution, terrestrial, habitat usually boggy and mossy.

The phylogeny presented in Fig. 1 supports 15 classes (11 in DISCUSSION Pezizomycotina, 1 in Saccharomycotina, 3 in Taphrinomycotina) with good statistical support (both ML bootstrap and Bayesian In keeping with the phylogeny presented in Fig. 1, we endorse posterior probability) for 14. Phylogenies with all lineages in the use of the -myceta suffix in order to circumscribe well-sup- analysed data matrix are included in Fig. 2. A run with 100 repeti- ported clades above class. The numbers of these clades are tions of RAxML under a gamma rate distribution option resulted limited, and the use of such taxa will continue to become more in a best scoring tree with a log likelihood of -111983. This tree practical as our biological knowledge base broadens. Use of shared the same supported nodes with the one presented in this suffix will also allow for the continued use of Leotiomyceta, Fig. 1 but had changes in poorly supported nodes regarding a taxon that has already been defined with a Latin diagnosis placement of the Eurotiomycetes and Dothideomycetes. The provided as a ranked superclass (Eriksson & Winka 1997) and two Bayesian runs produced trees with harmonic means of remains in use (Lumbsch et al. 2005, Wang et al. 2006a). We likelihood values of -112094 and -112076, respectively, with propose its continued use, but as a rankless taxon together similar topological differences in poorly supported nodes. with the newly proposed rankless taxa, ‘Saccharomyceta’, As can be seen in Fig. 1, we continue to find low bootstrap and ‘Dothideomyceta’ and ‘Sordariomyceta’. Since these taxa are posterior probability support for Leotiomycetes as a mono­ not currently accepted under the Code (McNeill et al. 2006), phyletic clade using a combined analysis of protein and nucleic we will refrain from formal designations. The relevant clades acids. In our analysis, this includes Neobulgaria pura as the are discussed below with the informal designations indicated earliest diverging lineage. The node internal from this lineage in single quotations. 132 Persoonia – Volume 22, 2009

96 Epichloë typhina 100 68 Claviceps purpurea 99 Elaphocordyceps capitata 80 Elaphocordyceps ophioglossoides Hypocreales 100 35 Cordyceps cardinalis 99 74 67 Nectria cinnabarina 100 100 Hypocrea lutea 90 100 Bionectria cf. aureofulva Melanospora tiffanyae Melanosporales Bertia moriformis Coronophorales 99 100 Gondwanamyces capensis 99 Ceratocystis fimbriata 53 100 Microascales 80 Halosphaeria appendiculata 94 100 Corollospora maritima Glomerella cingulata Verticillium dahliae Cryptosporella hypodermia 59 Gnomonia gnomon 100 Diaporthales Endothia gyrosa Sordariomycetes 98 Diaporthe eres 100 64 Calosphaeria pulchella Calosphaeriales herbarum 99 Magnaporthe grisea Magnaporthales heterostrophus 91 86 Gaeumannomyces medullaris 100 46 Allewia eureka 100 Ophiostoma stenoceras 87 Ophiostomatales 100 43 phaeocomes 52 Sordaria fimicola 100 Sordariales maculans Neurospora crassa herbarum 91 Xylaria hypoxylon 87 Xylariales 100 pisi 92 Lindra thalassiae Pleosporales Lulworthiales 45 minima Lulworthia grandispora 68 cylindrica 100 l l Stigmatomyces protrudens a’ siparia t 100 92 Hesperomyces virescens Laboulbeniales 92 38 nicotiae Pyxidiophora avernensis Pyxidiophorales 99 80 53 bilgramii Botryotinia fuckeliana 100 100 es enalia

t

a’ Monilinia laxa winteri

‘Leotio- myce t 72 Lambertella subrenispora Gloniopsis smilacis 99 97 Mitrula brevispora 87 100 Hysterographium mori Hysteriales 37 Mitrula elegans 100 100

aboulbenio- 97 100 Hysterium angustatum 90 Bryoglossum gracile

L 78 myce ‘Sordario- myce 42 Mytilinidion australe 92 98 35 90 Lachnum virgineum 40 Mytilinidion rhenanum Mytilinidiales 100100 Cudoniella cf. clavus 93 mytilinum Dermea acerina 95 94 Botryosphaeria tsugae 92 Neofabraea malicorticis 100 Leotiomycetes 100 Botryosphaeria dothidea 22 84 Microglossum rufum 2 Guignardia gaultheriae Botryosphaeriales 85 92 41 Microglossum rufum 1 74 Guignardia bidwellii Dothideomycetes 52 Rhytismatales 98 99 Thueminidium atropurpureum 1 paludosa 100 87 Thueminidium atropurpureum 2 34 100 Helicomyces roseus 100 Leotia lubrica 95 93 98 Mycosphaerella punctiformis 58 Microglossum olivaceum 100 Mycosphaerella graminicola

99 a’ 61 Spathularia velutipes 1 t 50 Mycosphaerella fijiensis 9 Spathularia velutipes 2 93 92 Capnodium coffeae Capnodiales Cudonia circinans 100 Scorias spongiosa 91 Neobulgaria pura Cladosporium cladosporioides 88 ‘Dothideo- 92 Geoglossum glabrum myce Davidiella tassiana 100 Geoglossum nigritum 91 92 92 Dothiora cannabinae Geoglossum umbratile Geoglossomycetes, cl. nov. 100 Dothidea sambuci Dothideales Trichoglossum hirsutum 1 79 Sydowia polyspora Trichoglossum hirsutum 2 Geoglossales, ord. nov. 100 Myriangium duriaei Myriangiales 99 Sarcoleotia globosa 2

Pezizo- mycotina Elsinoë veneta 9799 Sarcoleotia globosa 3 Schismatomma decolorans 100 Arthoniomycetes Sarcoleotia globosa 1 Roccella fuciformis Arthrobotrys elegans Orbiliomycetes cretacea Orbilia vinosa Orbiliales Lobaria scrobiculata 93 Aleuria aurantia 98 Lobariella pallida a’ 100 100 t Scutellinia scutellata 99 Peltigera degenii 98 100 Pyronema domesticum 94 Nephroma parile 99100 20 100 Eleutherascus lectardii 71 37 100 80 Coccocarpia erythroxyli Sarcosoma latahense 49 Lecidea fuscoatra

‘Saccharo- myce Gyromitra californica Pezizomycetes 54 Hypogymnia physodes 98 Verpa conica Canoparmelia caroliniana Lecanorales 97 100 Caloscypha fulgens 100 Pyxine subcinerea Ascobolus carbonarius 82 a Heterodermia vulgaris Teloschistales

t 100 85 Saccobolus dilutellus 99 Teloschistes exilis 99 Peziza vesiculosa 100 91 Diploschistes ocellatus crassa 94 100 Ostropales

Glomerobolus gelineus Lecanoromycetes Saccharomyces cerevisiae 100

Asco- myco Trapelia placodioides Agyriales Candida glabrata 97 Saccharomycetes 100 86 Icmadophila ericetorum Saccharo- mycotina Eremothecium gossypii 100 Pertusariales 45 Pertusaria dactylina

Rhizopus oryzae oryzae Rhizopus Saccharomycetales 62 Candida tropicalis 72 99 86 Ceramothyrium carniolicum Chaeto- Debaryomyces hansenii 96 100 Cyphellophora laciniata thyriales Taphrina wiesneri Taphrinomycetes 100 Exophiala salmonis 78 Taphrina deformans Taphrinales Staurothele frustulenta

57 100

(not shown) (not Outgroup Outgroup

Taphrino- mycotina Saitoella complicata Verrucariales 95 Schizosaccharomycetes, Agonimia sp. 74 Schizosaccharomyces pombe 72 Schizosaccharomycetales Coccidioides immitis Neolecta vitellina Neolectomycetes Ajellomyces capsulatum Onygenales a Neolecta irregularis t Neolectales Aspergillus protuberus 98 Fomitopsis pinicola Eurotiales 0.1 Eupenicillium limosum Eurotiomycetes 100 Grifola frondosa substi- Caliciopsis orientalis Coryneliales 99 Calostoma cinnabarinum tutions 100 per site Peltula umbilicata

Basidio- myco Calocera cornea Peltula auriculata Lichinomycetes Cryptococcus neoformans Lichinella iodopulchra Lichinales Fig. 2 A most likely tree obtained by RAxML for Ascomycota (as in Fig. 1). Phyla, subphyla, class, order and rankless taxa are indicated. Taxa designated as earth tongues are indicated in orange. The tree is displayed as two subtrees – orange arrows indicate where the subtrees were joined. The tree was rooted with outgroup Rhizopus oryzae (not shown). Bootstrap values are shown in orange above nodes and Bayesian posterior probabilities in blue below. Numbers were removed for nodes with 100 % bootstrap and 100 % posterior probability. C.L. Schoch et al.: Geoglossomycetes 133

Subphylum Taphrinomycotina have historically been classified in Geoglossaceae, and Sar­ As in recent studies using large multi-gene datasets (Spatafora coleotia has historically been classified in the Helotiaceae et al. 2006, Sugiyama et al. 2006, Liu et al. 2009, Schoch et al. (Leotiomycetes). These inoperculate Discomycetes produce 2009), we find ML bootstrap support here for the monophyly of terrestrial, stipitate, clavate ascomata, commonly referred to as the Taphrinomycotina. The addition of sequences from protein earth tongues, which include Leotia, Microglossum, Cudonia, coding genes has been vital to the establishment of statistical and Spathularia. In terms of ascomatal development, species support for this grouping. Recent work has shown that the short of Geoglossum, Trichoglossum, and Sarcoleotia possess a generation times characteristic of species in this group make hymenium that freely develops towards the base, while other phylogenetic analyses particularly susceptible to long branch earth tongue fungi feature a distinct ridge to their hymenium, attraction artefacts (Liu et al. 2009). The placement of Neolecta implying a developmental stage during which the hymenium is in this subclade is also confirmed here. The club-shaped apoth- enclosed (Schumacher & Sivertsen 1987, Spooner 1987, Wang ecia of the members of Neolecta share superficial similarity with et al. 2006b). An enclosed hymenium has been observed as those of the Geoglossaceae. Neolecta was long thought to be well in several other lineages, such as Cyttaria, Erysiphales included in the Geoglossaceae until molecular work proved oth- and Rhytismatales in the Leotiomycetes (Korf 1983, Gargas erwise (Landvik 1996). In support of its placement in this early et al. 1995, Johnston 2001). Although the name earth tongue diverging group, Neolecta has several presumably ancestral implies these fungi are terrestrial and have no direct associa- features, such as simplified non-poricidal asci without croziers tion found with other organisms, Trichoglossum, Geoglossum and the absence of paraphyses (Redhead 1979, Landvik et al. and Sarcoleotia globosa have often been recorded in boggy 2003). With additional sampling of both taxa and genes we find habitats abundant with bryophytes (Seaver 1951, Dennis 1968, here moderate support for the monophyly of Taphrinomycotina, Schumacher & Sivertsen 1987, Spooner 1987, Jumpponen et and thus demonstrate that the earliest diverging clade of the al. 1997, Zhuang 1998). Ascus apical morphology is one of Ascomycota was dimorphic, with both filamentous and yeast the major features in distinguishing higher ascomycetes, and growth forms. Nevertheless, it remains apparent that this part operculate ascomycetes as members of Pezizales have an of the Ascomycota tree remains under sampled. This lack of apical or subapical operculum which is thrown back at spore adequate sampling is supported by the recent description of a discharge while a definite plug is present in the thickened clade labelled ‘Soil Clone Group I’ (SCGI). SCGI is ubiquitous ‘inoperculate’ ascus apex as in species of the Helotiales (Korf in soil and is only known from environmental sequence data 1973). Ultrastructure of the ascus apical apparatus suggested (Porter et al. 2008). It appears possible that they form a novel no close relationship between Leotia lubrica and species of early diverging lineage outside of Taphrinomycotina. Very little Geoglossum and Trichoglossum. A structure known as a tractus remains known about their ecology, morphology and general connects the uppermost spore to the apical wall and the spores biology. to each other in Trichoglossum hirsutum, but is never found in other species of the Helotiales and is possibly homologous to Rankless taxon ‘Saccharomyceta’ structures in Sordariomycetes and Pezizomycetes (Verkley ‘Saccharomyceta’ includes the two remaining subphyla of 1994). Recent molecular phylogenetic analyses (Sandnes Ascomycota, Saccharomycotina and Pezizomycotina. Saccha­ 2006, Wang et al. 2006a, b) confirmed that the earth tongue romycotina comprises the ‘true yeasts’ (e.g., Saccharomyces fungi are not monophyletic. At least two origins occurred in cerevisiae), although hyphal growth has been documented in Leotiomycetes: in Leotia and allies in Helotiales, and in Cudo­ some taxa (e.g., Eremothecium). The Pezizomycotina consists nia and allies in Rhytismatales. Geoglossum, Trichoglossum. of the majority of filamentous, ascoma producing species, but Sarcoleotia (Geoglossomycetes as we define it) represent a numerous species are additionally capable of yeast and yeast- third, independent lineage of earth tongues, which we confirmed like growth phases. Thousands of species are only known does not belong within the Leotiomycetes. to reproduce asexually. These two subphyla form a well-sup- DNA-only and combined model analyses produced conflicting ported, monophyletic group that has been recovered in a large placements of Geoglossaceae within Pezizomycotina. Previ- number of studies across a diversity of character and taxon ous analyses applying nucleotide sequences only placed the sets. The recognition of ‘Saccharomyceta’ highlights the shared order as a sister group to the Lichinomycetes (Lutzoni et al. common ancestry of these two taxa and the inaccurate charac- 2004, Spatafora et al. 2006), which includes a small number terisation of Saccharomycotina as a primitive or basal lineage of lichenised species mainly associated with cyanobacteria of the Ascomycota. Rather, its small genome size (Dujon et al. (Reeb et al. 2004). Our sampling of Lichinomycetes includes 2004) and dominant yeast growth phase can be characterized two genera, Peltula and Lichinella that encompass at least as derived traits for this subphylum. some of the ascal diversity, i.e., rostrate and deliquescent, present in the class. In contrast, our combined amino acid and Rankless taxon ‘Leotiomyceta’ nucleotide model analyses resolved Geoglossaceae as an We apply ‘Leotiomyceta’ as a rankless taxon containing the isolated, unique lineage of ‘Leotiomyceta’ with no supported majority of fungi with a diversity of inoperculate asci (e.g., sister relationship, in agreement with Schoch et al. (2009). Dif- fissitunicate, poricidal, deliquescent). ‘Leotiomyceta’ excludes ferent levels of missing data underlie these two conflicting the earliest diverging classes of Pezizomycotina, Pezizomy­ topologies, and several phenomena can potentially explain this cetes and Orbiliomycetes. It was first defined as a superclass conflict, ranging from model misspecification to long-branch (Eriksson & Winka 1997). This definition has remained in attraction. Regardless of these concerns, our conclusion that use (Lumbsch et al. 2005, Spatafora et al. 2006). Included in the Geoglossaceae is a monophyletic lineage, unallied with this clade are the informal, rankless taxa ‘Dothideomyceta’, members of the Leotiomycetes and any of the other large fungal ‘Sordariomyceta’, as well as the classes Eurotiomycetes, Le­ classes remains strongly supported. canoromycetes, Lichinomycetes, and a newly proposed class, Eurotiomycetes and Lecanoromycetes are the two remaining Geoglossomycetes. classes in ‘Leotiomyceta’. Eurotiomycetes is arguably the most The type genus of Geoglossaceae, Geoglossum was initially ecologically diverse class within Ascomycota including - proposed by Persoon (1794). Persoon described it as club- ised species, saprobes and pathogens of animals and plants. shaped, with unitunicate, inoperculate asci, with the type As currently defined, this class incorporates several distinct species given as Geoglossum glabrum Pers. Trichoglossum orders and three subclasses spanning virtually all known fun- 134 Persoonia – Volume 22, 2009 DQ471090 AY885153 DQ883725 DQ767637 DQ677900 DQ677923 DQ677897 DQ677924 DQ497610 EF413612 DQ677913 Genome AY879117 Genome AY885152 DQ677914 DQ471092 FJ161091 FJ161111 DQ677928 DQ767641 DQ677910 DQ497607 DQ471111 EF411070 AY881019 DQ883733 DQ471089 DQ677898 DQ677918 DQ677920 DQ497606 DQ471107 DQ677899 FJ161104 DQ767638 FJ161110 FJ161092 DQ677883 DQ497603 DQ677922 DQ471062 DQ677909 DQ677921 TEF1 DQ247794 AY786057 DQ883715 DQ677944 DQ677982 DQ677954 DQ677976 DQ677950 DQ677977 DQ470925 EF413611 FJ238379 DQ677967 Genome AY780939 XM_570204 AY786056 DQ782866 DQ767644 DQ677983 DQ677987 DQ677993 DQ470920 FJ161114 FJ161129 DQ677981 DQ677963 DQ497614 DQ470939 EF411068 AY536286 DQ883713 DQ247788 DQ677952 DQ677971 DQ677973 DQ522854 DQ470936 DQ677953 DQ767643 FJ161128 FJ161115 DQ677938 DQ247790 DQ677975 DQ470894 DQ677962 DQ677974 EF413630 RPB2 DQ471163 AY864876 DQ883718 EU186063 DQ471168 EF413610

Genome AY857979 XM_570943 AY864874 DQ782825 DQ471165 DQ471185 AY857980 DQ471162 EU186064 DQ471182 DQ471136 EF413629 RPB1 FJ190610 AY548816 FJ190612 AY571389 FJ190624 AF346430 FJ225745 AY863212 FJ436112 EU704082 FJ190646 FJ190656 FJ190611 FJ190650 FJ190591 FJ190654 FJ772240 FJ190609 FJ190628 FJ190643 AY544739 FJ190636 FJ190631 AY544737 FJ190644 FJ190640 mSSU DQ247804 AY629318 AY548815 DQ678079 AY004343 EF413609 DQ678051 DQ678084 DQ678059 FJ161183 DQ678078 DQ678056 EF413619 FJ176897 DQ678070 AY213626 AY645054 Genome AY684164 AY584654 DQ767655 DQ678085 DQ678089 DQ678098 DQ470968 FJ161174 FJ161207 DQ678083 DQ767658 DQ678067 DQ499596 DQ470987 EF411064 AY701526 DQ883696 DQ247800 DQ678057 DQ678074 DQ678075 AY544681 DQ470984 DQ678058 FJ161196 DQ767654 FJ161203 FJ161175 DQ678044 AY544645 DQ678077 DQ470946 DQ678066 DQ678076 EF413628 nLSU DQ247812 AY705960 AY548809 DQ678028 AY016355 EF413608 DQ677998 DQ678033 AY016347 DQ678027 DQ678003 EF413618 FJ176842 DQ678018 AF113440 AY665773 Genome AY705967 AY584678 AF271127 DQ678034 DQ767652 DQ471017 FJ161135 FJ161167 DQ678032 DQ767651 DQ499595 DQ471039 EF411061 AY771610 DQ883705 DQ247808 DQ678004 DQ678022 DQ678024 AY544722 DQ479933 DQ678005 FJ161155 DQ767649 FJ161163 FJ161136 DQ677994 AY544727 DQ678026 DQ470993 DQ678014 DQ678025 EF413627 nSSU

herbarum

var. var.

pisi

var. var. Rhizopus oryzae Calocera cornea Calostoma cinnabarinum Cryptococcus neoformans Fomitopsis pinicola Grifola frondosa Roccella fuciformis Roccellographa cretacea Schismatomma decolorans Botryosphaeria dothidea Botryosphaeria tsugae Guignardia bidwellii Guignardia gaultheriae Capnodium coffeae Cladosporium cladosporioides Davidiella tassiana Mycosphaerella fijiensis Mycosphaerella graminicola Mycosphaerella punctiformis Scorias spongiosa Dothidea sambuci Dothiora cannabinae Sydowia polyspora Gloniopsis smilacis Hysterium angustatum Hysterographium mori Helicomyces roseus paludosa Tubeufia Elsinoë veneta Myriangium duriaei Lophium mytilinum Mytilinidion australe Mytilinidion rhenanum Allewia eureka Cochliobolus heterostrophus Delitschia winteri Lepidosphaeria nicotiae Pleomassaria siparia Pyrenophora phaeocomes Sporormiella minima Ulospora bilgramii enalia Verruculina cylindrica Westerdykella Ceramothyrium carniolicum Cyphellophora laciniata Exophiala salmonis Caliciopsis orientalis Aspergillus protuberus Eupenicillium limosum Taxon Taxon

1 GEL 5359 GEL 136 AW GB MB 03-036 DSH s.n. Diederich 15572 BG Printzen1981 DUKE 0047570 CBS 147.52 CBS 170.54 CBS 399.80 OSC 100622 CBS 292.38 CBS 113265 CBS 325.33 CBS 114601 EB 0324 EB 0249 CBS 283.51 CBS 245.49 CBS 304.66 CBS 454.72 CBS 658.74 CBS 339.97 Voucher GB CBS 115476 CBS 418.64 CBS 237.48 CBS 447.70 DAOM 231303 CBS 737.71 CBS 116.29 CBS 150.27 CBS 260.36 EB 0248 CBS 301.34 CBS 135.34 DAOM 195275 CBS 126.54 CBS 134.39 CBS 225.62 CBS 101341 DAOM 229267 CBS 276.37 CBS 279.74 CBS 541.72 DAOM 222769 CBS 524.50 CBS 110020 CBS 175.95 CBS190.61 CBS 157.67 CBS 138.64

Incertae sedis Incertae sedis Pleosporales Pleosporales Arthoniales Arthoniales Arthoniales Botryosphaeriales Botryosphaeriales Botryosphaeriales Botryosphaeriales Capnodiales Capnodiales Capnodiales Capnodiales Capnodiales Capnodiales Capnodiales Dothideales Dothideales Dothideales Hysteriales Hysteriales Hysteriales Myriangiales Myriangiales Mytilinidiales Mytilinidiales Mytilinidiales Pleosporales Pleosporales Pleosporales Pleosporales Pleosporales Pleosporales Pleosporales Pleosporales Pleosporales Pleosporales Pleosporales Pleosporales Chaetothyriales Chaetothyriales Chaetothyriales Coryneliales Eurotiales Eurotiales Order

outgroup outgroup outgroup outgroup outgroup outgroup Zygomycota Basidiomycota Basidiomycota Basidiomycota Basidiomycota Basidiomycota Arthoniomycetes Arthoniomycetes Arthoniomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Dothideomycetes Eurotiomycetes Eurotiomycetes Eurotiomycetes Eurotiomycetes Eurotiomycetes Eurotiomycetes Class Taxa and sequences used in this study. Taxa

54 93 283 126 946 942 940 671 438 770 701 939 277 439 307 274 267 1911 1586 1598 1037 1063 2014 1088 1618 1784 1615 1594 1580 1304 1256 1289 1591 2021 1359 1300 1613 1853 1583 1599 1576 1575 1600 1601 1033 1241 5007

Table 1 Table AFTOL no. C.L. Schoch et al.: Geoglossomycetes 135 Genome Genome DQ471133 DQ782889 DQ366251 DQ883768 FJ772246 DQ782897 DQ883775 DQ883730 DQ883773 DQ497602 DQ471057 DQ847414 FJ997862 DQ832327 DQ471109 DQ471045 DQ471041 FJ238397 DQ782917 DQ471044 DQ471049 FJ238412 DQ366258 DQ883772 DQ782899 DQ883777 DQ883764 DQ471091 DQ471101 DQ471104 DQ782919 DQ471056 Genome Genome AY548300 AY584683 DQ973091 DQ366253 DQ883749 DQ973075 AY584688 DQ883756 DQ883711 DQ883754 DQ470877 DQ470889 DQ470885 AY641033 FJ997863 DQ832328 AAF19058 DQ247786 DQ470876 FJ238350 DQ782874 DQ840560 DQ470879 DQ470881 FJ238377 DQ366260 DQ912381 DQ247793 DQ883753 DQ782868 DQ883758 DQ883759 DQ247791 DQ470930 DQ470933 DQ832331 DQ832335 DQ470888 Genome Genome DQ471133 DQ782817 DQ366252 DQ883736 DQ973061 DQ782826 DQ883743 DQ883723 DQ883741 DQ842030 FJ238425 DQ471124 DQ782857 DQ471116 DQ471113 FJ238434 DQ782853 DQ840553 DQ471115 DQ471119 DQ366259 DQ912355 DQ883740 DQ782828 DQ883745 DQ883779 DQ471164 DQ471176 DQ471179 DQ782856 DQ782855 DQ471128 AY584613 DQ972978 AY584621 46411390 AY584628 DQ912294 DQ986897 DQ912288 AY544745 AY544748 AY544751 AY584700 AY544732 AY544746 FJ225702 AY544740 AY544758 AF431962 DQ912275 DQ247784 DQ912297 DQ972973 DQ912292 FJ772245 DQ976373 DQ922954 FJ713604 AY789299 AY789409 AY789428 DQ973030 AY605077 AY544670 AY544662 AY533013 FJ997861 AY789357 DQ470986 DQ470985 Genome Genome AY548296 AY584634 AY584655 46411445 AY584657 DQ883800 DQ883694 DQ883798 AY544646 AY789397 AY789307 AY789305 AY789317 AY789321 AY544651 AY789420 AY544644 AY789293 FJ176865 DQ782913 DQ823098 AY544650 AY544653 AY789313 AF298235 AF298234 FJ176894 AF274103 DQ912332 DQ247803 DQ883797 DQ782907 DQ883802 AY584647 DQ247801 DQ470978 DQ257359 DQ470981 AY789335 DQ832330 DQ832334 DQ470944 AY789298 DQ973006 AF038877 AY544714 AY544706 AF107343 FJ997860 AY789356 DQ842040 DQ471037 Genome Genome AY548297 AY584658 AY584679 46411421 AY584681 DQ883791 DQ883704 DQ883789 AY544688 AY789396 AY789316 AY789302 AY544695 AY789419 AY544687 AY789292 DQ782885 DQ823105 AY544694 AY544697 AY789312 AF298233 AF298232 FJ176839 AF119500 DQ912310 DQ247811 DQ883788 DQ782880 DQ883793 AY584671 DQ247809 DQ471030 DQ257358 DQ471033 AY789334 DQ832332 DQ782887 DQ470992

clavus

cf. sp. Ajellomyces capsulatum Coccidioides immitis Agonimia Staurothele frustulenta Geoglossum glabrum Geoglossum nigritum Geoglossum umbratile Sarcoleotia globosa 1 Sarcoleotia globosa 2 Sarcoleotia globosa 3 hirsutum 1 Trichoglossum hirsutum 2 Trichoglossum Saitoella complicata Hesperomyces virescens Stigmatomyces protrudens Pyxidiophora avernensis placodioides Trapelia Lecidea fuscoatra Canoparmelia caroliniana Hypogymnia physodes Diploschistes ocellatus Glomerobolus gelineus Lobaria scrobiculata Lobariella pallida Nephroma parile Peltigera degenii Coccocarpia erythroxyli Icmadophila ericetorum Pertusaria dactylina Heterodermia vulgaris Pyxine subcinerea exilis Teloschistes Botryotinia fuckeliana Bryoglossum gracile Cudoniella Dermea acerina Lachnum virgineum Lambertella subrenispora Leotia lubrica Microglossum olivaceum Microglossum rufum 1 Microglossum rufum 2 Mitrula brevispora Mitrula elegans Monilinia laxa Neobulgaria pura Neofabraea malicorticis Thueminidium atropurpureum 1 Thueminidium atropurpureum 2 Cudonia circinans Spathularia velutipes 1 Spathularia velutipes 2 Lichinella iodopulchra Peltula auriculata Peltula umbilicata Neolecta irregularis Neolecta vitellina NYBG 808041 DUKE 0047959 OSC 60610 OSC 100009 Mycorec1840 HMAS 71956 OSC 63633 MBH 52476 OSC 100017 OSC 61726 GB GB CBS 657.82 GB DUKE 0047503 DUKE 0047520 DUKE 0047548 DUKE 0047504 DUKE 0047747 DUKE 0047641 DUKE 0047506 DUKE 0047507 DUKE 0047544 DUKE 0047925 OSC 100012 MBH 52481 OSC 100054 CBS 161.38 OSC 100002 CBS 811.85 OSC 100001 FH-DSH -97103 Ingo-Clark-Geo163 OSC 100641 ZW02-012 WZ-Geo47-Clark OSC 100063 CBS 477.97 OSC 100036 1100803 1136126 DUKE 0047585 OSC 100640 ZW Geo58 DAH-3 DAH-11 GB TIGR DUKE 0047522 IAM 12963 DUKE 0047740 DUKE 0047550 Lumbsch 995 JK 5548K Schultz16319a DUKE 0047648 DUKE 0047527

s.l. s.l.

Onygenales Onygenales Verrucariales Verrucariales Geoglossales Geoglossales Geoglossales Geoglossales Geoglossales Geoglossales Geoglossales Geoglossales Laboulbeniales Laboulbeniales Pyxidiophorales Agyriales Lecanorales Lecanorales Ostropales Ostropales Peltigerales Peltigerales Peltigerales Peltigerales Peltigerales Pertusariales Pertusariales Teloschistales Teloschistales Teloschistales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Helotiales Rhytismatales Rhytismatales Rhytismatales Lichinales Lichinales Lichinales Neolectales Neolectales Incertae sedis Incertae sedis

Eurotiomycetes Eurotiomycetes Eurotiomycetes Eurotiomycetes Geoglossomycetes Geoglossomycetes Geoglossomycetes Geoglossomycetes Geoglossomycetes Geoglossomycetes Geoglossomycetes Geoglossomycetes Laboulbeniomycetes Laboulbeniomycetes Laboulbeniomycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Lecanoromycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Leotiomycetes Lichinomycetes Lichinomycetes Lichinomycetes Neolectomycetes Neolectomycetes Incertae sedis 6 1 64 56 87 59 49 684 195 958 314 134 333 875 224 686 896 892 166 149 353 697 229 962 589 128 131 320 169 891 941 1084 1349 1262 1259 1362 2197 1292 1363 1083

136 Persoonia – Volume 22, 2009 DQ471054 DQ471088 FJ238402 DQ471047 FJ238389 Genome Genome Genome Genome FJ238421 AF543778 DQ522325 AF543785 AF543773 AY489632 DQ471065 Genome FJ238415 DQ471134 DQ862029 DQ466085 DQ471066 DQ471094 AY489615 DQ497608 DQ836912 Genome DQ518175 DQ471097 FJ238395 DQ471071 DQ471059 DQ471093 FJ238392 Genome Genome DQ862034 DQ479931 DQ471096 AY489618 AF543777 AF543781 FJ238390 DQ471042 TEF1 DQ247787 DQ470918 FJ238353 DQ247796 XM_448959 Genome SCYOR151C D13337 AY780151 DQ522417 DQ522422 DQ522456 DQ522441 DQ522468 DQ470897 Genome FJ238372 FJ238381 FJ238373 AY548298 DQ862013 DQ247785 DQ470898 DQ470922 DQ522421 DQ518181 DQ836891 XM_324476 DQ470927 FJ238349 DQ470891 DQ247795 CR382139 AE016819 DQ862018 DQ470919 DQ470926 DQ522429 DQ522440 DQ522446 AY015637 DQ470878 RPB2 FJ238423 DQ471126 DQ471160 FJ238436 DQ479935 XM_447415 Genome X96876 X56564 AY489648 DQ522370 AY489666 AY489659 AY489673 DQ471139 Genome DQ471134 DQ471120 DQ471140 FJ238430 DQ471167 AY489649 XM_959004 DQ471170 DQ471145 DQ471130 DQ471166 FJ238424 XM_456921 NM_209535 DQ471161 DQ471169 AY489652 AY489662 DQ471114 RPB1 FJ190606 FJ190587 AY544761 AF442281 X54421 EF469007 FJ713622 FJ190626 FJ713630 FJ190593 FJ190660 AY548291 FJ713625 FJ190615 FJ713628 FJ190595 FJ190618 FJ713610 AY544741 FJ190613 AF442353 FJ190607 FJ713629 FJ713624 FJ713620 AY544760 mSSU AY544677 DQ470966 DQ247806 AY544666 AY198398 Genome AF543789 U00748 AF543786 DQ470945 DQ470947 FJ176854 FJ176888 DQ247799 FJ176870 SCYLR154C Z19136 AY761075 AY695260 AY184962 AB026819 U17401 FJ176901 AY548292 DQ862027 AY544654 DQ470948 AY544668 AF408361 AY489721 DQ522856 DQ836904 AF286411 AY545724 DQ470973 FJ176864 DQ470952 AY544673 DQ247805 FJ176860 AF485980 AE016820 DQ862028 AF408350 DQ470972 AY489723 U17396 AF543791 AY015630 U46885 AY544648 nLSU AY544720 DQ471014 DQ247814 AY544710 AY198398 M55527 AF543765 U32412 AF543762 AY489705 DQ470994 FJ176801 FJ176834 DQ247807 FJ176814 SCYLR154C X54866 AY761071 AY184973 AB026819 U32418 FJ176846 AY548293 DQ862044 AY544698 DQ470995 AY544712 DQ471019 AY489689 DQ522855 DQ836897 X04971 AY545728 DQ471024 FJ176810 DQ471000 AY544717 DQ247813 FJ176806 DHA508273 AE016820 DQ862049 DQ471015 DQ471023 AY489691 U32405 AF543768 AY015619 U46872 AY544692 nSSU

aureofulva cf. Arthrobotrys elegans Orbilia vinosa Aleuria aurantia Ascobolus carbonarius Caloscypha fulgens Eleutherascus lectardii Gyromitra californica Peziza vesiculosa Pyronema domesticum Saccobolus dilutellus Sarcosoma latahense Sarcosphaera crassa Scutellinia scutellata conica Verpa Candida glabrata Candida tropicalis Debaryomyces hansenii Eremothecium gossypii Saccharomyces cerevisiae Schizosaccharomyces pombe Calosphaeria pulchella Bertia moriformis Cryptosporella hypodermia Diaporthe eres Endothia gyrosa Gnomonia gnomon Bionectria Claviceps purpurea Cordyceps cardinalis Elaphocordyceps capitata Elaphocordyceps ophioglossoides Epichloë typhina Hypocrea lutea Nectria cinnabarina Glomerella cingulata dahliae Verticillium Lindra thalassiae Lulworthia grandispora Gaeumannomyces medullaris Magnaporthe grisea Melanospora tiffanyae Ceratocystis fimbriata Corollospora maritima Gondwanamyces capensis Halosphaeria appendiculata Ophiostoma stenoceras Neurospora crassa Sordaria fimicola Xylaria hypoxylon deformans Taphrina wiesneri Taphrina Taxon Taxon

1 CBS 397.93 CBS 917.72 OSC 100018 KH-00-08 OSC 100062 CBS 626.71 OSC 100068 TL-6398 CBS 666.88 CBS 472.80 CBS 733.68 OSC 100049 OSC 100015 22338 NRRL GB GB GB GB GB GB CBS 115999 SMH4320 GJS 71-328 GAM 12885 OSC 93609 OSC 71233 OSC 106405 56429 ATCC 208838 ATCC CBS 114055 553 FAU 16535 ATCC 15515 ATCC TCH C89 728a CBS 122611 CBS 197.60 CBS 139.51 Broad CBSC 15-5973 OSC 100004 Voucher CBS 171.69 CBS 109767 CBS 112915 CBS 199.53 JK 5090A JK 4686 JK 5528S Broad CBS 356.35 IAM 14515

Orbiliales Orbiliales Pezizales Pezizales Pezizales Pezizales Pezizales Pezizales Pezizales Pezizales Pezizales Pezizales Pezizales Pezizales Saccharomycetales Saccharomycetales Saccharomycetales Saccharomycetales Saccharomycetales Schizosaccharomycetales Calosphaeriales Coronophorales Diaporthales Diaporthales Diaporthales Diaporthales Hypocreales Hypocreales Hypocreales Hypocreales Hypocreales Hypocreales Hypocreales Hypocreales Incertae sedis Incertae sedis Lulworthiales Lulworthiales Magnaporthales Magnaporthales Melanosporales Microascales Microascales Microascales Microascales Ophiostomatales Sordariales Sordariales Xylariales Taphrinales Taphrinales Order

Orbiliomycetes Orbiliomycetes Pezizomycetes Pezizomycetes Pezizomycetes Pezizomycetes Pezizomycetes Pezizomycetes Pezizomycetes Pezizomycetes Pezizomycetes Pezizomycetes Pezizomycetes Pezizomycetes Saccharomycetes Saccharomycetes Saccharomycetes Saccharomycetes Saccharomycetes Schizosaccharomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Sordariomycetes Taphrinomycetes Taphrinomycetes Class (cont.)

74 62 70 51 65 voucher GB = obtained from GenBank, or genome databases without clear numbers. 152 933 176 954 162 192 237 413 747 409 189 949 187 193 734 216 265 905 507 153 935 952 163 156 159

1199 5011 1073 1269 1077 5086 2124 1265 1081 1907 1078 1299 1072 1069 1223 1906 1038 1234 1252

AFTOL 1 Table 1 Table no. C.L. Schoch et al.: Geoglossomycetes 137 gal ecological niches (Geiser et al. 2006). Lecanoromycetes layer of wall cells, and long perithecial necks that release their contain the majority of the lichenised fungi (Miadlikowska et ascospores passively in droplets at the tips of their necks; this al. 2006). Earlier large-scale phylogenies (e.g. Lutzoni et al. mechanism is repeatedly derived within Ascomycota for insect 2004) have suggested a sister relationship between these two dispersal of ascospores (Blackwell 1994). For this reason, classes, but we find that such a relationship remains without they have been likened to other insect-dispersed perithecial strong statistical support (Fig. 1). Despite this, internal nodes ascomycetes (e.g., Ophiostomatales) that now are strongly are well supported enough to provide good support for the supported as members of Sordariomycetes. Laboulbeniales hypothesis that lichenisation evolved multiple times in the includes ectoparasites of insects and displays morphologi- Ascomycota, with losses being rare (Gueidan et al. 2008, cal traits not found elsewhere in the Ascomycota. They form Schoch et al. 2009). apomorphic ascomata produced by the division and enlarge- The remaining classes are discussed in relation to their respec- ment of ascospores that are difficult to characterize in existing tive rankless taxa listed below. ascomatal terms. Laboulbeniales feature an ostiole, how- ever, which is consistent with perithecia produced by hyphal Rankless taxon ‘Dothideomyceta’ growth. Determinate growth of the ascospore with a series of predictable cell divisions produces a thallus of a finite number This taxon is well supported, with ML bootstrap of 91 % and of cells that is characteristic at the genus and species level a moderate Bayesian posterior probability of 92 %. It includes (Tavares 1979). The analyses presented here strongly support two classes of fungi which produce fissitunicate asci, Artho­ Laboulbeniomycetes as sister to Sordariomycetes. This place- niomycetes and Dothideomycetes. Arthoniomycetes consists ment corresponds with the terminology originally applied to of ± 1 600 species of lichenised and lichenicolous fungi with this group (Thaxter 1896). It is interesting to note that while fissitunicate asci and exposed hymenia (Grube 1998, Ertz et al. species of Pyxidiophorales are endowed with a diverse group 2009). Unlike other species with fissitunicate asci, these taxa of anamorphs, members of Laboulbeniales are mainly known have ascohymenial development, prompting their placement to reproduce sexually. in a transitory group, or ‘Zwischengruppe’ that is intermediate between ascohymenial and ascolocular development (Henssen Summary & Jahns 1974). The class is resolved as sister to Dothideo­ In conclusion, we propose two monotypic formal taxa and mycetes, consistent with recent studies (Lutzoni et al. 2004, describe continued support for four informal rankless taxa. Spatafora et al. 2006, Wang et al. 2006a). Dothideomycetes Important improvements in the resolution of deep nodes within is a large class containing two subclasses, Dothideomycetidae the Ascomycota may be attributed to multi-gene sequence data and (Schoch et al. 2006). Our analysis produced by AFTOL and other projects during the last 5 years. contains members of all known orders in the class, including The accelerating accumulation of genome-scale sequence data recent additions (Boehm et al. 2009). This broad representa- will continue to challenge and improve existing phylogenetic tion yields increased resolution in the placement of an order hypotheses. However, in order to direct limited resources previously labelled incertae sedis, Botryosphaeriales (Schoch towards under-sampled areas in the fungal phylogeny, an ac- et al. 2006). Placement of Botryosphaeriales within subclass curate, up-to-date classification is required. By placing three Pleosporomycetidae is well supported, as is a close relationship earth tongue genera in a separate newly described class, we with the unplaced family (Fig. 2). underscore and communicate the genetic diversity that is found in the fungi producing these very convergent morphologies. Rankless taxon ‘Sordariomyceta’ ‘Sordariomyceta’ contains three classes, Leotiomycetes, Laboul­ Acknowledgements We are grateful to Kentaro Hosaka, Dan Luoma and beniomycetes and Sordariomycetes. We find similar resolution Zhuliang Yang for the photographs used. We acknowledge funding provided for this clade as for the ‘Dothideomyceta’. These three classes by NSF through a grant (DEB 0717476) to J.W.S. and C.L.S. (while at Ore­ are characterised by the production of unitunicate, poricidal gon State). C.L.S. was also supported in part by the Intramural Research asci, or derivatives of such asci (e.g., deliquescent asci). Leotio­ program of the National Institutes of Health. Z.W. was supported by the Yale mycetes and Sordariomycetes include numerous fungi associ- University Anonymous Postdoctoral Fellowship. ated with plants as pathogens, endophytes and epiphytes. The sordariomycete phylogeny is comparatively well resolved with References 15 orders and 3 subclasses named (Zhang et al. 2006, Kirk et al. 2008). In contrast, the leotiomycete classification still poorly Altekar G, Dwarkadas S, Huelsenbeck JP, Ronquist F. 2004. Parallel Me- matches its inferred phylogeny. A recent class-wide effort to tropolis coupled Markov chain Monte Carlo for Bayesian phylogenetic assess morphological and ecological data in a phylogenetic inference. Bioinformatics 20: 407–415. Blackwell M. 1994. 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