In Press at Mycologia, preliminary version published on June 8, 2012 as doi:10.3852/11-430 Short title: Three new ascomycetes Three new ascomycetes from freshwater in China Dian-Ming Hu State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China International Fungal Research & Development Center, the Research Institute of Resource Insects, Chinese Academy of Forestry, Bailongsi, Kunming 650224, China Lei Cai1 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Kevin D. Hyde1 International Fungal Research & Development Center, the Research Institute of Resource Insects, Chinese Academy of Forestry, Bailongsi, Kunming 650224, China, and Institute of Excellence in Fungal Research and School of Science, Mae Fah Luang University, Chiang Rai, Thailand. Abstract: Three new freshwater ascomycetes, Diaporthe aquatica sp. nov. (Diaporthaceae), Ophioceras aquaticus sp. nov. (Magnaporthaceae) and Togninia aquatica sp. nov. (Togniniaceae), are described and illustrated based on morphological and molecular data (ITS, 18S, 28S rDNA sequences). Diaporthe aquatica is characterized by globose to subglobose, black ascomata with long necks, broadly cylindrical to obclavate asci, and small, ellipsoidal to fusiform, one-septate, hyaline ascospores; it is unusual among Diaporthe species in the fact that it lacks a stroma and has freshwater habitat. Ophioceras aquaticus is characterized by globose ascomata with a long beak, cylindrical, eight-spored asci with J- subapical rings and 3–5-septate filiform ascospores with slightly acute ends. Togninia aquatica is Copyright 2012 by The Mycological Society of America. characterized by globose ascomata with long necks, clavate and truncate asci clustered on distinct ascogenous hyphae, and small, reniform, hyaline ascospores. Differences among the new taxa and similar species are discussed. Key words: aquatic fungi, Diaporthaceae, Magnaporthaceae, systematics, Togniniaceae INTRODUCTION Freshwater ascomycetes are a highly diverse group of fungi, which mainly include Eurotiales (25 spp.), Halosphaeriales (24 spp.), Helotiales (101 spp.), Hypocreales (14 spp.), Pleosporales (121 spp.), Sordariales (114 spp.) and Xylariales (19 spp.) (Shearer et al. 2007). The taxonomic affinities of many other freshwater fungal taxa are unclear. For example, Annulatascaceae (Sordariomycetidae) is a typical freshwater family, with 75 estimated species belonging to 21 genera (Kirk et al. 2008, Lumbsch and Huhndorf 2010), but its ordinal placement is unclear. It is difficult to determine phylogenetic relationships among freshwater taxa based solely on morphology (Shearer et al. 2009); therefore, molecular phylogenetic studies are critical to an understanding of freshwater fungi. During our investigation of freshwater fungi in China (Cai et al. 2008; Cai and Hyde 2007; Hu et al. 2007, 2010a, b), we collected two taxa belonging to Diaporthales and another taxon belonging to Magnaporthaceae. Morphological comparisons and molecular analyses suggested that these are new taxa of Diaporthe Nitschke, Togninia Berl. and Ophioceras Sacc. Eight species and three genera of Diaporthales have been reported from freshwater habitats. Ho et al. (2001) reported Diaporthe beckhausii Nitschke, Gnomoniella rubicola Pass. and G. microspora M. Monod in streams in Hong Kong, Brunei and Malaysia respectively. Gnomonia petiolorum (Schwein.) Cooke and G. papuana Sivan. & D.E. Shaw were reported from freshwater habitats in USA (Fallah and Shearer 2001) and Papua New Guinea (Sivanesan and Shaw 1977). Jobellisia viridifusca K.M. Tsui & K.D. Hyde and J. luteola (Ellis & Everh.) M.E. Barr were reported in Hong Kong (Ranghoo et al. 2001) and USA (Raja et al. 2009). Thailandiomyces Pinruan, Sakayaroj, K.D. Hyde & E.B.G. Jones and Phruensis Pinruan are new genera reported from freshwater habitats in Thailand (Jones et al. 2008, Pinruan et al. 2004) and Hyalorostratum Raja & Shearer was reported as a new genus from freshwater habitats in the USA (Raja et al. 2010). Nineteen Magnaporthaceae species, including nine Ophioceras, have been reported from freshwater (Shearer and Raja 2010). MATERIALS AND METHODS Sample collection and specimen examination.—Unidentified submerged wood from streams, lakes, ponds, reservoirs and ditches were collected in southern China and incubated in moist chambers at room temperature (ca. 25 C). Samples were examined for fungal fruiting bodies under a dissecting microscope (Leica MZ16A). Observations and photographs were prepared from materials mounted in water and examined with a compound microscope (Nikon E800) (Hu et al. 2012). The single-spore isolation method outlined by Chomnunti et al. (2011) was used to obtain pure cultures with potato dextrose agar (PDA). The species found are deposited as herbarium specimens in International Fungal Research and Development Center (IFRD). DNA extraction.—Total DNA was extracted from pure cultures with a Biospin Fungus Genomic DNA Extraction Kit (BioFlux®) following the manufacturer's protocol. The cultures used to extract DNA are provided (TABLE I). DNA amplification and sequencing.—Internal transcribed spacer (ITS) rDNA and fragments of the partial large subunit (LSU) and small subunit (SSU) rDNA were amplified by the polymerase chain reaction (PCR). Primers ITS4 and ITS5 (White et al. 1990) were used for PCR amplification of ITS rDNA sequences. Primers LROR and LR6 (Rehner and Samuels 1995, Vilgalys and Hester 1990) and NS1 and NS4 (White et al. 1990) were used respectively for PCR amplification of LSU and SSU rDNA sequences. The amplification of all the three genes was performed in a 50 µL reaction volume (buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 0.8 µM of each primer and 1 unit Taq DNA polymerase). All the three genes were amplified with the same thermal cycle. Thermal-cycling parameters included an initial denaturation at 95 C for 2 min, followed by 35 cycles each consisting of denaturation at 94 C for 1 min, annealing at 50 C for 1 min, and extension at 72 C for 1 min. A final extension at 72 C for 10 min was included at the end of thermal cycling. The PCR products of ITS, LSU and SSU genes were purified and sequenced respectively with ITS4 and ITS5, LROR an LR6 and NS1 and NS4 primers in a sequencer (ABI-PRISM3730) at Sangon Biotech (Shanghai, China). Sequences alignment and phylogenetic analyses.—Sequences were aligned with BioEdit (Hall 1999). Eight novel sequences (TABLE I) from the new taxa, together with reference sequences obtained from GenBank, were aligned with Clustal X (Thompson et al. 1997). Alignment was adjusted manually to allow maximum alignment and minimize gaps. Phylogenetic analyses were performed with maximum parsimony as implemented in PAUP* 4.0b10 (Swofford 2002). Characters were equally weighted, and gaps were treated as missing data. Trees were inferred with the heuristic search option with TBR branch swapping and 1000 random sequence additions. MAXTREES were unlimited, branches of zero length were collapsed and all parsimonious trees were saved. Clade stability was assessed using a bootstrap (BT) analyses with 1000 replicates, each with 10 replicates of random stepwise of taxa. Kishino-Hasegawa tests (KH Test) (Kishino and Hasegawa 1989) were performed to determine whether trees were significantly different. Trees were drawn with TreeVIEW (Page 1996). The model of evolution was estimated with MrModeltest 2.2. Posterior probabilities (PP) (Rannala and Yang 1996, Zhaxybayeva and Gogarten 2002) were determined by Markov chain Monte Carlo sampling (BMCMC) in MrBayes 3.0b4 (Huelsenbeck and Ronquist 2001). Six simultaneous Markov chains were run 1 000 000 generations, and trees were sampled every 100th generation (resulting 10 000 total trees). The first 2000 trees, which represented the burn-in phase of the analyses, were discarded, and the remaining 8000 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree. RESULTS Phylogenetic analyses.—The ITS rDNA dataset of Diaporthe/Phomopsis included 44 sequences (32 of them from type strains) from 43 Diaporthe and Phomopsis species, with Valsa ambiens (Pers.) Fr. (Diaporthales, Valsaceae) as an outgroup taxon. The final dataset comprised 514 characters after alignment without ambiguous regions. Parsimony analysis resulted in 832 trees. One of the most parsimonious trees (TL = 511, CI = 0.429, RI = 0.675, RC = 0.289, HI = 0.571) is illustrated (FIG. 1). In the tree the two strains of Diaporthe aquatica clustered in the Diaporthe/Phomopsis clade and were related most closely to Diaporthe strumella var. longispora Wehm. The ITS rDNA dataset of Togoninia/Phaeoacremonium included 16 sequences (six of them from type strains) from eight Togoninia species and three Phaeoacremonium species, with Gnomonia betulina Vleugel (Diaporthales, Valsaceae) as an outgroup taxon. The final dataset comprised 547 characters after alignment without ambiguous regions. Parsimony analysis resulted in two trees without significant difference. One of the most parsimonious trees (TL = 239, CI = 0.862, RI = 0.840, RC = 0.724, HI = 0.138) is illustrated (FIG. 2). In the tree Togninia aquatica clustered in Togoninia/Phaeoacremonium clade and is related most closely to T. parasitica L. Mostert, W. Gams & Crous. The 28S rDNA dataset included sequences from 19 Magnaporthaceae strains representing three genera, with Ophiostoma piliferum (Fr.) Syd. & P. Syd. (Ophiostomatales, Ophiostomataceae) as an outgroup taxon.