Molecular Phylogenetics and Evolution 41 (2006) 295–312 www.elsevier.com/locate/ympev Evolution of helotialean fungi (Leotiomycetes, Pezizomycotina): A nuclear rDNA phylogeny Zheng Wang a,¤, Manfred Binder a, Conrad L. Schoch b, Peter R. Johnston c, Joseph W. Spatafora b, David S. Hibbett a b a Department of Biology, Clark University, 950 Main Street, Worcester, MA 01610, USA Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA c Herbarium PDD, Landcare Research, Private bag 92170, Auckland, New Zealand Received 5 December 2005; revised 21 April 2006; accepted 24 May 2006 Available online 3 June 2006 Abstract The highly divergent characters of morphology, ecology, and biology in the Helotiales make it one of the most problematic groups in traditional classiWcation and molecular phylogeny. Sequences of three rDNA regions, SSU, LSU, and 5.8S rDNA, were generated for 50 helotialean fungi, representing 11 out of 13 families in the current classiWcation. Data sets with diVerent compositions were assembled, and parsimony and Bayesian analyses were performed. The phylogenetic distribution of lifestyle and ecological factors was assessed. Plant endophytism is distributed across multiple clades in the Leotiomycetes. Our results suggest that (1) the inclusion of LSU rDNA and a wider taxon sampling greatly improves resolution of the Helotiales phylogeny, however, the usefulness of rDNA in resolving the deep relationships within the Leotiomycetes is limited; (2) a new class Geoglossomycetes, including Geoglossum, Trichoglossum, and Sarcoleotia, is the basal lineage of the Leotiomyceta; (3) the Leotiomycetes, including the Helotiales, Erysiphales, Cyttariales, Rhytismatales, and Myxotrichaceae, is monophyletic; and (4) nine clades can be recognized within the Helotiales.  2006 Elsevier Inc. All rights reserved. Keywords: Ascomycota; Ecology; Endophytic symbiosis; Life history; Plant pathogens 1. Introduction The Ascomycota is the largest clade of Fungi and is characterized by the production of asci (sac-like meiosporangia producing ascospores), although asexual reproduction is common. Most species in this group are lichen-forming fungi, some are saprotrophs and parasites, and a few enter mycorrhizal associations. The classiWcation of Ascomycota was historically based on their fruiting bodies (sporocarps or ascomata). The “discomycetes” was one of the largest and most species rich groups, but it is no longer recognized * Corresponding author. Present address: 310 Biology Building, Roy J. Carver Center for Comparative Genomics, Department of Biological Sciences, University of Iowa, Iowa City, IA 52242-1324, USA. Fax: +1 508 793 8861. E-mail address: zhengwangV@yahoo.com (Z. Wang). 1055-7903/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2006.05.031 as a formal taxon (Alexopoulos et al., 1995; Kirk et al., 2001). Discomycetes develop open spore producing fruiting bodies known as apothecia, which often take on the forms of cups, saucers, cushions or clubs, and produce their asci in an exposed hymenium. Two groups of discomycetes are recognized on the basis of ascus dehiscence, those with operculate asci and those with inoperculate asci. Apothecia of inoperculate discomycetes are usually small and produce asci with an apical perforation or pore, through which the spores are discharged. Apothecia of operculate discomycetes are generally large and produce asci with a hinged capor lid-like structure that opens to release ascospores. Inoperculate discomycetes along with other ascomycetes producing inoperculate asci are classiWed in the superclass Leotiomyceta (Eriksson and Winka, 1997; Lumbsch et al., 2005), including both non-lichen- and lichen-forming fungi. These fungi colonize a large variety of habitats, and act as 296 Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 saprobes, or form parasitic associations with a wide range of other organisms. Besides parasites and saprobes, the group includes endophytes that are symbionts of a wide range of plants (Grünig and Sieber, 2005; Monreal et al., 1999; Read et al., 2000; Wilson et al., 2004). The Helotiales in a traditional sense, which is the focus of this study, includes a polyphyletic assemblage of morphologically diverse inoperculate fungi that usually produce their ascomata not embedded in host tissue. A number of recent molecular studies have helped to improve our understanding of phylogenetic relationships in the Helotiales. For example, Neolecta, a former member of the Geoglossaceae that produces club-shaped sporocarps, was shown to be placed in the basal branch of Ascomycota composed of dimorphic Taphrinales parasitizing angiosperms in their mycelial stage, Wssion yeasts, and the mammalian pathogen Pneumocystis carinii (Landvik, 1996; Landvik et al., 2001; Liu and Hall, 2004). In addition, the genus Orbilia was shown to form a separate lineage from other inoperculate discomycetes (Baral et al. in Eriksson et al., 2003; Gernandt et al., 2001; PWster, 1997), despite having similarly shaped fruiting bodies. Eriksson (2005) has compiled data from a wide range of recent studies, which suggest that the helotialean fungi might be closely related to several macroscopically distinct groups. He includes the Cyttariales, Erysiphales, Thelebolales, Myxotrichaceae, and Rhytismatales along with the Helotiales in the class Leotiomycetes, although these relationships remain poorly resolved (Gernandt et al., 2001; Landvik, 1996; Ogawa et al., 1997; PWster and Kimbrough, 2001; Saenz et al., 1994). The Helotiales includes 13 families and 395 genera, within which 92 genera are of uncertain position (Eriksson, 2005). It is the largest and the most diverse group in the Leotiomycetes, and it has already been subject to several nomenclatural reinterpretations (Carpenter, 1988; Dennis, 1968; Korf and Lizon, 2000, 2001). Most helotialean species produce small apothecia that possess relatively few characters that are diagnostic at the level of family. Morphological characters such as shape and color of the apothecia, ecological characters such as terrestrial or aquatic lifestyle, and biological characters such as parasitic or saprobic nutritional mode, have been used to deWne the families in the Helotiales. Species in the Helotiales, however, show extraordinary variation in these characters, and classiWcations based on these characters are not always consistent with cellular, ultrastructural, and molecular characters (Gernandt et al., 2001; Lutzoni et al., 2004; Verkley, 1994; Wang et al., 2005). The morphological diversity of the Helotiales has led to the recognition of form groups, which have dominated the classiWcation for decades (Dennis, 1968; Korf, 1973; Kirk et al., 2001). In the Helotiales, the current classiWcation uses morphological characters such as shape and color of apothecium, hymenium, and ascospore, ontogeny of apothecia, reaction of asci to Melzer’s Reagent (iodine), and ultrastructure of asci (Korf, 1973). ClassiWcations based on apothecial morphology in this group of fungi are not always reliable, and it is likely that similar morphologies may have evolved multiple times. A good example is the Geoglossaceae, a family that includes genera with clavate or spathulate apothecia. Based on recent morphological and molecular studies, the genera of the Geoglossaceae are distributed in Wve diVerent families, and the placement of the Geoglossaceae in the Helotiales has been disputed (Gernandt et al., 1997, 2001; Imai, 1941; Korf, 1973; Landvik, 1996; Lutzoni et al., 2004; Platt, 2000; Spooner, 1987; Verkley, 1994; Wang et al., 2002). Characteristic reactions of the ascus to Melzer’s reagent are usually consistent within a genus but are too variable for use in higher level classiWcation (e.g., Stone and Gernandt, 2005). The ultrastructure of asci could provide clues for inferring early relationships among ascomycetes (e.g., Baral, 1987; Verkley, 1992, 1994). However, the study of ultrastructure is technically challenging and the lack of knowledge of functions associated with observed structures limits the potential of this technique. The systematics of the Helotiales is further hampered by a limited knowledge about interconnections between anamorphs (asexual forms) and teleomorphs (sexual forms). Many helotialean fungi are only known from a teleomorphic stage, and their anamorphs are either not yet discovered or have been lost in evolution. Anamorphs in various environmental samples including some root endophytes have been suggested to belong to the Helotiales, but without any clear teleomorph connections. In addition, there is little correlation between the classiWcations of helotialean teleomorphs and their anamorphs (Marvanova, 1997; Sutton and Hennebert, 1994; Raja and Shearer, http:// fm5web.life.uiuc.edu/fungi/). The overall diversity in the Helotiales makes it a focus for phylogenetic studies in the Leotiomycetes—one of the more problematic classes of Ascomycota (Lutzoni et al., 2004). Discovering more informative characters and achieving broader taxon sampling are two major challenges in phylogenetic studies of the Helotiales. Sequence data from ribosomal DNA (rDNA) have been used in phylogenetic reconstructions of major groups of ascomycetes (e.g., Berbee and Taylor, 1992; Gargas and Taylor, 1995; Eriksson and Strand, 1995; Spatafora and Blackwell, 1993) and protein-coding gene phylogenies involving helotialean fungi are slowly emerging (e.g., Landvik et al., 2001; Liu et al., 1999; Liu and Hall, 2004; Lutzoni et al., 2004). Most contemporary results suggest that the Helotiales and currently delimited families are not monophyletic, and that the highly conserved small subunit (SSU) rDNA is not informative enough to resolve these lineages with conWdence (Gernandt et al., 2001). Another ribosomal locus, the internal transcribed spacers (ITS) and the 5.8S rDNA gene, has also been used to infer relationships within the Helotiales (e.g., Abeln et al., 2000; Goodwin, 2002). Closely related fungi usually form strongly supported clades in ITS phylogenies, whereas alignment diYculties make the application of ITS problematic for higher level phylogenies. For these reasons, we combined large subunit (LSU) rDNA sequences with SSU rDNA and 5.8S rDNA to estimate the phylogenetic relationships of the Helotiales. Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 The goals of this study were threefold: (1) to investigate the evolutionary relationships of the Cyttariales, Erysiphales, Rhytismatales, and Helotiales within the superclass Leotiomyceta using an overlapping SSU, LSU, and 5.8S rDNA data set; (2) to explore the phylogenetic structure within the Helotiales by using a diverse sample of taxa; (3) to investigate the phylogenetic distribution of morphological, biological, ecological, and biogeographic characters among the clades of the Helotiales. 2. Materials and methods 2.1. Taxon sampling A data matrix containing 99 taxa of Pezizomycotina, 50 of them from the Helotiales, was constructed with sequences from SSU rDNA, LSU rDNA, and 5.8S rDNA genes. The data for this study were generated in laboratories at Clark University and Oregon State University, and are available from GenBank or the AFTOL database (http://ocid.nacse.org/research/aftol/data.php). Eleven of the 13 currently accepted families in the Helotiales (Eriksson, 2005) were included, excluding only the Phacidiaceae and the Ascocorticiaceae. To examine the monophyly of the Leotiomycetes and the Helotiales, species belonging to the Myxotrichaceae, Cyttariales, Rhytismatales, Erysiphales, Sordariomycetes, and Dothideomycetes were also included. Peziza species (Pezizomycetes), Orbilia species, and two budding yeasts were also sampled to address outgroup diversity. Previous studies suggested that lichen-forming inoperculate discomycetes form clades distantly related to the Leotiomycetes, thus representatives of major lichen groups were included in this study (Liu and Hall, 2004; Lumbsch et al., 2005; Lutzoni et al., 2004). Neolecta irregularis was suggested having a basal position in the Ascomycota (Landvik et al., 2001; Liu and Hall, 2004), and was therefore used to root the trees. 2.2. Molecular techniques DNA was isolated from dried fruiting bodies as described in Wang et al. (2005). Crude DNA extracts were puriWed with GeneClean (Bio 101, La Jolla). Cleaned DNA samples were diluted with distilled water up to 500-fold for use as PCR templates. Sequence data were generated from three regions: (1) partial nuclear small subunit (SSU) rDNA bounded by primers PNS1 and NS41 (Hibbett, 1996; White et al., 1990); (2) partial nuclear large subunit (LSU) rDNA bounded by primers JS-1 and LR5 (Landvik, 1996; Vilgalys and Hester, 1990); (3) complete internal transcribed spacers 1 and 2 and the 5.8S rDNA (ITS rDNA) bounded by primers ITS-1F and ITS4 (White et al., 1990). Sequences generated in this study were submitted to GenBank, and additional sequences were downloaded from GenBank and the AFTOL database or were kindly provided by others (Table 1). 297 PCR mixes (Promega Corp., Madison, Wisconsin) contained 2.5 L 10£ PCR buVer, 5 M dNTP, 12.5 pM of each PCR primer, and 5 L DNA in 25 L. The ampliWcation program included 40 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min. PCR products were puriWed using Pellet Paint (Novagen, Madison, Wisconsin) and sequenced using the ABI Prism BigDye-terminator cycle sequencing kit 1.1 (Applied Biosystems, Foster City, California) according to the manufacturer’s protocols. Primers used for sequencing were PNS1, NS19bc, NS19b, NS41, JS-1, LR3, LR3R, LR5, ITS1F, and ITS4. Sequencing reactions were puriWed using Pellet Paint and were run on an Applied Biosystems 377XL automated DNA sequencer. Sequences were edited with Sequencher version 3.1 (GeneCodes Corporation, Ann Arbor, Michigan). 2.3. Phylogenetic analyses Two data sets were prepared based on sequences of 99 taxa from three nuclear genes, SSU rDNA (950 bp), LSU rDNA (914 bp), and 5.8 S rDNA (156 bp). Four isolates of Sordariomycetes that formed a clade with very long branches in parsimony analysis (results not shown) were excluded from the Wnal data sets. Data set one included 95 taxa and was used to resolve the phylogenetic relationships within the Helotiales and between the Helotiales and other major groups in the Leotiomycetes (widerrange analyses). Data set one contains some missing data, as follows: the SSU rDNA sequences of Ciboria batschiana, Bisporella citrina, and Scleromitrula shiraiana were about 360–560 base pairs (bp) shorter than sequences of the other taxa, and no SSU rDNA sequence of Sarcoleotia globosa was available. The LSU rDNA sequence of Rutstroemia bolaris was 527 bp shorter than in other taxa. No 5.8S rDNA sequences of Hemiphacidium longisporum, Roccella fuciformis, Peltula umbilicata, and Dibaeis baeomyces were available. Thirteen species were placed on conspicuously long branches and their placements were not consistent in diVerent analyses. These problematical species include Bisporella citrina, Hyaloscypha daedaleae, Cordierites frondosa, Chlorociboria species, Cyttaria darwinii, three species of the Myxotrichaceae, Byssoascus striatisporus, Myxotrichum deXexum, Pseudogymnoascus roseus, and Pseudeurotium zonatum (Pseudeurotiaceae), and three species in the Erysiphales, Arthrocladiella mougeotii, Blumeria graminis, and Uncinula septata. Consequently, these 13 taxa and the four isolates of Sordariomycetes were excluded from data set two. Thus, data set two included 82 taxa, and was used to focus on the relationships within the Helotiales (narrower-range analyses). Sequences were aligned with ClustalX using default setting (Thompson et al., 1997) and further adjusted by eye in the data editor of PAUP¤ 4.0b (SwoVord, 1999). Introns were deleted and ambiguously aligned positions were excluded from the data sets before performing the analyses. All data sets were analyzed in PAUP¤ 4.0b 298 Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 Table 1 Species studied with information on GenBank Accession numbers by DNA locus Species SSU-rDNA LSU-rDNA 5.8S rDNA Arthrocladiella mouqeotii (Lév.) Vassilkov Arthonia sp. Ascocoryne calichnium (Tul.) Korf Ascocoryne sarcoides (Jacq.) J.W. Groves and D.E. Wilson Ascocoryne turWcola (Boud.) Korf Berlesiella nigerrima (R.P. Bloxam ex Curr.) Sacc. Bisporella citrina (Batsch.) Korf Blumeria graminis (DC.) Speer Botryosphaeria ribis Grossenb. and Duggar Bryoglossum gracile (P. Karst.) Redhead Bulgaria inquinans (Pers.) Fr. Byssoascus striatisporus (G.L. Barron 7 C. Booth) Arx Candida albicans (C.P. Robin) Berkhout Capronia mansonii (Schol-Schwarz) E. Müll., Petrini, Fisher, Samuels, and Rossman Chlorencoelia versiformis (Pers.) Dixon Chlorociboria aeruginosa (Oeder) Seaver ex C.S. Ramamurthi, Korf, and L.R. Batra Chlorociboria sp. Chloroscypha sp. Chlorovibrissea sp. Ciboria batschiana (Zopf) N. F. Buchw Cladonia caroliniana (Schwein.) Tuck Cordierites frondosa (Kobayasi) Korf Cudonia sp. Cudoniella clavus (Alb. and Schwein.) Dennis Cudoniella clavus (Alb. and Schwein.) Dennis Cyttaria darwinii Berk Dermea acerina (Peck) Rehm Dibaeis baeomyces (L. f.) Rambold and Hertel Dothidea sambuci (Pers.) Fr. Dothidea sp. Eupenicillium javanicum (J.F.H. Beyma) Stolk and D.B. Scott Eurotium amstelodami L. Mangin Fabrella tsugae (Farl.) Kirschst Geoglossum glabrum Pers. Geoglossum umbratile Sacc. Gremmeniella abietina (Lagerb.) M. Morelet Hemiphacidium longisporum Ziller and A. Funk Heyderia abietis (Fr.) Link Heyderia abietis Holwaya mucida (Schulzer) Korf and Abawi Hyaloscypha daedaleae Velen Hydrocina chaetocladia Scheuer Hymenoscyphus scutula (Pers.) W. Phillips Hypocrea lutea (Tode) Petch Lachnum bicolor (Bull.) P. Karst Lachnum virgineum (Batsch) P. Karst Lecanora concolor Ramond Leotia lubrica (Scop.) Pers. Lophodermium pinastri (Schrad.) Chevall Loramyces juncicola W. Weston Meria laricis Vuill. Microglossum olivaceum (Pers.) Gillet Microglossum rufum (Schwein.) Underw Microglossum sp. Mitrula brevispora Zheng Wang Mitrula paludosa Fr. Mollisia cinerea (Batsch) P. Karst Monilinia laxa (Aderh. and Ruhland) Honey Mycocalicium poplyporaeum (Nyl.) Vain Myxotrichum deXexum Berk Neobulgaria pura (Pers.) Petr Neofabraea malicorticis H.S. Jacks Neofabraea alba (E. J. Guthrie) Velkley AB033477 AY571379 AY789393 AY789387 AY789276 AY541478 AY789324 AB033476 AF271129 AY789419 AY789343 AJ315170 X53497 X79318 AY789350 AY544713 DQ257348 AY544700 DQ257351 DQ257354 AY584664 AY789353 AF107343 AY789340 AY789372 U53369 UNPUBL. AF085473 AY544722 AY016343 U21298 AB002076 AF106015 AY789316 AY789302 AF203456 UNPUBL. AY789288 AY789295 DQ257355 AY789414 AY789411 AY789430 AF543768 AY544690 AY544688 AY640993 AY789358 AF106014 UNPUBL. AF106017 AY789396 DQ257358 DQ257361 AY789292 AY789422 UNPUBL. UNPUBL. AY789361 AB015777 DQ257364 AY544706 N/A AB022379 AY571381 AY789394 AJ406399 AY789277 AY350579 AY789325 AB022362 AY004336 AY789420 AY789344 AB040688 L28817 AY004338 AY789351 AY544669 DQ257349 AY544656 DQ257352 AY789322 AY584640 AY789354 AF279379 AY789341 AY789373 UNPUBL. UNPUBL. AF279385 AY544681 AY016360 AF263348 AY213699 AF356694 AY789317 AY789303 UNPUBL. UNPUBL. AY789289 AY789296 DQ257356 AY789415 AY789412 AY789431 AF543791 AY544674 AY544646 AY640954 AY789359 AY004334 UNPUBL. UNPUBL. AY789397 DQ257359 DQ257362 AY789293 AY789423 UNPUBL. UNPUBL. AY789362 AY541491 DQ257365 AY544662 AY064705 AF073358 AF138813 AY789395 AY789388 AY789278 AF050251 AY789326 AJ313142 AF027744 AY789421 AY789345 AF062817 AY672930 AF050247 AY789352 AY755360 DQ257350 U92311 DQ257353 AY526234 AF456408 AY789355 AF433149 AY789342 AY789374 UNPUBL. UNPUBL. N/A AY883094 AF027764 U18358 AY213648 U92304 AY789318 AY789304 U72259 N/A AY789290 AY789297 DQ257357 AY789416 AY789413 AY789432 AF359264 U59005 U59004 AF070037 AY789360 AF775701 UNPUBL. U92298 AY789398 DQ257360 DQ257363 AY789294 AY789424 UNPUBL. AF150676 AY789363 AF062814 DQ257366 AF281386 AY359236 299 Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 Table 1 (continued) Species SSU-rDNA LSU-rDNA 5.8S rDNA Neolecta irregularis (Peck) Korf and J.K. Rogers Neurospora crassa Shear and B.O. Dodge Ochrolechia parella (L.) A. Massal Ombrophila violacea P. Karst Orbilia auricolor (A. Bloxam ex Berk.) Sacc. Orbilia delicatula (P. Karst.) P. Karst Peltigera aphthosa (L.) Willd Peltigera degenii Gyeln. Peltula umbilicata (Vain.) Swinscow and Krog Peziza phyllogena Cooke Peziza varia (Hedw.) Fr. Phialocephala fortinii C.J.K. Wang and H.E. Wilcox Phoma herbarum Westend. Piceomphale bulgarioides (Rabenh.) Svrcek Pilidium acerinum (Alb. and schwein.) Kunze Pilidium concavum (Desm.) Höhn Pseudogymnoascus roseus Raillo Pseudeurotium zonatum J.F.H. Beyma Rhytisma sp. Roccella fuciformis (L.) DC. Roccella tuberculata Vain Rutstroemia bolaris (Batsch) Rehm Saccharomyces cerevisiae Meyen ex E.C. Hansen Sarcoleotia globosa (Sommerf. ex Fr.) Korf Sarcoleotia cf. globosa Scleromitrula shiraiana (Henn.) S. Imai Sclerotinia sclerotiorum (Lib.) de Bary Sordaria Wmicola (Roberge ex Desm.) Ces. and De Not Spathularia Xavida Pers Trapelia placodioides Coppins and P. James Trichoglossum hirsutum (Pers.) Boud Uncinula septata E.S. Salmon Vibrissea albofusca G.W. Beaton Vibrissea Xavovirens (Pers.) Korf and J.R. Dixon Vibrissea truncorum (Alb. and Schwein.) Fr. Xylaria hypoxylon (L.) Grev AY789379 AY046271 AF274109 AY789364 AJ001986 U72603 AY424225 AY584681 AF356688 AY789327 AY789390 AY524846 AY293777 Z81388 AY487093 AY487099 AB015778 AF096184 U53370 AY584678 AF110351 UNPUBL. J01353 N/A AY789298 AY789406 AY789346 UNPUBL. AY789356 AF119500 AY789312 AB183530 AY789382 AY789425 AY789401 U20378 AY789380 AF286411 AF274097 AY789365 AJ261148 AY261178 AF286759 AF356689 AF356689 AY789328 AY789391 AF269219 AY293790 Z81415 AY487092 AY487098 AB040690 AF096198 UNPUBL. AY584654 AY779329 UNPUBL. J01355 AY789409 AY789299 AY789407 AY789347 UNPUBL. AF433142 AF274103 AY789313 AB183532 AY789383 AY789426 AY789402 AF132333 AY789381 AF388914 AF329174 AY789366 U51952 U72595 AF158645 AY257904 N/A AY789329 AY789392 AY347413 AY293802 Z81441 AY487091 AY487097 AF062819 AY129286 AY465516 N/A AJ634045 UNPUBL. AY247400 AY789410 AY789300 AY789408 AF455526 UNPUBL. AF433152 AF274081 AY789314 AB183533 AY789384 AY789427 AY789403 AF163035 Information about unpublished sequences is available from the AFTOL website. (SwoVord, 1999) and MrBayes 3.1.1 (Huelsenbeck and Ronquist, 2001), with gaps treated as missing data. Parsimony analyses were performed using equal weighting of characters and transformations. Heuristic searches were performed with one thousand replicate searches, each with one random taxon addition sequence, MAXTREES set to autoincrease, and TBR branch swapping. Robustness of individual branches was estimated by maximum parsimony bootstrap proportions (BP), using 500 replicate, each consisting of a single heuristic search with 50 random taxon addition sequences, MAXTREES set to autoincrease, and TBR branch swapping. Bayesian phylogenetic analyses were performed using the Metropolis-coupled Markov chain Monte Carlo method (MCMCMC) under the GTR++I model, which was identiWed as the optimal model using Modeltest version 3.5 (Posada and Crandall, 1998), in MrBayes 3.1.1 by running four chains with 2,000,000 generations. Trees were sampled every 100th generation. Likelihoods converged to a stable value after ca. 500,000 generations in the wider-range analyses and after ca. 100,000 generations in the narrower-range analysis, and all trees obtained prior to con- vergence were discarded before computing a consensus tree in PAUP¤. Bayesian posterior probabilities (PP) were obtained from the 50% majority-rule consensus of the remaining trees, and clades with PP 7 0.95 were considered to be signiWcantly supported. 3. Results 3.1. Phylogenetic inference from data set one (wider-range analyses) Relationships among the Helotiales and other groups in the Leotiomycetes were investigated using three rDNA regions (LSU + SSU + 5.8S) from 95 taxa. The combined genes had an aligned length of 2020 bp (14 positions were excluded from the analyses) with 266 uninformative variable positions and 647 parsimony-informative positions. Equally weighted parsimony analysis yielded 35 equally parsimonious trees of 4557 steps with a consistency index CID0.323 (Fig. 1). Although the inoperculate discomycetes were supported (BPD70%), the backbone of the Leotiomycetes received no support. The Leotiomycetes was not 300 Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 Arthrocladiella mouqeotii Blumeria graminis Uncinula septata 100 Chlorociboria sp. Chlorociboria aeruginosa * Cordierites frondosa Peltula umbilicata Cyttaria darwinii Data set one, * 65 Byssoascus striatisporus Myxotrichum deflexum parsimony analyses 85 Leotia lubrica 96 Microglossum rufum Microglossum olivaceum Microglossum sp * Bulgaria inquinans * * Holwaya mucida 95 Pseudogymnoascus roseus Pseudeurotium zonatum 78 Neofabraea malicorticis 81 Neofabraea alba Dermea acerina * Chlorencoelia versiformis B--Bulgariaceae 57 * Hemiphacidium longisporum D--Dermateaceae 97 100 Heyderia abietis Heyderia abietis H1--Helotiaceae Fabrella tsugae H2--Hemiphacidiaceae Meria laricis 90 Ciboria batschiana * 97 H3--Hyaloscyphaceae 100 67 Sclerotinia sclerotiorum 53 G--Geoglossaceae Monilinia laxa 96 Scleromitrula shiraiana L1--Leotiaceae 95 Rutstroemia bolaris L2--Loramycetaceae Piceomphale bulgarioides Bisporella citrina R--Rutstroemiaceae 94 Ascocoryne calichnium S--Sclerotiniaceae 89 Ascocoryne sarcoides 85 Ascocoryne turficola V--Vibrisseaceae Chloroscypha sp. A--Anamorphic ascomycete Neobulgaria pura 59 Chlorovibrissea sp. Vibrissea albofusca * 58 Lachnum virgineum Lachnum bicolor Bryoglossum * 99 Cudoniella clavus gracile 87 Cudoniella clavus Ombrophila violacea Hymenoscyphus scutula * 97 Vibrissea flavovirens 82 Vibrissea truncorum 99 * Phialocephala fortinii 98 Loramyces juncicola Leotiomycetes Mollisia cinerea (excluding the Hyaloscypha daedalae 100 Mitrula brevispora Geoglossum clade) 56 Mitrula paludosa Gremmeniella abietina Hydrocina chaetocladia 100 Cudonia sp. Spathularia flavida 100 Rhytisma sp. Lophodermium pinastri 100 Pilidium acerinum Pilidium concavum 100 Peltigera aphthosa Peltigera degenii 82 Ochrolechia parella Trapelia placodioides Dibaeis baeomyces 62 Lecanora concolor Cladonia caroliniana Leotiomyceta 100 Berlesiella nigerrima 61 Capronia mansonii 100 Eupenicillium javanicum Eurotium amstelodami 70 Mycocalicium polyporaeum 100 Roccella tuberculata 100 Roccella fuciformis Arthonia sp. Phoma herbarum 100 Dothidea sp. Dothidea sambuci 55 Botryosphaeria ribis 93 Geoglossum glabrum 89 Geoglossum umbratile 98 Trichoglossum hirsutum 99 72 Sarcoleotia globosa Sarcoleotia cf. globosa 100 Orbilia delicatula Orbilia auricolor 100 Peziza phyllogena Peziza varia 100 Saccharomyces cerevisiae Candida albicans Neolecta irregularis 84 96 Erysiphales H1 H1 H1 LICHINOMYCETES Cyttariales Myxotrichaceae L1 G G Leotia-Bulgaria clade G B H1 Myxotrichaceae ? D Dermea clade D D H1 H2 H1 Hemiphacidium clade H1 H2 H2 S S S Sclerotinia clade R R R H1 H1 H1 Ascocoryne clade H1 H1 L1 V V H3 Lachnum clade H3 H1 H1 H1 Hymenoscyphus clade H1 H1 V V A Vibrissea-Loramyces clade L2 D H3 S S Mitrula clade H1 H1 Rhytismatales Pilidium clade LECANOROMYCETES EUROTIOMYCETES ARTHONIOMYCETES DOTHIDEOMYCETES G G Geoglossum clade G H1 H1 ORBILIOMYCETES PEZIZOMYCETES SACCHAROMYCETES NEOLECTOMYCETES 10 changes Fig. 1. Phylogenetic relationships among Helotiales and Leotiomycetes based on three rDNA regions (data set one) using parsimony analyses. ClassiWcations follow Eriksson (2005) and family names are abbreviated and listed next to the corresponding genus. Clades discussed in this study are in boldface type. One of the 35 most parsimonious trees (Length D 4557, CI D 0.323, RI D 0.517). Bootstrap values greater than 50% are indicated along nodes, branches that collapse in the strict consensus tree are marked with asterisks. Exceedingly long branches are dashed. Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 monophyletic due to the placement of the Geoglossum clade (BP D 98%) including species of Geoglossum, Trichoglossum, and Sarcoleotia. Except for Peltula umbilicata (Lichinales), which apparently groups with Corderites frondosa possibly due to long branch attraction, members of the Dothideomycetes, Lecanoromycetes, Eurotiomycetes, and Arthoniomycetes were placed in between the Geoglossum clade and the remaining Leotiomycetes. Excluding the Geoglossum clade, the other helotialean fungi of the Cyttariales, Erysiphales, Rhytismatales, and Myxotrichaceae formed a clade with Pilidium (anamorph)/ Discohaninesia (teleomorph) species as a basal branch without bootstrap support. Here, we regard this clade as the Leotiomycetes, and the Geoglossum clade was excluded from both the Helotiales and the Leotiomycetes. The monophyly of the Helotiales was not strongly supported. Overall, the tree was not well resolved and support for the backbone of the tree was weak. Species of Chlorociboria (Helotiaceae) formed a clade with the Erysiphales, and Cordierites frondosa (Helotiaceae) formed a clade with Peltula umbilicata (Lichinales) and Cyttaria darwinii (Cyttariales), however, these relationships were not supported by bootstrap values. Relationships among the Helotiales, Erysiphales, Cyttariales, and Myxotrichaceae were not resolved. Although most families in the Helotiales were not monophyletic, some clades can be recognized with substantial support within the Helotiales: (1) the Dermea clade, including three species in the Dermateaceae, Neofabraea malicorticis, N. alba, and Dermea acerina, formed a lineage (BP D 81%) with an unresolved position in the strict consensus tree. (2) The Hemiphacidium clade, including three species of the Hemiphacidiaceae, Hemiphacidium longisporum, Fabrella tsugae, and Meria laricis, and two species of the Helotiaceae, Chlorencoelia versiformis and Heyderia abietis, was strongly supported (BP D 97%). (3) The Sclerotinia clade, including a subclade (BP D 100%) of three species of the Sclerotiniaceae, Ciboria batschiana, Sclerotinia sclerotiorum, and Monilinia laxa, and two species in the Rutstroemiaceae, Scleromitrula shiraiana and Rutstroemia bolaris, and Piceomphale bulgarioides, received strong support (BP D 95%). (4) The Ascocoryne clade included species of Ascocoryne, Chloroscypha, and Neobulgaria pura on a long branch, but Ascocoryne and Chloroscypha species were closely related (BP D 85%). (5) The Lachnum clade composed of two Lachnum species (BP D 58%) and Bryoglossum gracile was not supported. (6) The Hymenoscyphus clade including Cudoniella clavus and Ombrophila violacea, was supported (BP D 87%), with Hymenoscyphus scutula as the sister group. (7) The Vibrissea-Loramyces clade was strongly supported (BP D 99%), and within the clade, close relationships between Vibrissea and Phialocephala (BP D 82%), and between Loramyces and Mollisia (BP D 98%) received support. (8) The Mitrula clade included a weakly supported group (BP D 56%) of Mitrula species and Gremmeniella abietina, and Hydrocina chaetocladia. (9) The Leotia-Bulgaria clade, including species of Leotia, Microglossum, Bulgaria, and Holwaya, collapsed in 301 the strict consensus tree, however, these four genera and two species of the Myxotrichaceae were grouped together by all analyses. A clade including Leotia lubrica, Microglossum rufum, and M. olivaceum collected from the Northern Hemisphere was supported (BP D 96%), with a Microglossum species from New Zealand as the sister group (BP < 50%). Relationships among those nine clades were not resolved, except for a sister relationship between the Hemiphacidium clade and the Sclerotinia clade (BP D 97%). There was no signiWcant conXict between the results of the Bayesian analysis of data set one (Fig. 2) and the results from parsimony analyses, however, support for the clades and deeper nodes of the tree from Bayesian analyses were generally higher. The Geoglossum clade received strong support (PP D 1.0), and its basal position within the superclass Leotiomyceta was upheld (PP D 1.0). The Lecanoromycetes, Eurotiomycetes, Arthoniomycetes, and Dothideomycetes were all supported as monophyletic groups (PP D 1.0), but the relationships among those groups received no support (PP D 0.53–0.77). The Leotiomycetes were supported as monophyletic with PP D 1.0. The Helotiales was not resolved as monophyletic. Chlorociboria species shared a clade with the Cyttariales and the Erysiphales (PP D 0.98), and Cordierites frondosa shared a clade with the Myxotrichaceae (PP D 1.0). Within the Helotiales, clades recognized in the parsimony analysis were recovered in the Bayesian analysis, even though support for the backbone of this part of the tree was weak (PP D 0.53–0.88). Contents of the Dermea clade (PP D 1.0), Hemiphacidium clade (PP D 1.0), Lachnum clade (PP D 0.95), Ascocoryne clade (PP D 1.0), Sclerotinia clade (PP D 1.0), and Mitrula clade (PP D 0.98), were the same as in the parsimony analysis, but received much stronger support. The Vibrissea-Loramyces clade was strongly supported (PP D 1.0), and within the clade, close relationships between Vibrissea and Phialocephala, and between Loramyces and Mollisia were conWrmed with PP D 1.0. Two New Zealand isolates, Chlorovibrissea sp. and Vibrissea albofusca, formed a lineage sister to the Vibrissea-Loramyces clade without strong support (PP D 0.86). The Leotia-Bulgaria clade was not resolved in the Bayesian analysis, and there was no support for a clade including Leotia lubrica and all Microglossum species. Relationships among the helotialean clades were not resolved with statistic support, except for the sister relationship between the Hemiphacidium clade and the Sclerotinia clade (PP D 1.0). 3.2. Phylogenetic inference from data set two (narrowerrange analyses) Relationships within the Helotiales were examined using three rDNA regions (LSU + SSU + 5.8S) from 82 taxa, with an aligned length of 2020 bp (14 were excluded from the analyses) including 242 uninformative variable positions and 628 parsimony-informative positions. 302 Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 Data set one, Bayesian analyses B--Bulgariaceae D--Dermateaceae H1--Helotiaceae H2--Hemiphacidiaceae H3--Hyaloscyphaceae G--Geoglossaceae L1--Leotiaceae L2--Loramycetaceae R--Rutstroemiaceae S--Sclerotiniaceae V--Vibrisseaceae A--Anamorphic ascomycete Leotiomycetes Leotiomyceta V Vibrissea flavovirens V Vibrissea truncorum A Phialocephala fortinii Vibrissea-Loramyces clade L2 Loramyces juncicola D Mollisia cinerea Chlorovibrissea sp. V V Vibrissea albofusca H1 Cudoniella clavus H1 Hymenoscyphus clade Cudoniella clavus H1 Ombrophila violacea H1 Hymenoscyphus scutula Lachnum virgineum H3 Lachnum bicolor H3 Lachnum clade Bryoglossum gracile H1 Mitrula brevispora S Mitrula paludosa S H1 Mitrula clade Gremmeniella abietina Hydrocina chaetocladia H1 Arthrocladiella mouqeotii Blumeria graminis Erysiphales Uncinula septata H1 Chlorociboria sp. Chlorociboria aeruginosa H1 Cyttariales Cyttaria darwinii D Neofabraea malicorticis Neofabraea alba D Dermea clade Dermea acerina D Hyaloscypha daedalae H3 Chlorencoelia versiformis H1 Hemiphacidium longisporum H2 H1 Hemiphacidium clade Heyderia abietis H1 Heyderia abietis H2 Fabrella tsugae H2 Meria laricis S Ciboria batschiana S Sclerotinia sclerotiorum Monilinia laxa S Sclerotinia clade Scleromitrula shiraiana R Rutstroemia bolaris R Piceomphale bulgarioides R Ascocoryne calichnium H1 Ascocoryne sarcoides H1 Ascocoryne turficola H1 Ascocoryne clade Neobulgaria pura L1 Chloroscypha sp H1 Cordierites frondosa H1 Myxotrichum deflexum Myxotrichaceae Byssoascus striatisporus Bisporella citrina H1 Cudonia sp Spathularia flavida Rhytismatales Lophodermium pinastri Rhytisma sp. Pilidium acerinum Pilidium clade Pilidium concavum L1 Leotia lubrica Microglossum rufum G Microglossum olivaceum G Leotia-Bulgaria clade Microglossum sp. G Pseudogymnoascus roseus Myxotrichaceae ? Pseudeurotium zonatum Holwaya mucida H1 Bulgaria inquinans B Peltigera aphthosa Peltigera degenii Ochrolechia parella Trapelia placodioides LECANOROMYCETES Dibaeis baeomyces Lecanora concolor Cladonia caroliniana Berlesiella nigerrima Capronia mansonii EUROTIOMYCETES Eupenicillium javanicum Eurotium amstelodami Mycocalicium polyporaeum Peltula umbilicata Roccella tuberculata Roccella fuciformis ARTHONIOMYCETES Arthonia sp. Phoma herbarum Botryosphaeria ribis DOTHIDEOMYCETES Dothidea sp Dothidea sambuci G Geoglossum glabrum Geoglossum umbratile G Geoglossum clade Trichoglossum hirsutum G Sarcoleotia globosa H1 Sarcoleotia cf. globosa H1 Peziza phyllogena PEZIZOMYCETES Peziza varia Orbilia delicatula ORBILIOMYCETES Orbilia auricolor Saccharomyces cerevisiae SACCHAROMYCETES Candida albicans Neolecta irregularis NEOLECTOMYCETES Fig. 2. Phylogenetic relationships among the Helotiales and the Leotiomycetes inferred from three rDNA regions (data set one) using Bayesian approaches under the GTR++I model. ClassiWcations follow Eriksson (2005), and family names are abbreviated and listed next to the corresponding genus. Majority-rule consensus tree of 19,000 MCMCMC-sampled trees. Group frequencies greater than 0.95 are indicated as bold branches. Equally weighted parsimony analysis yielded 69 equally parsimonious trees of 3964 steps and consistency index CI D 0.349 (Fig. 3). The strict consensus tree based on the 69 trees was much better resolved than the one based on the 35 trees in the wider-range analyses. The Geoglossum clade (BP D 99%) formed the basal branch within the Leotiomyceta (BP D 67%). The Helotiales was monophyletic (BP < 50%). The Hemiphacidium clade (BP D 97%) was composed of a subclade (BP D 87%) of Fabrella tsugae and Meria laricis, a subclade (BP D 53%) of Chlorencoelia versiformis and Heyderia abietis, and Hemiphacidium longisporum. The Sclerotinia clade (BP D 93%) included species of Scleromitrula, Rutstroemia, Piceomphale, and a subclade of Ciboria batschiana, Sclerotinia sclerotiorum, and Monilinia Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 303 Heyderia abietis Heyderia abietis Chlorencoelia versiformis Hemiphacidium clade 97 Hemiphacidium longisporum Fabrella tsugae 87 Meria laricis 67 Ciboria batschiana 98 100 Sclerotinia sclerotiorum 53 Monilinia laxa 95 Sclerotinia clade Scleromitrula shiraiana 93 Rutstroemia bolaris Piceomphale bulgarioides 77 Neofabraea malicorticis Neofabraea alba Dermea clade 85 Dermea acerina 94 Ascocoryne calichnium 89 Ascocoryne sarcoides 94 Ascocoryne turficola Ascocoryne clade Chloroscypha sp. 63 Neobulgaria pura 95 Vibrissea flavovirens 79 Vibrissea truncorum 99 Phialocephala fortinii 99 Loramyces juncicola Vibrissea-Loramyces clade Mollisia cinerea 62 Chlorovibrissea sp. Vibrissea albofusca * 99 Cudoniella clavus 86 Cudoniella clavus Hymenoscyphus clade Ombrophila violacea * Hymenoscyphus scutula 55 Lachnum virgineum Lachnum bicolor Lachnum clade Bryoglossum gracile 100 Mitrula brevispora 51 Mitrula paludosa Mitrula clade Gremmeniella abietina Hydrocina chaetocladia 94 Leotia lubrica 100 Microglossum rufum 58 Microglossum olivaceum Leotia-Bulgaria clade Microglossum sp. Bulgaria inquinans Holwaya mucida Botryosphaeria ribis Phoma herbarum 100 Dothidea sp. Dothidea sambuci 100 Pilidium acerinum Pilidium concavum 100 Cudonia sp. 100 Spathularia flavida Lophodermium pinastri Rhytisma sp. * 100 Berlesiella nigerrima 55 Capronia mansonii 100 Eupenicillium javanicum * Eurotium amstelodami Mycocalicium polyporaeum 100 Roccella tuberculata 100 Roccella fuciformis * Arthonia sp. Peltula umbilicata 80 Ochrolechia parella Trapelia placodioides Dibaeis baeomyces 56 Lecanora concolor Cladonia caroliniana 100 Peltigera aphthosa Peltigera degenii 84 Geoglossum glabrum 92 Geoglossum umbratile Trichoglossum hirsutum Geoglossum clade 73 Sarcoleotia globosa Sarcoleotia cf. globosa Orbilia delicatula Orbilia auricolor Peziza phyllogena Peziza varia Saccharomyces cerevisiae Candida albicans 100 53 Data set two, parsimony analyses Helotiales Leotiomyceta 67 54 99 99 100 100 100 Neolecta irregularis * 10 changes Fig. 3. Phylogenetic relationships within the Helotiales inferred from three rDNA regions (data set two) using parsimony analysis. One of the 69 most parsimonious trees (Length D 3964, CI D 0.349, RI D 0.539). Bootstrap values greater than 50% are indicated along nodes, branches that collapse in the strict consensus tree are marked with asterisks. laxa (BP D 100%). The Dermea clade (85%) included a subclade of Neofabraea species (77%) and Dermea acerina. The Ascocoryne clade (BP D 63%) was weakly supported with Neobulgaria pura as the sister lineage to the core clade including species of Ascocoryne and Chloroscypha (BP D 94%). The Vibrissea-Loramyces clade (BP < 50%) was composed of a southern lineage (99%) of Chlorovibrissea sp. and Vibrissea albofusca, and a northern lineage (99%) including two clades: one clade of Vibrissea species and Phialocephala fortinii (BP D 79%), and another of 304 Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 Loramyces juncicola and Mollisia cinerea (99%). The Hymenoscyphus clade collapsed in the strict consensus tree, but the close relationship between Cudoniella and Ombrophila species was supported (BP D 86%). The Lachnum clade was not supported (BP < 50%) and Lachnum species received weak support (BP D 55%). The Mitrula clade, including species of Mitrula, Gremmeniella, and Hydrocina chaetocladia, received no support (BP < 50%). The Leotia-Bulgaria clade was resolved in the strict consensus tree, and the New Zealand Microglossum species clade was weakly supported (BP D 51%) as the sister group to a subclade including the northern collections of Leotia and Microglossum (BP D 100%). The sister group relationship between the Hemiphacidium clade and the Sclerotinia clade was highly supported (98%). The Leotia-Bulgaria clade was positioned as the basal branch in the Helotiales in this analysis without support. The Vibrissea-Loramyces clade, Hymenoscyphus clade, Lachnum clade and the Mitrula clade formed a monophyletic group without support (BP < 50%). 4. Discussion 4.1. Limits and relationships of the Helotiales in the Leotiomycetes Both the wider-range and narrower-range analyses suggest that Geoglossum species and related fungi form a basal lineage in the Leotiomyceta, and that the relationship between this lineage and other members of the Leotiomycetes is distant. This result agrees with previous studies in separating the Geoglossum clade from other Leotiomycetes (e.g., Lutzoni et al., 2004; Reeb et al., 2004). However, conXicts in the systematic position of the Geoglossum clade remain. The remainder of the Leotiomycetes, which includes the Cyttariales, Helotiales, Erysiphales, Rhytismatales, and the Myxotrichaceae, was supported as a monophyletic group in both wider- and narrower-range analyses. Although the majority of relationships within the Leotiomycetes were not resolved with strong statistical support, the Erysiphales and the Rhytismatales were strongly supported as monophyletic. Studies based on ascocarp development and rDNA phylogenies suggested a placement of the Myxotrichaceae in the inoperculate ascomycetes (Sugiyama and Mikawa, 2001; Tsuneda and Currah, 2004), and our results support including this family in the Leotiomycetes. However, monophyly of the Myxotrichaceae is not supported in this study, and more data are needed to examine the relationships between the Myxotrichaceae, Pseudeurotiaceae, and saprotrophic helotialean fungi. The most surprising relationship within the Leotiomycetes is a clade including the Erysiphales, Cyttariales, and Chlorociboria species (Figs. 1 and 2). Given the striking diVerence in macromorphology between these fungi, this relationship could be an artifact of insuYcient informative characters, and/or unbalanced taxon sampling. Nevertheless, some signiWcant aspects of these fungi are worth men- tioning here. The Erysiphales is one of the most intensively studied groups of the Leotiomycetes since they are obligate plant pathogens, causing powdery mildew diseases on plant species (Matsuda and Takamatsu, 2003). Species of the Erysiphales reproduce sexually by means of ascospores within asci in completely closed, minute ascocarps on leaves, and there are no morphological features supporting the molecular data linking these fungi to the Leotiomycetes (Gargas and Taylor, 1995). Some lineages of the Erysiphales apparently have a geographic origin in the Southern Hemisphere, with subsequent dispersal throughout the Northern Hemisphere (Bremer, 1994; Takamatsu and Matsuda, 2004). The Cyttariales, containing a single genus, Cyttaria, is composed of about a dozen species. Cyttaria species are parasites on the Southern Hemisphere beech, Nothofagus, in southern South America, Australia, and New Zealand (Gamundí, 1991). The systematic position of the Cyttariales remains unclear, with inconsistent results from morphological studies and molecular phylogenies (Carpenter, 1976; Korf, 1983; Landvik, 1996). Chlorociboria species generally produce a blue-green staining on fallen wood. Fifteen species, including 13 new species, were reported from New Zealand based on morphological characters and ITS sequence data, and a possible Asian/Australasian center of diversity for the Chlorociboria was suggested (Johnston and Park, 2005). With the Erysiphales, Cyttariales, Myxotrichaceae, and species of Chlorociboria and Cordierites frondosa excluded, results from the narrower-range analyses supported the Helotiales as a monophyletic group with the Rhytismatales and Discohaninesia/Pilidium (traditionally placed in the Helotiales family Dermateaceae) as the sister group (Figs. 3 and 4). 4.2. Phylogenetic and ecological diversity of the Helotiales The limited sampling and the poorly resolved phylogenetic relationships in this study make it premature to present a revised taxonomy of the Helotiales. ClassiWcation is an important prerequisite for the ecological and biological study of organisms, and the major purpose of this study is to provide a framework for future phylogenetic classiWcations. With a few exceptions, our results are more or less congruent with the current classiWcation of the Helotiales at a higher level (Eriksson, 2005). Some clades are not strongly supported by molecular characters, and in these cases, characters of morphology, ecology, and biology are used to deWne the clade. 4.2.1. Phylogenetic distribution of ecological and biological characters Biological relationships of helotialean fungi in ecosystems are diverse, and members of the Helotiales have been described as plant pathogens, endophytes, nematode-trapping fungi, mycorrhiza-forming (including ectomycorrhizae and ericoid mycorrhizae), ectomycorrhizal parasites, fungal parasites, terrestrial saprobes, aquatic saprobes, root Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 305 Fig. 4. Phylogenetic relationships within the Helotiales based on three rDNA regions (data set two) using Bayesian approaches under the GTR++I model. The majority-rule consensus of 19,000 MCMCMC-sampled trees. The resulting posterior probabilities (PP) greater than 0.90 are shown above branches. symbionts, and wood rot fungi (Boddy, 2001; Grünig et al., 2002; Grünig and Sieber, 2005; Hosoya and Otani, 1995; Johnston and Park, 2005; Monreal et al., 1999; Platt, 2000; Pöder and Scheuer, 1994; Shoemaker et al., 2002). Endophytes represent putative symbiotic interactions between fungi and plants and live within plant tissues with- out producing noticeable symptoms. Endophytic fungi have been found in various vegetative organs and from a broad range of plant hosts, and they can inXuence the distribution, ecology, and biology of plants (Arnold et al., 2003; Carroll, 1988; Sridhar and Raviraja, 1995). Fungi termed endophytes have a wide range of lifestyles 306 Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 (Stone et al., 2004) and amongst the Leotiomycetes include the Sclerotiniaceae, Rutstroemiaceae, Hemiphacidiaceae, Phacidiaceae, the Hyaloscyphaceae, Dermateaceae, Bulgariaceae, and Helotiaceae in the Helotiales, as well as the Rhytismatales, Erysiphales, and probably the Cyttariales (Fig. 4) (Egger and Sigler, 1993; Johnston, 1989; Platt, 2000; Rossman et al., 2004; Vrålstad et al., 2002a,b; Wilson et al., 2004). Such a broad distribution of the endophytic lifestyle suggests it could be plesiomorphic in the Helotiales, but inadequate information of biology and poorly resolved phylogeny within the Leotiomycetes make it premature to reconstruct the ancestral lifestyle in the Helotiales. Fungal endophytes are mainly ascomycetes, and the endophytic lifestyle may play an important role in the evolution of the higher ascomycetes. Endophytes are able to colonize host tissue early and occupying the habitat puts them in a good position to make a shift to parasitism (when hosts are under stress) or to saprophytism (after hosts die). Many helotialean fungi are collected from fallen leaves, dead ferns, and herbaceous debris, and were recorded as saprobes, whereas current studies using molecular probes suggest that at least some of them have endophytic stages or are closely related to endophytes (Abeln et al., 2000; Cabral, 1985; Johnston, 1998; Monreal et al., 1999). 4.2.2. Clades The clades discussed below are named after the representative genera as well as important morphological, ecological, and/or biological characters (in parentheses). Biological relationships among helotialean clades are discussed on the basis of the rDNA phylogeny. 4.2.2.1. Geoglossum clade (black terrestrial saprobe clade)—Geoglossomycetes. Species of the genera Geoglossum, Trichoglossum, and Sarcoleotia are included in this clade. The concept of the Geoglossaceae has been changed and modiWed recently (Eriksson, 2005; PWster and Kimbrough, 2001; Platt, 2000; Spooner, 1987; Wang et al., 2002, 2005). The separation of the Geoglossaceae from other helotialean fungi has been suggested in previous studies, and paraphyses with dark pigments and dark ascospores with multiple septa were considered as unique characters deWning this group (Platt, 2000; Lutzoni et al., 2004). Our results suggest a clade including species of Geoglossum, Trichoglossum, and Sarcoleotia are holding the basal position in the superclass Leotiomyceta with strong support, and a new class, the Geoglossomycetes, is proposed for this clade. Color and the number of septa in the ascospores of Geoglossum and Trichoglossum are variable among species and with ascus age (Zhuang, 1998), and thus should not be considered as a consistent morphological character for this clade. Sarcoleotia globosa produces pileate, black apothecia, and hyaline ascospores with 0–5 septa, and has been included in the Helotiaceae (Schumacher and Sivertsen, 1987). Paraphyses (or homologous structures) cover the stipe surface in Geoglossum and Trichoglossum, and obscure the boundary of the fertile hymenium (Spooner, 1987), a phenomenon not known from other inoperculate ascomycetes. Similar to Geoglossum, the pileate apothecia in S. globosa have a hymenium that is continuous with the stipe at an early stage, and then recedes from the stipe to form a pileate like fruit body (Schumacher and Sivertsen, 1987). This diVers from other helotialean fungi with pileate apothecia such as species of Cudoniella and Leotia, which have a hymenium that is bounded by the edge of the excipulum. Species of Microglossum, Thuemenidium, and Bryoglossum also have a distinct hymenium boundary, and this shared morphological character supports the molecular evidence that these genera should be removed from the Geoglossaceae. Asexual stages are unknown for most species in this clade, and apothecia of these fungi are most commonly found in associated with mosses (Imai, 1941; Jumpponen et al., 1997; Schumacher and Sivertsen, 1987). Species of both Geoglossum and Trichoglossum have a worldwide distribution, while Sarcoleotia globosa is so far mainly known from temperate areas in the Northern Hemisphere (Schumacher and Sivertsen, 1987; Spooner, 1987; Zhuang and Wang, 1998). 4.2.2.2. Ascocoryne clade (gelatinous endophyte clade). Species of three small genera Ascocoryne, Neobulgaria, and Chloroscypha are included in this clade, a lineage not previously recognized in the Helotiales. The presence of gelatinous tissue seems of limited use in recognizing phylogenetic relationships. Moore (1965) studied the gelatinous tissue in the Leotiomycetes and suggested four diVerent developmental types: the coryneoid type, the cudonioid type, the leotioid type, and the bulgarioid type. Ascocoryne species have the coryneoid type while Neobulgaria and Leotia species have the leotioid type. The apothecia of Chloroscypha are only slightly gelatinous, and a gelatinous substance maybe also excreted from the paraphyses (Dennis, 1968; Petrini, 1982; Seaver, 1931, 1951). Baral (1987) studied the ring-like amyloid structures of ascus apices using light microscopic techniques and suggested that species of Chloroscypha, Neobulgaria, and perhaps Ascocoryne have an ascus apparatus similar to species of Sclerotiniaceae. Apothecia of Chloroscypha species can be induced in vitro from the foliage of host plants, but ascospores collected from the apothecia fail to develop after germination (Petrini, 1982). This indicates that after successfully colonizing the host tissue and establishing the endophytic lifestyle, species of Chloroscypha may be capable of completing their life cycles as saprobes. Ascocoryne sarcoides has been considered to be protective against decay fungi as an endophyte and is found more frequently in roots than in stems (Basham, 1973; Whitney, 1995; Whitney et al., 2002). Fungi in this clade have a worldwide distribution. 4.2.2.3. Dermea clade (bark endophyte clade). Three species of Dermea and Neofabraea in the Dermateaceae are included in this clade. The Dermateaceae is a large, poorly Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 studied, and heterogeneous family (PWster and Kimbrough, 2001; Raitviir and Spooner, 1994). Previous studies based on the ITS region suggest that our Dermea clade may also including species of Pezicula, Ocellaria, Dermea, and Neofabraea (Abeln et al., 2000; De Jong et al., 2001; Goodwin, 2002; Verkley, 1999). The only other member of the Dermateaceae we sampled, Mollisia, is not in this clade. Morphologically, species of the Dermea clade produce erumpent or superWcial, Xeshy and small apothecia on plants, with an excipulum consisting of rounded cells with often dark walls. The hymenium in several genera in the Dermateaceae are covered by an ‘epithecium’, a gelatinized structure composed of tips of the paraphyses and extracellular material (Verkley, 1999). Many species of the Dermea clade produce two types of conidia, i.e., macro and microconidia, and both anamorphic and teleomorphic stages can be observed on the same stroma. Most species of Pezicula are pioneers that colonize twigs and branches just before they die back, typically while they remain held oV the ground (Verkley, 1999). Such species are probably endophytes living in inner bark. Mature ascospores in Pezicula usually are septate and thick walled and embedded in the gelatinized epithecium, and they could be transferred and dispersed via feeding activities of insects or in the insects gut. Insects are well-known vectors of fungal pathogens (Saikkonen et al., 1998; Vega and Blackwell, 2005), and there are several lineages of endosymbionts in beetles’ guts having independent origins in pathogenic ascomycetes (Suh et al., 2001). Data from Pezicula show that some species have a narrow host range, and some are even only known from a single host species (Taylor, 1983; Verkley, 1999), and this again raises the issue about vectors, particularly insects. There are some plant pathogens as well in this family, for instance, Diplocarpon rosae, which causes a very serious rose black-spot disease. Although poorly studied from the Southern Hemisphere, genera in this clade are world wide in distribution. At least six species of Pezicula or Neofabraea occur in New Zealand, and most of these are undescribed and some are known only from culture from studies of plant endophytes (P.R. Johnston and S. Joshee, unpublished data). 4.2.2.4. Hemiphacidium clade (gymnosperm leaf endophyte clade). The genera Heyderia and Chlorencoelia and three genera in the Hemiphacidiaceae, Fabrella, Hemiphacidium, and Meria (anamorph of Rhabdocline, Gernandt et al., 1997) are included in this clade. The Hemiphacidiaceae, proposed by Korf (1962), has been thought to be a small family in the Helotiales, but our results suggest it may need expanding to include more genera previously placed in the Helotiaceae. Stone and Gernandt (2005) proposed Sarcotrochila as the valid name for Hemiphacidium, but they were undecided about the limits of the family Hemiphacidiaceae sensu Korf, so we retain the traditional names to limit confusion. All members of the Hemiphacidiaceae sensu Korf produce small, simple apothecia beneath the surface of leaves, 307 and the apothecia are erumpent and push the covering host tissue back as a small scale (Korf, 1962). The ectal excipulum in these apothecia is highly reduced. In contrast, Heyderia abietis and Chlorencoelia versiformis form large, well-developed apothecia. Species in the Hemiphacidiaceae are plant pathogens or endophytes, and typically cause needle-blight or needle-cast disease. Species of Heyderia and Chlorencoelia have been regarded as saprobes, and H. abietis has been thought of as a decomposer of spruce needles in Europe. Endophytic stages of two Heyderia species have been discovered recently using molecular markers (Jean Bérubé, per. comm.). Chlorencoelia species can be found from wood of conifers and rotting wood of Quercus and Salix (Dennis, 1968) and they are also common on Nothofagus in New Zealand (http://www.landcareresearch.co.nz). If our results reXect true evolutionary relationships among these fungi, then this suggests a correlation between morphology and biology: i.e., a highly reduced apothecium is associated with a parasitic and endophytic lifestyles as in Hemiphacidium species, while the larger and fully developed apothecium is associated with a saprobic lifestyle as in Chlorencoelia species. A similar adaptation has been reported in the Rhytismatales; pathogens such as Rhytisma and Lophodermium produce simple and small apothecia on host tissues, while saprobes such as Spathularia and Cudonia produce large and complex apothecia on duV (Wang et al., 2002). 4.2.2.5. Hymenoscyphus clade (ericoid root-endophyte— aquatic saprobe clade). The genera Cudoniella, Ombrophila, and Hymenoscyphus included here do not always form a monophyletic group (they were weakly supported as a clade in Wang et al., 2005), and the genus Hymenoscyphus and similar taxa form a morphological group without obvious unifying characters (regarded as a “wastebasket” by Korf, 1973). Diverse ericoid mycorrhizal fungi have been found to be closely related to Hymenoscyphus based on rDNA sequences (Egger and Sigler, 1993; Monreal et al., 1999), but this relationship was not supported by recent studies using ITS (Vrålstad et al., 2002a,b; Zhang and Zhuang, 2004). Although morphologically simple, these fungi are among the most common helotialean taxa in the Weld which have been found on various substrates. Species of Cudoniella, Ombrophila, and many species of Hymenoscyphus produce apothecia on submerged woody substrates or decaying wood in boggy places (Abdullah et al., 1981; Dennis, 1968; Descals et al., 1984; Fisher and Spooner, 1987; Fisher and Webster, 1983; Webster et al., 1995). Members of the H. ericae aggregate form both ecto- and ericoid mycorrhizal symbioses, and have diverse ecological attributes. Some aquatic hyphomycetes have been documented as root endophytes (Sati and Belwal, 2005). Our study provides evidence that root endophytes, saprobic teleomorphs, and aquatic teleomorphs form a clade. However, our conWdence about this relationship is not strong due to the poorly resolved phylogeny. The anamorphs of fungi in this group have been well documented for aquatic species. Various 308 Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 forms of conidia have been recorded from Cudoniella and Hymenoscyphus species, often stressed as evidence for the poor correlation between the classiWcations of teleomorphs and anamorphs (Abdullah et al., 1981; Descals et al., 1984; Fisher and Spooner, 1987; Fisher and Webster, 1983; Marvanova, 1997; Webster et al., 1995). 4.2.2.6. Lachnum clade (hairy endophyte- saprobe clade). Two Lachnum and one Bryoglossum species appear in this clade. Lachnum is a large genus in the Hyaloscyphaceae, which is probably polyphyletic. Abeln et al. (2000) recognized 2 clades of hairy discomycetes, Lachnoideae (equivalent to our Lachnum clade) and Hyaloscyphoideae. Data from other studies (Cantrell and Hanlin, 1997; Wang et al., 2005) suggested that several other genera, such as Hyaloscypha, Trichopezizella, Neodasyscypha, Trichopeziza, Solenopezia, Perrotia, Proliferodiscus, and Lachnellula, belong to this clade, along with Lachnum and Bryoglossum. There are no genera in our study that represent the Hyaloscyphoideae clade of Abeln et al. (2000). Morphologically, fungi in this group are diverse but they are all characterized by various hairs as cellular extensions from the ectal excipulum of the apothecium. Subgroups or tribes have been suggested within the family based on characters of the hairs, excipulum, paraphyses, and asci. Placing Bryoglossum gracile in the Geoglossaceae due to its clubshaped apothecia is artiWcial, since hairs are also present on the stalk of this fungus (Kankainen, 1969; Wang et al., 2005). These fungi occur on various substrates and have been treated as saprobes (e.g., Huhtinen, 1990), but this Wnding may need reassessment in many cases, especially in taxa with a high degree of substrate speciWcity. For example, Bryoglossum gracile is moss-inhabiting (Redhead, 1977), while some Lachnellula and Lachnum species are known to be pathogens on conifers, or are consistently associated with diseased ferns (Spooner, 1987). The life histories of these fungi are barely known, and various conidia, including Phialophora-like conidia, have been reported in Hyaloscypha and allied genera (Huhtinen, 1990). The distribution of hairy helotialean fungi is worldwide, but no collections from the Southern Hemisphere or the tropical regions were included in this study. 4.2.2.7. Leotia-Bulgaria clade (wood and litter decomposer clade). Species of Bulgaria, Holwaya, Microglossum, and Leotia form a clade in the narrower-range analyses. Microglossum species have been included in the Geoglossaceae along with Geoglossum and Trichoglossum primarily based on morphology, and this placement has been supported by ultrastructural studies of the ascus apex (Verkley, 1994). Close relationships between Leotia and Microglossum have been suggested by previous studies based on rDNA or protein-coding gene sequences (e.g., Gernandt et al., 2001; Landvik, 1996; Liu and Hall, 2004). Analyses based on LSU rDNA data also place Thuemenidium in this clade (Z. Wang, unpublished data), a genus traditionally placed in the Geoglossaceae on the basis of morphology. The fungi in this clade are morphologically very diverse. Gelatinous structures are present in both Leotia and Bulgaria but they are classiWed as diVerent types based on anatomy (Moore, 1965). Species of Microglossum, Thuemenidium, and Holwaya produce long, multiseptate, and hyaline ascospores. Characters of ascospores in B. inquinans link this fungus to the Sordariales (Döring and Triebel, 1998), which probably is the sister group of the Leotiomycetes. The biology of these fungi is barely known. In the Northern Hemisphere, Bulgaria inquinans is frequently collected on bark of hardwoods in the Fagaceae, and it may be a weak plant pathogen (Itzerott, 1967 cited by Döring and Triebel, 1998 therein), while Holwaya mucida is mostly found on wood and bark of Tilia (Korf, 1973). Species of Leotia, Microglossum, and Thuemenidium are found usually on humus rich ground, sometimes on decaying wood, but rarely on leaf litter. There are no reports of Holwaya from the Southern Hemisphere, but the genera Leotia, Microglossum, Thuemenidium, and Bulgaria are globally distributed. One New Zealand collection of Microglossum is placed outside of the clade including northern collections of Microglossum and Leotia, which suggests a long isolation period from other Microglossum species. 4.2.2.8. Mitrula clade (leaf parasite-aquatic saprobe clade). Three small genera, Mitrula, Gremmeniella, and Hydrocina are included in this clade without strong bootstrap support. This relationship has not been discovered in previous studies. The position of Mitrula in the Helotiales has been controversial (Eriksson, 2005; Kirk et al., 2001; Wang et al., 2005). Hydrocina chaetocladia and Gremmeniella abietina both produce tiny disc-shaped apothecia with a cream-white hymenium. The receptacle of H. chaetocladia is colorless, with the stalk embedded in a gelatinous substance, while the receptacle of G. abietina is heavily pigmented and sclerotized (Puninthalingam and Gibson, 1973; Webster et al., 1991). The apothecia of Mitrula species are club-shaped with a bright yellow, pinkish-yellow to beige hymenium and have a reduced receptacle. Species of Hydrocina and Mitrula are known as aero-aquatic saprobes, i.e., they live on submerged substrates but produce apothecia above water level (Redhead, 1977; Wang et al., 2005; Webster et al., 1991). Gremmeniella abietina is known as a pathogen of conifers, and causes serious diseases especially to seedlings of pines. G. abietina grows also on artiWcial media (Petrini et al., 1989), implying that this fungus is capable of living as a saprobe. The biology of Mitrula species is still somewhat unclear. Conidia have been induced in vitro and may be adapted to environments such as slow moving water and vernal forest pools (Wang et al., 2005). Hydrocina chaetocladia produces two types of conidia, of which the macroconidia (Tricladium chaetocladium) are adapted to an aquatic environment. G. abietina causes Scleroderrisdisease and produces conidia within a dark-colored stromatic pycnidium. These conidia are able to infect young shoots to start an initial infection (Gremmen, 1968, 1972). Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 Discharge of both conidia and ascospores in G. abietina requires the presence of free water (Skilling, 1969). Fungi in this clade are only known from temperate areas in the Northern Hemisphere. 4.2.2.9. Sclerotinia clade (stromatic pathogen-saprobe clade). Fungi in this clade have been well investigated in previous studies using diVerent rDNA regions (Holst-Jensen et al., 1997a,b, 1998, 1999; Schumacher and Holst-Jensen, 1997), and several pathogenic species are amongst the best studied in the Helotiales (e.g., Dennis, 1968; Dumont, 1971; Dumont and Korf, 1971; Holst-Jensen and Schumacher, 1994; Kohn, 1979, 1982; Kohn and Schumacher, 1984; Korf, 1973; Novak and Kohn, 1991; Spooner, 1987; Zhuang, 1998). Holst-Jensen et al. (1997b) recognized two closely related stromatic (stroma producing) groups, viz. Sclerotiniaceae (sclerotial stromata) and Rutstroemiaceae (substratal stromata), and this relationship is conWrmed in this study. Holst-Jensen et al. (1997b) suggested that Piceomphale bulgarioides should be excluded from the Rutstroemiaceae, but our results suggest a basal position of this spruce endophyte in this clade. Sister relationships between this clade and the Hemiphacidium clade, mostly conifer endophytes with highly reduced apothecia, are strongly supported for the Wrst time. The wider host range in the Sclerotinia clade compared to species in the Hemiphacidium clade suggests that major lineages in the Sclerotinia clade have shifted or expanded from conifer hosts to angiospermous hosts. Except for a few well-known pathogens, the lifestyles of most fungi in this clade are unknown, and they have been described as necrotrophs, opportunistic parasites, saprotrophs, and endophytes. A study of a chestnut pathogen Sclerotinia pseudotuberosa ( D Ciboria batschiana) showed that the fungus occurred asymptomatically in diVerent tissues of the host, and the endophytic behavior may represent a adaptive strategy of the pathogen for rapid and massive host colonization in favorable situations (Vettraino et al., 2005). Representatives of the Rutstroemiaceae are worldwide in distribution, whereas the Sclerotiniaceae may be primarily a northern temperate group. 4.2.2.10. Vibrissea-Loramyces clade (dark septate root endophyte-aquatic saprobe clade). Aero-aquatic Vibrissea, Chlorovibrissea, aquatic Loramyces, dark septate endophyte Phialocephala fortinii, and the wood inhabiting Mollisia are included in this clade. Gernandt et al. (2001) and Wilson et al. (2004) also used molecular evidence to link fungi isolated as root endophytes with aquatic fungal teleomorphs. The family Vibrisseaceae, including Vibrissea and Chlorovibrissea, is not monophyletic. Based on previous studies using data of ITS and or SSU rDNA sequences, two other endophytic genera, Acephala and Rhexocercosporidium, and one plant pathogenic Tapesia species, also belong in this clade (Goodwin, 2002; Grünig and Sieber, 2005; Shoemaker et al., 2002; Wilson et al., 2004). 309 Species of Chlorovibrissea, typically found on submerged or partly submerged wood in streams, and aquatic species of Vibrissea are morphologically similar, except that the former ones are green and probably restricted to the Southern Hemisphere (Kohn, 1989; Korf, 1990). Some species of Vibrissea are not aquatic and produce smaller and sessile apothecia on various substrates with a brown, sclerotium-like base (Iturriaga, 1997). A similar sclerotium-like base in Mollisia places this large, problematic, and probably polyphyletic genus in the Dermateaceae (Dennis, 1968; Korf, 1973). The morphology of Loramyces species is unique and highly adapted to an aquatic environment. Dark cells present at the base of the Loramyces apothecium and the hyphal structure of the apothecia are analogous to those of the Dermateaceae (Digby and Goos, 1987). Dark septate endophytes, Phialocephala species, are characterized by dark-colored and septate hyphae, and are associated with various plant hosts (Grünig et al., 2002). Most genera in this clade include some species, which produce conidia putatively adapted to an aquatic lifestyle. At least one Mollisia species has an aquatic anamorph, producing Helicodendron macroconidia (strongly coiled conidia designed to capture air for Xoating) along with Phialophora-like microconidia (Fisher and Webster, 1983). Two types of conidia are produced in Vibrissea Xavovirens as well (Hamad and Webster, 1988). Fungi in this clade may have a worldwide distribution, except for species of Loramyces (in the Northern Hemisphere) and Chlorovibrissea. Vibrissea albofusca from New Zealand and a Chlorovibrissea species form a weakly supported clade outside of the clade that includes two Northern Hemisphere collections of Vibrissea. Convergent evolution in aquatic environments rather than geographic isolation would be the best explanation for the distant relationships within the Vibrisseaceae. 5. Conclusions Studies of symbiotic relationships between fungi and higher plants have focused mainly on mycorrhizae, plant pathogens or endophytes and their host plants (Saikkonen et al., 1998; Allen et al., 2003). How these relationships aVect the evolution of higher fungi and the diversity of woody plant endophytes, especially higher ascomycetes, has not received much attention. Analyses of data from three rDNA regions with a wide taxonomic sampling in this study improves our understanding of evolutionary relationships within the Helotiales, and provide a framework for future phylogenetic studies of this group. Our study suggests that lifestyle and ecological factors are critical in shaping the evolutionary history of the helotialean fungi. Plant endophytism is a widespread strategy used by members of the Leotiomycetes. Transformations among endophytes, parasites, and saprobes, and shifts between terrestrial and aquatic habitats may be important factors driving the high morphological diversity observed 310 Z. Wang et al. / Molecular Phylogenetics and Evolution 41 (2006) 295–312 in this group of fungi. However, more data from the rDNA regions analyzed here as well as protein-coding genes, and wider sampling from all families recognized in the Helotiales and the Leotiomycetes are required to generate a robust phylogenetic classiWcation and to estimate the ancestral lifestyles of the Helotiales and related fungi. In addition, molecular data from environmental samples, such as plant leaves and roots, insects, soil, and water are needed for a more comprehensive view of the ecology and evolutionary relationships within the Helotiales. Acknowledgments We thank Dr. Ove E. Eriksson, Dr. R.P. Korf and two anonymous reviewers for their very constructive suggestions and comments. Kristin R. Peterson (Harvard University, Massachusetts, USA) kindly provided sequences of Cyttaria species. This study was supported by National Science Foundation Grants DEB-0228657 to D.S.H. and DEB-0128925 to D.S.H. and M.B. and a National Geographic Society Grant 7192-02 to Z.W. and D.S.H. We thank S. Redhead, David Mitchel, Donna Mitchell, D. PWster, H. Knudsen, A. Holst-Jensen, Ch. Scheuer, P.B. Matheny, T. Schumacher, D. Hewitt, A. Wilson, J. Slot, M. Takahasi, S. O. Khattab, and the curators of DAOM, PDD, UWH, CUP, FH, HMAS, HKAS, OSC, NIFG, MBH, and WTU for providing collections. 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