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Home , Ambrosiella, Annulatascaceae, Balansia, Bertia (fungus), Bionectria, Bionectriaceae

Mycologia, 98(6), 2006, pp. 1076–1087. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897

An overview of the systematics of the based on a four- phylogeny

Ning Zhang of 16 in the Sordariomycetes was investigated based Department of , NYSAES, Cornell on four nuclear loci (nSSU and nLSU rDNA, TEF and University, Geneva, New York 14456 RPB2), using three of the Leotiomycetes as Lisa A. Castlebury outgroups. Three subclasses (i.e. , Systematic Botany & Laboratory, USDA-ARS, and Xylariomycetidae) currently Beltsville, Maryland 20705 recognized in the classification are well supported with the placement of the in either Andrew N. Miller a group of the Sordariomycetes or a sister group Center for Biodiversity, Illinois Natural History Survey, of the Hypocreomycetidae. Except for the Micro- Champaign, Illinois 61820 ascales, our results recognize most of the orders as Sabine M. Huhndorf monophyletic groups. species form Department of Botany, The Field Museum of Natural a clade outside of the and are recognized History, Chicago, Illinois 60605 as a distinct in the Hypocreomycetidae. Conrad L. Schoch is excluded from the Department of Botany and Plant Pathology, Oregon and placed in Hypocreomycetidae . In State University, Corvallis, Oregon 97331 the Sordariomycetidae, the is a strongly supported clade and occurs within a well supported Keith A. Seifert clade containing the and Chaetosphaer- Biodiversity (Mycology and Botany), Agriculture and iales. Aspects of morphology, ecology and evolution Agri-Food Canada, Ottawa, Ontario, K1A 0C6 Canada are discussed. Amy Y. Rossman Key words: classification, ecology, evolution, Systematic Botany & Mycology Laboratory, USDA-ARS, Hypocreomycetidae, Sordariomycetidae, Xylariomy- Beltsville, Maryland 20705 cetidae Jack D. Rogers Department of Plant Pathology, Washington State University, Pullman, Washington 99164 INTRODUCTION Jan Kohlmeyer Brigitte Volkmann-Kohlmeyer The class Sordariomycetes is one of the largest Institute of Marine Sciences, University of North monophyletic clades in the with more Carolina at Chapel Hill, Morehead City, North than 600 genera and 3000 known species (Kirk et al Carolina 28557 2001). It includes most nonlichenized ascomycetes Gi-Ho Sung1 with perithecial (flask-shaped) or less frequently Department of Botany and Plant Pathology, Oregon cleistothecial (nonostiolate) ascomata and inopercu- State University, Corvallis, Oregon 97331 late unitunicate or prototunicate asci (Alexopoulos et al 1996). The term ‘‘pyrenomycetes’’ was used to unite fungi with perithecial ascomata and unitunicate Abstract: The Sordariomycetes is one of the largest asci (Luttrell 1951). Its use was discontinued based on classes in the Ascomycota, and the majority of its the placement of perithecial species outside the clade species are characterized by perithecial ascomata and and the inclusion of species with prototunicate asci inoperculate unitunicate asci. It includes more than (e.g. and ) to avoid confusion. 600 genera with over 3000 species and represents In the current classification sensu Eriksson (2006) the a wide range of ecologies including and Sordariomycetes comprises 16 orders in three sub- of plants, animal pathogens and myco- classes (i.e. the Hypocreomycetidae, Sordariomyceti- parasites. To test and refine the classification of the dae and Xylariomycetidae) based on rDNA phyloge- Sordariomycetes sensu Eriksson (2006), the phyloge- nies. netic relationship among 106 taxa from 12 orders out Ecology and its importance.—Members of the Sordar- Accepted for publication 27 September 2006. iomycetes are ubiquitous and cosmopolitan and 1 Corresponding author. E-mail: [email protected] function in almost all ecosystems as pathogens and

1076 ZHANG ET AL:SORDARIOMYCETES SYSTEMATICS 1077 endophytes of plants, arthropod and mammals, and other alkaloids produced by Claviceps and mycoparasites and saprobes involved in decomposi- Epichloe¨. also are a good source of tion and nutrient cycling. Most plant pathogens in the novel bioactive compounds (Bugni and Ireland Sordariomycetes are distributed in the , 2004). Hypocreales, , , Phylla- chorales and . These include the best known Morphology and classification.—The majority of Sor- plant pathogens (e.g. parasitica [the dariomycetes produce perithecial ascomata (FIG. 1D– causal agent of blight], grisea G).Theshape,size,pigmentation,textureand [the cause of rice blast], and O. novo- position of ascomata were characters used in tradi- ulmi [Dutch disease causal agents], tional . For example the position of species and Rosellinia species [Alexopoulos et al 1996, ascomata in relation to substrates was used in family Samuels and Blackwell 2001]). In addition to patho- delimitation of the Diaporthales by Barr (1978) but gens of plants the Sordariomycetes includes endo- this classification was not supported by phylogenetic phytes that live inside the above-ground parts of analyses using molecular characters (Castlebury et al apparently healthy plants (Alexopoulos et al 1996). 2002, Zhang and Blackwell 2001). A phylogenetic The best studied endophytes belong to the Hypo- study by Miller and Huhndorf (2005) suggested that creales (e.g. and Epichloe¨), Xylariales (e.g. ascomal wall morphology is a better character than Nemania and Xylaria)andColletotrichum.Their morphology in defining genera in the infected plants benefit from increased drought Sordariales. Nannfeldt (1932) and Luttrell (1951) resistance, reduced feeding by insects and limited first applied ontogenetic characters (FIG. 1F, G) in infections. filamentous ascomycete classification. Although the Members of the Hypocreales, Ophiostomatales and ontogenetic characters do not always correspond well Microascales often are associated with opportunistic with molecular phylogenies, they are still informative infections of humans and other animals (e.g. characters in ordinal classification within the Sordar- schenkii, species complex iomycetes (Samuels and Blackwell 2001). The typical and spp.) (Gugnani et al 1976, Summer- arrangement of asci in the Sordariomycetes is basal or bell 2003). As symbionts of arthropods the Sordar- peripheral in a hymenium (FIG. 1E). The presence or iomycetes comprise a diverse assemblage of species absence of an apical ring (amyloid or nonamyloid) is that range from antagonistic to mutualistic (Samuels an important feature in Sordariomycetes classification and Blackwell 2001). Members of the Microascales (FIG. 1H–J). Asci of the Sordariomycetes typically are and Ophiostomatales (e.g. Ceratocystis, Ophiostoma octosporous (FIG. 1K, M, N). Ascospore wall orna- and ) are associated with bark beetles in mentation has been used to delimit certain genera, fungal dispersal. Species of the Hypocreales but phylogenetic studies on the and (e.g. and ) directly parasitize Sordariales show that this character is prone to a broad range of arthropods. The Hypocreales also convergence and hence is phylogenetically uninfor- is known to be rich in mycoparasites. Most species of mative. The Sordariomycetes is an anamorph-rich are parasitic on fleshy fungal bodies class, with significant diversity represented by hypho- such as and large apothecia (Rogerson mycete and coelomycete species. Many species of the and Samuels 1989). Some species of Trichoderma, Hypocreales, Ophiostomatales and used alone or combined with other biocontrol agents, have two or more distinguishable anamorphs (syna- have been applied in agriculture for plant disease namorphs). occur throughout the management as an alternative to pesticides (Harman class, but coelomycete anamorphs also occur, most et al 2004). notably in the Glomerellaceae and Diaporthales. In Saprobic Sordariomycetes function in the decom- common with teleomorph characters many character- position and nutrient cycling of plant litter including istics used to delimit anamorph genera (e.g. varia- wood, herbaceous stems and dung. Important taxa tions in conidiomata, pigmentation, conidiophore include crassa, the model organism widely branching and conidial septation) are homoplasic in used in molecular and genetic studies, and Chaeto- the Sordariomycetes. Despite this, recognizable pat- mium, an important cellulolytic organism responsible terns of anamorph morphological characters often for the destruction of paper and fabrics. The allow recognition of phylogenetic groups (Seifert and Sordariomycetes also contains species known as Gams 2001). producers of some of the most important fungal Recent comprehensive taxonomic studies of fami- secondary metabolites. These include the trichothe- lies and higher taxa of the Sordariomycetes were cene mycotoxins produced by many members of the published by Barr (1990), Samuels and Blackwell and the clade and the (2001) and Eriksson (2006). The classification 1078 MYCOLOGIA

FIG. 1. Morphology of species in the Sordariomycetes. A. lutea BPI 744483, perithecia embedded in stromata, Hypocreales, bar 5 1 mm. B. Xylaria longipes, mature stromata growing on a moss covered hardwood log, Xylariales, bar 5 1 cm. C. Cordyceps scarabaeicola, mature stromata growing on a scarabaeid pupa, Hypocreales, bar 5 5 mm. D. salicis BPI 748461, longitudinal section of perithecia, Diaporthales, bar 5 100 mm. E. leucoplaca SMH 2578, longitudinal section of perithecium, , bar 5 40 mm. F. DAOM 83684, apical paraphyses in perithecium, Hypocreales, bar 5 90 mm. G. Hypomyces lactifluorum DAOM 83684, periphyses in perithecium, Hypocreales, bar 5 11 mm. H. luteola ANM 233, apical ring of , Diaporthales, bar 5 10 mm. I. Cainia desmazierei DAOM 89953(a), apical ring of ascus, Xylariales, bar 5 5 mm. J. graminis DAOM 19596, apical ring of ascus, , bar 5 9 mm. K. Coniochaeta leucoplaca SMH 2578, asci with , Coniochaetales, bar 5 10 mm. L. Hypocrea lutea BPI 744503, asci and ascospores, Hypocreales, bar 5 20 mm. M. ovina SMH 1538, asci with globose subapical globules and ascospores, Sordariales, bar 5 40 mm. N. moriformis ANM 264, ascus, , bar 5 10 mm. O. thalassiae JK 2728 (IMS), septate ascospore without apical chambers, Lulworthiales, bar 5 25 mm. P. Varicosporina ramulosa JK 3907 (IMS), , Halosphaeriales, bar 5 5 mm. ZHANG ET AL:SORDARIOMYCETES SYSTEMATICS 1079 proposed by Barr (1990) was based on morphology assessed by nonparametric bootstrapping (Felsenstein while the other two were based on both morpholog- 1985). Nodal supports in the Bayesian analyses were ical characters and sequences from the nuclear small examined by posterior probabilities from a 10 000 000 subunit ribosomal RNA gene (nSSU rDNA). The generation analysis. To examine potential impact of and Coryneliales sensu Barr (1990) were saturation in third codons of TEF and RPB2 on nodal supports in higher relationships, bootstrap proportions and excluded from the Sordariomycetes (Samuels and posterior probabilities were reassessed after excluding the Blackwell 2001). Eriksson (2006) placed the Erysi- third codon positions of protein-coding . phales in the Leotiomycetes, a sister taxon of the Sordariomycetes (see also Spatafora et al on Pezizo- mycotina, this issue). The extralimital pyrenomycete RESULTS AND DISCUSSION Laboulbeniales (Samuels and Blackwell 2001) has Data analyses.—The alignment for the phylogenetic been recognized as a separate class (Eriksson 2006, analyses, after excluding ambiguously aligned re- Weir and Blackwell 2001). The main goals of this gions, comprised 4514 base pairs, 1697 of which were paper are twofold: to define monophyletic groups parsimony informative. The reciprocal comparisons within the class and to infer the evolutionary relation- of 70% bootstrap did not reveal any incongru- ships among major orders in the Sordariomycetes. ence (data not shown). The heuristic search in MP and WP analyses yielded four most parsimonious trees MATERIALS AND METHODS (MPTs) with 17 501 steps (CI 5 0.1858, RI 5 0.5361) and one MPT with 29 222.44 steps, respectively. ML Character and taxon sampling.—Genomic DNA extraction from specimens or cultures was performed according to heuristic search analysis resulted in a of standard methods as in Castlebury et al (2004). Amplifica- 281 968.65 log-likelihood. In all Bayesian analyses tions and sequencing were performed for four nuclear gene the log-likelihood was converged at approximately regions (NS1-NS6 region of nSSU rDNA, LR0R-LR7 region 30 000 generations and a 50% majority rule consensus of nLSU rDNA, 983f-2218r region of TEF, and 5–7 region of tree was generated from the 10 000 000 generation RPB2) under standard conditions using primers in analysis with the exclusion of the first 500 trees. The the AFTOL Website (http://aftol.biology.duke.edu/pub/ tree from WP analyses is provided (FIG. 2) with all primers/viewPrimers). A total of 106 taxa were sampled to bootstrap proportions (i.e. MP-BP, WP-BP and ML- represent 12 orders of the Sordariomycetes following the BP) as well as the Bayesian posterior probabilities classification of Eriksson (2006). Due to the lack of data (PP) and the tree from the Bayesian analyses is from protein-coding genes, the members of other orders (i.e. the , Meliolales, Phyllachorales and presented (SUPPLEMENTARY FIG. 1). Internodes were ) are not represented in this study. (For considered strongly supported if they received all of voucher information and GenBank accession numbers see bootstrap proportions (MP-BP, WP-BP and ML-BP) $ SUPPLEMENTARY TABLE I). 70% and posterior probabilities (PP) $ 95% (Lutzoni et al 2004). Phylogenetic analyses.—Sequences were edited with the Seqmerge program in GCG software (Accelrys, San Diego, Systematics of the Sordariomycetes.—The four-gene California). Sequences of each gene were aligned manually phylogenetic analyses (FIG. 2) provide support for and the concatenated alignment was deposited in Tree- three major clades in the Sordariomycetes, corre- BASE (study No. SN2723). To detect incongruence among genes a 70% reciprocal MP bootstrap comparison (Mason- sponding to the three subclasses of Eriksson (2006). Gamer and Kellogg 1996) was used as a dataset of the 61 The Xylariomycetidae is placed as a basal group of the core taxa for which all four genes were available. Maximum Hypocreomycetidae and the Sordariomycetidae, and weighted parsimony (MP and WP) analyses were which are strongly supported after the exclusion of performed as described in Lutzoni et al (2004) on third codons in protein-coding genes. The Lul- a combined dataset of the 106 taxa including taxa that worthiales is basal to other Sordariomycetes accord- were complete for at least two genes. Maximum likelihood ing to the MP, WP and ML tree, but it is resolved as (ML) analyses were conducted with Phyml (Guindon and a sister taxon of the Hypocreomycetidae based on the Gascuel 2003) with the GTR+I+C model. Bayesian analyses Bayesian analyses (SUPPLEMENTARY FIG. 1). Because with Markov chain Monte Carlo (MCMC) algorithm were performed with the GTR+I+C model for each of eight the four-gene phylogeny is largely consistent with the partitions using MrBayes 3.0b4 (Huelsenbeck and Ronquist current classification, the ordinal relationships and 2001). Bayesian analysis with 10 000 000 generations was characteristics of each clade are discussed below conducted in four chains with sampling every 100 genera- according to the classification of the Outline of tions. Ten independent analyses with 1 000 000 generations Ascomycota (Eriksson 2006). from different starting trees were performed to reconfirm the adequate log-likelihood convergence and mixing of Xylariomycetidae.—This well supported monophyletic chains. Nodal supports in MP, WP and ML analyses were subclass contains a single order, the Xylariales, which 1080 MYCOLOGIA

FIG. 2. Phylogenetic tree of the Sordariomycetes. The phylogeny is constructed from single most parsimonious tree from weighted parsimony analyses. Bootstrap proportions (MP-BP, WP-BP and ML-BP) of $ 70% and posterior probabilities (PP) of $ 95% are provided for the corresponding internodes. Bootstrap proportions and posterior probabilities in circles were calculated with the exclusion of the third codon positions in TEF and RPB2. The internodes that are supported by all bootstrap proportions and posterior probabilities are shown as a thicker line. The name of taxa (e.g. subclasses, orders, families) is provided to the right of species names. ZHANG ET AL:SORDARIOMYCETES SYSTEMATICS 1081 is characterized by well developed stromata, dark Graphostromatace contains Graphostroma. Although colored perithecia, persistent asci often with amyloid species have a stroma reminiscent of the apical rings and true paraphyses. Most species of the and ascospores somewhat suggestive of that family, its Xylariales are saprobes or plant parasites in terrestrial affinities are clearly with the (see FIG. 2). habitats. While most marine species are classified in Family Diatrypaceae has long been recognized as the Halosphaeriales and Lulworthiales (Spatafora et a natural assemblage allied with the Xylariaceae. A al 1998) the sampled species, Anthostomella torosa, cardinal feature is the pale brown allantoid ascospore. represents marine species in the Xylariales (Kohl- The anamorphs of the Diatrypaceae also have meyer and Volkmann-Kohlmeyer 2002). The Xylar- sympodially proliferating as well as percurrently iales is one of the largest orders in the Sordariomy- proliferating conidiogenous cells (sometimes on the cetes and includes more than 800 species in eight same conidiophore) but lack conspicuous scars and families according to the Dictionary of the Fungi (Kirk often have tiny conidiogenous cells and conidia et al 2001). Some of its families are larger and more densely packed into rudimentary coelomycetous diverse than small orders in the class. Therefore we conidiomata, sometimes little more than cavities in will discuss each family to not oversimplify the stroma (Glawe and Rogers 1982). Although circum- picture. scription of the family is clear and widely accepted Family Xylariaceae is a large assemblage and the (Barr 1990) the delimitation of genera is in part type family of the Xylariales. Perithecia are embedded artificial (Acero et al 2004). Family in stromata, asci usually possess amyloid apical rings, as circumscribed by Barr (1990) contains 13 genera. and mature ascospores are usually unicellular with The assemblage is probably polyphyletic. The type a prominent germ slit. A number of recent reports Hyponectria falls within the Xylariales in the have dealt with phylogenetics within the family (e.g. two-locus phylogeny of Lutzoni et al (2004). Family Sa´nchez-Ballesteros et al 2000, Triebel et al 2005 and Myelostromataceae is monotypic. It generally is Hsieh et al 2005). Anamorphs of the Xylariaceae have assigned to the Xylariales, but its true affinities sympodially proliferating conidiogenous cells with remain uncertain. characteristic scars, and holoblastic, single-celled Hypocreomycetidae.—This sublass is recognized here conidia with detachment scars corresponding with as a strongly supported monophyletic clade and those on the conidiogenous cells. Variation in co- includes its four orders (i.e. the Coronophorales, nidiophore branching and the nature of the con- Halosphaeriales, Hypocreales and Microascales) in idiogenous cell proliferation (ampulliform, irregular- the classification sensu Eriksson (2006). The members ly swollen, geniculate) allows the distinction of of the subclass typically possess light colored perithe- anamorph genera such as Nodulisporium and Genicu- cia. Asci are nonamyloid or amyloid, or lack apical losporium and several others, which often delimit rings, and true paraphyses are lacking in most monophyletic groups (Ju and Rogers 1996). The members (FIG. 2). While the Coronophorales, Halo- Amphisphaeriaceae in the sense of Mu¨ller and von sphaeriales and Hypocreales are recognized as mono- Arx (1973) included taxa that greatly resemble phyletic groups, the Microascales is rendered para- members of the Xylariaceae but with ascospores that phyletic due to the placement of the Halosphaeriales. (mostly) lack defined germination sites. Barr (1990) A new finding is that the sister group of the presented a much more restricted circumscription of Melanospora clade is the Coronophorales and this the family, and Kang et al (2002) published a molec- long branch internode (SUPPLEMENTARY TABLE I) is ular-based phylogeny including the restricted group. strongly supported without third codon positons. The versicolored, multiappendaged conidia of the Therefore Melanospora (type species: M. zamiae)is Pestalotia complex seem to be a synapomorphic excluded here from the Hypocreales and recognized character for these coelomycetous anamorphs of the as a distinct order. The remaining clade in the Amphisphaeriaceae (Kang et al 2002). subclass comprises and an Family Cainiaceae was established for a small anamorphic species, . The Glomer- assemblage of monocot inhabitants with two-celled ellaceae is validated with G. cingulata and placed ascospores ornamented with striations or multiple within the Hypocreomycetidae. germination slits and asci with amyloid apical rings. The two-locus phylogeny of Lutzoni et al (2004) Hypocreales.—The Hypocreales is a strongly sup- places Cainia among the Xylariales. Family Clypeo- ported monophyletic order that includes virulent sphaeriaceae sensu Barr (1990) includes genera with plant and insect pathogens as well as useful mycopar- mostly iodine-negative ascal apices and germination asitic, endophytic and saprobic species (FIG. 2, Ross- sites, when present, that are pores. Important genera man et al 1999). This order is defined by species with include Clypeosphaeria and .Family soft textured, generally light to bright colored 1082 MYCOLOGIA perithecial fruiting bodies and unitunicate asci that genera of so-called Ingoldian hyphomycetes. The develop among apical (and centripetal in some hyphomycetous anamorph Varicosporina ramulosa species) paraphyses that often dissolve at maturity. (FIG. 1P) produces conidia and sclerocarps, sclero- The colorless ascospores vary from nonseptate, tium-like fruiting bodies, lacking sexual capacity. It globose to one- to multiseptate, ellipsoidal to filiform. was postulated that V. ramulosa is an ascomycete that The Hypocreales include more than 70 genera in four has lost its ability to reproduce sexually (Kohlmeyer major families (Rossman et al 1999). The - and Charles 1981). Sclerocarps are morphologically ceae and Nectriaceae consist primarily of species similar to ascomata of Corollospora species and well previously placed in the large and artificial genus adapted to extreme conditions of sandy beaches, having nonstromatic, uniloculate perithecia. where they may be exposed to long periods of drying The genus Nectria (anamorph: ) now is and extreme high or low temperatures. restricted to a related group of 23 species. The Microascales.—This order is recognized here as Nectriaceae (e.g. , Nectria and Pseudo- a paraphyletic group with respect to the Halosphaer- nectria in FIG. 2) is defined by the orange to red, iales (FIG. 2). However taxonomic revision is pre- KOH+ perithecia. The (e.g. Bionectria, mature due to poorly supported internal nodes and and in FIG. 2) consists the need for greater taxon sampling. The Microscales primarily of pallid, nectria-like species such as is a small order of primarily saprobic fungi in soil, Bionectria (anamorph: ), some of which rotting vegetation and dung. A few species of this are used to control greenhouse pathogens. The order cause plant diseases such as Ceratocystis includes Hypocrea (anamorph: Tricho- fimbriata, transmitted by beetles to living trees and derma), increasingly useful in the biological control of causing cacao wilt and many other economically plant pathogens (Samuels 2006), and Hypomyces with important diseases. Other members such as species diverse anamorphic states. The pro- of cause incurable diseases of hu- duces a wide range of secondary metabolites and mans. The order is characterized by nonstromatic insect-associated genera including Cordyceps, Hypo- black perithecial ascomata with long necks or rarely crella and Torrubiella. The anamorphs of the Hypo- with cleistothecial ascomata that lack paraphyses, and creales are generally phialidic producing slimy con- globose and evanescent asci, developing singly or in idia; -like anamorphs also are chains. Nonseptate, colorless ascospores often bear produced by many species. The anamorphs include ornamenting ridges or wings. The anamorphs of the the well known phialidic genera , Tricho- produce percurrently proliferating derma (Hypocreaceae), Clonostachys, Stilbella (Bionec- conidiogenous cells (annellides) and sometimes triaceae), , , Fusarium, chlamydospore-like or aleurioconidial synanamorphs Tubercularia (Nectriaceae, Rossman et al 1999), and that are classified mostly in the hyphomycete genera , , , and , and (Abbott et (Clavicipitaceae, Hodge 2003). The ‘‘toxic ’’ al 1998, Okada et al 1998). fungus, , represents part of a lineage separate from any of the defined families Coronophorales and Melanosporales.—The Corono- within the Hypocreales (Castlebury et al 2004). phorales is composed of primarily wood-inhabiting taxa and defined by species with erumpent to Halosphaeriales.—This order is strongly supported superficial ascomata that often collapse upon drying (FIG. 2) as shown in Spatafora et al (1998). The order and that sometimes possess an extensive hyphal is characterized by usually submerged perithecial subiculum or basal stroma. Many taxa contain Munk ascomata; an interascal tissue is absent, but the pores (small pores each of which is surrounded by centrum is filled by a thin-walled pseudoparenchyma a ring or thickening) in their ascomatal cell walls and that dissolves or breaks up to form filamentous a Quellko¨rper (a gelatinous mass of cells in the apical catenophyses. A small number of species in this order region of the ascoma, believed to function in are found in freshwater, but the majority consists of rupturing the ascoma) in their centrum (Huhndorf marine species. The ascus wall in most species et al 2004a). While Munk pores are found in a few deliquesces releasing the ascospores, which are forced taxa outside the order, the Quellko¨rper is a character into the neck of the by production of unique to the Coronophorales. Filiform paraphyses additional asci and ascospores. Within the order the are absent in the group, and in most species the asci distinctive ascospore appendages are important char- are thin-walled, clavate, stipitate and lack an apical acters for delineating most genera. Several ana- ring. Ascospores usually are hyaline, small and morphs have been attributed to the Halosphaeriales, allantoid. Anamorphs are hyphomycetous when including chlamydospore-like anamorphs and several known. The typical characteristics of the Melanospor- ZHANG ET AL:SORDARIOMYCETES SYSTEMATICS 1083 ales include translucent perithecial or cleistothecial Colletotrichimodo phialidica; conidia unicellularia, ascomata, pseudoparenchymatous centra, absence of mucida, guttulata. paraphyses in development, clavate evanescent asci and dark colored ascospores. Previous phylogenetic Typus Glomerella Spauld. & H. Schrenk studies based on rDNA sequences suggested that Melanospora is within or near Hypocreales (Spatafora Sordariomycetidae.—Members of this subclass possess and Blackwell 1994, Zhang and Blackwell 2002). light to dark colored perithecia. Asci are nonamyloid, When analyzed with more taxa of the Sordariomy- amyloid or lack apical rings, and true paraphyses are cetes, Melanospora formed a distinct clade outside present in some members. The subclass consists of six Hypocreales (Castlebury et al 2004). The four-gene orders, all of which are recognized as monophyletic phylogeny (FIG. 2) supports the exclusion of Mela- groups in this study (FIG. 2). The clade representing nospora from the Hypocreales. Furthermore a close the Coniochaetales is well supported and the Con- relationship between the Coronophorales and the iochaetales remains as an unsupported sister group of Melanosporales (FIG. 2) is recognized. Similar mor- the clade containing the Sordariales, Boliniales and phological and ecological features of the two orders Chaetosphaeriales. The internode uniting the Chae- include a pseudoparenchymatous ascomal wall, cla- tosphaeriales with the Boliniales also is supported vate, deliquescent asci, lack of paraphyses (with a few strongly. The Diaporthales forms a sister group exceptions in the Coronophorales) and often a myco- relationship with the economically important family parasitic habit. Magnaporthaceae (e.g. and Gaeu- Glomerellaceae.—The Glomerellaceae is a so far mannomyces graminis). The Ophiostomatales is an monotypic family characterized by black, nonstro- unsupported sister group of the clade containing the matic perithecia, well developed periphysate ostioles, representative taxa of the Annulatascaceae and abundant thin-walled paraphyses, clavate asci with J- Papulosaceae. apical ring, and hyaline, smooth, often curved Boliniales.—Members of the Boliniales occur primar- ascospores. Anamorphs known are ily on wood. It is the only order in the Sordariomy- species. Glomerella had been placed in the Phyllachor- cetidae that contains taxa with large, stromatic, ales (see the orders of uncertain positions) but some carbonaceous ascomata. Other taxa in the group of its features are clearly distinct from those of other may possess soft-textured stromata that may be Phyllachorales, such as its lack of stromatic tissue and brightly colored. Perithecia may be polystichous or its exclusively Colletotrichum anamorphs. Molecular monostichous wherein they often will be distinctively phylogenetic analyses (Wanderlei-Silva et al 2003) vertically elongate. Members of this order possess confirmed these distinctions and they recognized small asci and brown, ellipsoidal, frequently flattened Glomerella as a family in the Hypocreomycetidae. ascospores that often have polar germ pores. No Further investigation is required to test whether anamorphs are known. The Boliniales in the four- Verticillium dahliae, a sister taxon of G. cingulata in gene phylogeny is represented by four species of the phylogeny presented here (FIG. 2), is actually encompassing disparate morphologies and a member of the Glomerellaceae. The anamorphic more strongly allied with Lasiosphaeriella nitida and genus Verticillium apparently is related closely to the holomorphic genus Plectosphaerella (Zare et al 2004), Linocarpon appendiculatum (taxa that currently are which together might represent an undescribed placed in Sordariomycetidae inc. sed.) (FIG. 2). family (Gams, pers comm). The family was invalidly Chaetosphaeriales.—The Chaetosphaeriales includes published by Locquin (1984) and is validated here. saprobic, often wood-inhabiting fungi. Small, dark perithecial ascomata that are often setose are found Glomerellaceae Locq. 1984 ex Seifert & W. Gams, in a number of genera in the group. Ascomata fam. nov. typically are associated with dematiaceous, hyphomy- Glomerellaceae Locq., Mycol. Ge´n. Struct.: 175. 1984, cetous anamorphs. Members of the Chaetosphaer- nom. inval., Art. 36 iales have filiform paraphyses and cylindrical asci, Stromata absentia. Ascomata perithecia, nigra, usually with a pronounced apical refractive ring. saepe sclerotiformia, pariete crasso, ostiolum periph- Ascospores are often hyaline. Ascospores can range ysatum. Textura interascalis paraphysibus veris, tenui- from ellipsoidal, nonseptate to elongate, almost tunicatis. Asci clavati, brevi-stipitati, tenuitunicati non filiform, and septate in species of , fissitunicati, plerumque annulo parvo apicali praediti, with fusiform, 1–3-septate ascospores being typical of qui iodo non reagit. Ascosporae hyalinae, unicellu- most members of the order (Ferna´ndez and Huhn- lares, leves, nonnumquam curvatae. Anamorphe dorf 2005). 1084 MYCOLOGIA

Sordariales.—This order consists of mostly wood- and (), or elongated () to large, dung-inhabiting species with relatively large, erum- multiseptate, and black (). Commonly pent or superficial ascomata with large celled, encountered anamorphs are coelomycetes such as membraneous or coriaceous ascomatal walls. Filiform (), (Diaporthe)and paraphyses are present in some taxa as are subapical (Melanconis), producing pycnidia with globules within the asci. Ascospores in this group phialidic conidiogenous cells, often on the same show variation on a distinct developmental theme and stroma as the sexual state. range from cylindrical, hyaline ascospores to ellip- soid, brown ascospores, often with appendages or Ophiostomatales.—This order includes fungi associat- sheaths. Many of the species in this order lack ed with wood and bark, such as the agents of Dutch anamorphs, but some have lightly pigmented, phia- elm disease (Ophiostoma ulmi and O. novo-ulmi) and lidic, Phialophora-like anamorphs, or the ascospores blue stain of hard- and softwood timber (O. germinate by directly producing phialides (Gams piliferum). Most species of the order however are 2000, Re´blova´ and Winka 2000). The most important saprobic and usually are associated with beetle discovery in these four-gene analyses is that the dispersers, while related anamorphs (i.e. Sporothrix internode representing the Sordariales is strongly schenckii) are reported to cause diseases in humans supported by both bootstrap and posterior probabil- (Summerbell 1999). The order is characterized by ity, which was not shown in previous studies (Huhn- solitary, black perithecia, and most species have long dorf et al 2004b, Miller and Huhndorf 2005). The necks from which sticky ascospores ooze and are present taxon sampling encompasses some of the transported by insects to new substrates. The asci are great morphological variation within the group, but globose and dissolve early in development, while the the use of four genes does not distinguish any well ascospores often have ornamenting sheaths, ridges or supported new lineages. Previous studies uncovered wings. Many of the hyphomycete genera once well supported clades that correlated with distinct associated with the Ophiostomatales have been ascomal wall characters (Miller and Huhndorf 2005). simplified by the recognition that the apparent variety The (e.g. Neurospora and Sordaria) in conidium ontogeny represents minor variation of remains a well supported monophyletic group, and a common pattern. In genera such as Leptographium this is the only traditional family recognized in the and Pesotum a percurrent proliferation of the order. While Chaetomium is monophyletic with strong conidiogenous cells and delayed secession of conidia support, the genera and are gives a false impression of sympodial proliferation. In polyphyletic. addition some species have exclusively sympodially proliferating, denticulate conidiogenous cells that are Coniochaetales.—Membersofthisordertypically classified in Sporothrix (Wingfield et al 1993). possess small, setose perithecial ascomata and occur Ophiostoma once was considered synonymous with on wood, dung or soil. The asci are more or less Ceratocystis in the Microascales and their long-necked, clavate without an apical ring. Ascospores are brown, fleshy perithecia with sticky ascospores associated with ellipsoidal, fusoid or discoid, and most possess a germ insects in wood represent a morphological homoplasy slit. The relatively simple ascomatal morphology can (Cain and Weresub 1957, Samuels and Mu¨ller 1978, cause members of the group to be confused with taxa Spatafora and Blackwell 1994). in the Chaetosphaeriales or the Sordariales. The known anamorphs of this order are classified as Magnaporthaceae, Annulatascaceae and Papulosa- species of Lecythophora. These are phialidic hypho- ceae.—These families, each with one representative mycetes with the phialide often reduced to a collarette species included in this phylogenetic study (FIG. 2), lateral on a hyphal cell (Gams 2000). were grouped in the Sordariomycetidae. Their ordi- nal placement is unsettled due to their distinctive Diaporthales.—The Diaporthales is a strongly sup- morphological characters and paucity of sequence ported monophyletic order (FIG. 2) and includes data. The Magnaporthaceae is characterized by primarily plant-associated fungi such as the agent of nonstromatic black perithecia, usually with long hairy (Cryphonectria parasitica) and dog- necks, tapering paraphyses, persistent asci and fili- wood anthracnose ( destructiva). The order is form ascospores. The anamorphs are hyphomycetous. characterized by black perithecial fruiting bodies that Members of the Annulatascaceae are found in rotten may or may not be aggregated and immersed in wood in tropical freshwater habitats. Nonstromatic a stroma, evanescent interthecial elements, and asci perithecia are dark brown to black, sometimes long- that float free at maturity and often have conspicuous necked, paraphyses are persistent, asci are typically refractive apical rings. The ascospores vary from cylindrical with large nonamyloid apical rings, and colorless, allantoid (Valsa), one-septate, ellipsoidal ascospores often have polar appendages or a ZHANG ET AL:SORDARIOMYCETES SYSTEMATICS 1085 gelatinous sheath (Wong et al 1998). The obligate Sordariomycetes, although the relationships among marine fungus Papulosa amerospora grows in leaves of the three subclasses still are not resolved confidently. the halophyte Juncus roemerianus. It is characterized Most of the sampled taxa of the Hypocreomycetidae by dark brown, nonstromatic perithecia, long periph- have light colored perithecia, nonamyloid apical rings ysate necks, lateral paraphyses, persistent cylindrical when apical rings are present and the absence of true asci with apical amyloid rings and aseptate, ellipsoi- paraphyses. dal, verruculose ascospores covered by a gelatinous The majority of members of the Sordariomycetes sheath. are terrestrial, and life in aquatic habitats is consid- ered a derived character for the class (Samuels and Order of uncertain position.—The placement of the Blackwell 2001). The Halosphaeriales, Sordariales, Lulworthiales, although taxon sampling has been Diaporthales, Xylariales and Magnaporthaceae con- limited, suggests a new subclass lineage and is treated tain freshwater species, while most marine species are as an order of uncertain position in this study. This classified in the Halosphaeriales and Lulworthiales. recently described order was segregated from the Most of these fungi break down and Halosphaeriales based on molecular data (Kohlmeyer from plant debris in intertidal and subtidal zones, et al 2000). The Lulworthiales is characterized by dark very rarely also in the deep sea. All the major lineages ostiolate ascomata, with or without a subiculum, and in the Sordariomycetes contain aquatic species the absence of interascal tissue although the centrum (Shearer 1993, Spatafora et al 1998). This study is filled initially with a thin-walled pseudoparenchy- further supports the conclusion that the move to ma. Asci are thin-walled and deliquesce early, and aquatic environments occurred many times in the ascospores are usually filamentous (FIG. 1O), mostly class. The Xylariomycetidae comprises saprobes and with mucus-containing apical chambers or appen- plant pathogens, which are also abundant in the dages. The members of the Lulworthiales degrade other two subclasses. Therefore the saprobic and wood and marsh plants in marine and estuarine plant parasitic habits might be the ancestral states of environments. Two species of an enigmatic genus the Sordariomycetes. Most mycoparasites and insect Spathulospora, obligate parasites on red algae, re- associates are derived from the Hypocreomycetidae, cently were placed in this order based on nuclear and the Sordariomycetidae is rich in coprophilous ribosomal data (Inderbitzin et al 2004). It was noted taxa. by Inderbitzin et al (2004) that Spathulospora shares the apical mucus-filled chambers of the ascospores CONCLUSION with several lulworthialean species. However major We have tested the hypotheses of the systematics of features of Spathulospora species, absent in other the Sordariomycetes based on multigene phylogenet- members of Lulworthiales, are presence of sterile ic analyses. While the phylogeny is largely consistent ascomatal hairs, antheridia and trichogynes. Halo- with the subclass and ordinal classification of Eriksson guignardia, another genus with algae-inhabiting (2006), the Melanosporales is recognized as a distinct species and ascospores characterized by mucus-filled order in the Hypocreomycetidae. The phylogenetic apical chambers, also is nested within the Lul- analyses strongly support monophyly of the three worthiales (Campbell et al 2005). The only reported subclasses, and 12 orders in the study are recognized anamorphs in this order are the hyphomycetes, as monophyletic groups except for the Microascales. Zalerion maritimum (teleomorph unisep- Results from this study, coupled with 400 000 000 y tata, Nakagiri 1984), with helically coiled conidia, and old fossil record (Taylor et al 2005), also provide an Anguillospora marina (teleomorph Lindra obtusa, insight into the long and diverse evolution of the Nakagiri and Tubaki 1983) with filiform conidia. nutritional mode/fungal symbioses in the Sordario- Evolution of morphology and ecology.—A synapomor- mycetes. Because its ancestral nutritional mode is phy of the Sordariomycetes is the perithecial ascoma, hypothesized to be saprobic or plant pathogenic, our which is evolved from the apothecium of ancestral data indicate multiple independent origins of other (see Spatafora et al this issue). nutritional ecologies (e.g. insect associates and However taxa in a number of unrelated lineages of mycoparasites) and provide a foundation for de- the Sordariomycetes have lost ostioles, which usually veloping hypotheses on the dynamic process of is associated with the loss of forcible discharge of evolutionary patterns, such as marine colonization. ascospores (Malloch 1981, Suh and Blackwell 1999). Most members of the Xylariomycetidae and some of ACKNOWLEDGMENTS the Sordariomycetidae have dark perithecia, amyloid We thank Meredith Blackwell and Joseph W. Spatafora for asci, true paraphyses and periphysate ostioles. We suggestions in the improvement of this manuscript and hypothesize that these traits are plesiomorphies in the Walter Gams for assistance with the Latin diagnosis. We are 1086 MYCOLOGIA grateful to Robert Shoemaker for photographs of Cainia, ically little-differentiated anamorphs of divergent asco- Gaeumannomyces and Hypomyces,GaryJ.Samuelsfor mycetes. Stud Mycol 45:187–199. photographs of Hypocrea, and David Farr for the photo- Glawe D, Rogers J. 1982. Observations on the anamorphs of graph of Valsella. We also appreciate assistance and advice six species of Diatrype and Diatrypella. Can J Bot 60: with computational resources by Scott Givan and Chris 245–251. Sullivan at the Center for Gene Research and Biotechnology Gugnani HC, Talwar RS, Njoku-Obi AN, Kodilinye HC. of Oregon State University. Ben O’Rourke was great 1976. Mycotic keratitis in Nigeria. A study of 21 cases. assistance to us in generating sequence data. 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