State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of

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1 In Press at Mycologia, preliminary version published on June 8, 2012 as doi: / 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 , China International Fungal Research & Development Center, the Research Institute of Resource Insects, Chinese Academy of Forestry, Bailongsi, Kunming , China Lei Cai 1 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China Kevin D. Hyde 1 International Fungal Research & Development Center, the Research Institute of Resource Insects, Chinese Academy of Forestry, Bailongsi, Kunming , 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.

2 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.

3 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,

4 1.5 mm MgCl 2, 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 generations, and trees were sampled every 100th generation (resulting 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

5 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. The final dataset comprised 872 characters after alignment without ambiguous regions. Parsimony analysis resulted in two trees without significant difference. One of the most parsimonious trees (TL = 337, CI = 0.721, RI = 0.844, RC = 0.608, HI = 0.279) is illustrated (FIG. 3). In the tree Ophioceras aquaticus clustered in the Ophioceras (Magnaporthaceae) clade and is related most closely to Ophioceras commune Shearer, J.L. Crane & W. Chen. The 18S rdna dataset included sequences from 23 Magnaporthaceae strains representing three genera, with Ophiostoma piliferum (Fr.) Syd. & P. Syd.

6 (Ophiostomatales, Ophiostomataceae) as an outgroup taxon. The final dataset comprised 1263 characters after alignment without ambiguous regions. Parsimony analysis resulted in 15 trees without significant difference. One of the most parsimonious trees (TL = 130, CI = 0.877, RI = 0.933, RC = 0.818, HI = 0.123) is illustrated (FIG. 4). In the tree Ophioceras aquaticus clustered in the Ophioceras (Magnaporthaceae) clade and is most closely related to Ophioceras tenuisporum Shearer, J.L. Crane & W. Chen. TAXONOMY Diaporthe aquatica D.M. Hu, L. Cai & K.D. Hyde, sp. nov. FIG. 5 MycoBank MB Etymology: aquatica, referring to the aquatic habitat of the fungus. Stroma absent. Ascomata µm high, µm diam, globose to subglobose, black, coriaceous, immersed to semi-immersed, single to clustered. Neck µm, cylindrical, black. Peridium µm thick, comprising compressed cells of textura angularis, outer layers composed of black brown, thickwalled cells, inner layers composed of pale brown to hyaline thin-walled cells. Paraphyses ca. 6 µm diam, longer than asci, hyaline, cylindrical, septate. Asci µm, eight-spored, unitunicate, thin-walled, apedicellate, broad cylindrical to obclavate, with a minute apical ring, ca. 1 µm high and 2 µm diam. Ascospores µm (x = µm, n = 30), overlapping biseriate, ellipsoidal to fusiform, hyaline, one-septate, slightly constricted at the septum, thin-walled, smooth-walled, containing four small globules. Specimens examined: CHINA. GUIZHOU PROVINCE: Guiyang City, Tianhetan Park, on wood submerged in a small ditch, 22 May 2009, D.M. Hu (IFRD , HOLOTYPE), ex-type living culure: IFRDCC 3051; ibid (IFRD ), related living culture: IFRDCC Ophioceras aquaticus D.M. Hu, L. Cai & K.D. Hyde, sp. nov. FIG. 6

7 MycoBank MB Etymology: aquaticus referring to the aquatic habitat. Ascomata µm diam, globose, superficial to submerged, solitary to gregarious, black, coriaceous; beak ca µm long, 60 µm diam, cylindrical, black, fragile, periphysate. Peridium thick, blackened, composed of large cells of textura angularis. Paraphyses µm long, µm wide at the base, hypha-like, hyaline, septate, numerous, broad at the base, tapering distally, apically free, not embedded in a gelatinous matrix. Asci µm, eight-spored, cylindrical, apedicellate, unitunicate, persistent, with a J-, thimble-shaped, subapical ring, ca. 2 µm high, 1.5 µm diam, asci becoming detached from the ascogenous hyphae and lying free in the ascomatal cavity. Ascospores µm (x = µm, n = 50), fasciculate, filiform, slightly acute at each end, falcate or sigmoid, 3 5-septate, not constricted at the septa, hyaline, thin-walled, smooth-walled, guttulate. Specimen examined: CHINA. YUNNAN PROVINCE: Mengla County, Dashaba Reservoir (N21 36, E ), on submerged wood, 24 Mar 2010, D.M. Hu ML20 (IFRD , HOLOTYPE), ex-type culture: IFRDCC 3091; ibid DSB26. A KEY TO THE SPECIES OF FRESHWATER OPHIOCERAS 1. Periphyses and paraphyses absent O. leptosporum 1. Periphyses and paraphyses present 2 2. Ascospores heavily guttulate, µm O. guttulatum 2. Ascospores not heavily guttulate 3 3. Ascospores consistently narrow, µm wide O. tenuisporum 3. Ascospores mostly wider than 1.5 µm 4 4. Ascospores µm long 5 4. Ascospores mostly over 110 µm long 8 5. Ascospores 3 6-septate 6

8 5. Ascospores up to seven-septate 8 6. Ascospores µm O. fusiforme 6. Ascospores mostly wider than 3 µm 7 7. Ascospores µm O. hongkongense 7. Ascospores µm O. aquaticus 8. Ascospores consistently 2 µm wide O. commune 8. Ascospores µm wide O. dolichostomum 9. Ascospores 2 4 µm wide, 5(4 6) septate O. vennezuelense 9. Ascospores 4 7 µm wide, 5 12 septate O. arcuatisporum Togninia aquatica D.M. Hu, L. Cai & K.D. Hyde, sp. nov. FIG. 7 MycoBank MB Etymology: aquatica, referring to the aquatic habitat of the fungus. Ascomata ca µm diam, scattered, submerged, globose, black, coriaceous. Neck up to 1450 µm long, ca. 35 µm diam, one per ascoma, erect or lying on substrata, straight or curved, cylindrical, black. Peridium µm thick, comprising 8 10 layers cells of textura angularis, cells of outer layers black-brown to pale brown, inner layers hyaline. Paraphyses hyaline, septate, hypha-like, cylindrical, narrowing toward the tip, thread-like at the apex, longer than asci. Asci µm, eight-spored, unitunicate, clavate, apex truncate, appearing spicate when mature, apedicellate, with truncate bases. Ascogenous hyphae hyaline, septate, simple, smooth-walled, 2 3 µm at the base. Ascospores µm (x = µm, n = 30), biseriate, reniform with rounded ends, unicellular, hyaline, thin-walled, smooth-walled, often containing small guttules at the ends. Specimens examined: CHINA. YUNNAN PROVINCE: Mengla County, in a small stream, on submerged wood, 3 Apr 2009, D.M. Hu (IFRD , HOLOTYPE), ex-type living culture IFRDCC 3035; Beijing, Yanqin County, Songshan Forestry Park, on wood submerged in stream, 8 Jul 2011, F. Liu YQ05.

9 DISCUSSION Diaporthe was established by Nitschke (1870) to accommodate a group of species with stromatic ascomata, ellipsoid to fusiform spores and enclosed unilocular pycnidia anamorphs (Wehmeyer 1933). Wehmeyer (1933) revised the genus and segregated the species into five genera (i.e. Apioporthe, the species with unequally two-celled ascospores; Diaporthe, the species with blackened zones present in the substratum and equally two-celled ascospores; Diaporthopsis, the species with one-celled ascospores; Diaporthella, the species with strongly developed, widely erumpent I-like disks, lacking blackened zones in the substratum and equally two-celled ascospores; and Cryptodiaporthe, the species without strongly developed disks, lacking blackened zones in the substratum and equally two-celled ascospores). The anamorph is in Phomopsis, which has more than 900 recorded names with few linked to the teleomorphs (Udayanga et al. 2011). To date 829 names are listed in Diaporthe (Index Fungorum: thus resolving the phylogenetic relationships among all the species of the genus is challenging and has not been attempted. Some reports have focused on resolving the phylogenetic relationship within the species complex on certain hosts (Diogo et al. 2010, Santos and Phillips 2009) and the anamorphic genus Phomopsis (Udayanga et al. 2011). The anamorphic state is important in the systematics of Diaporthe (Diogo et al. 2010, Santos and Phillips 2009), but we have not observed conidial formation in pure culture after 3 mo. Diaporthe aquatica is characterized by globose to subglobose, black ascomata with long necks, broad cylindrical to obclavate asci, and hyaline, small, one-septate, ellipsoidal to fusiform ascospores. The morphological characters of D. aquatica fit

10 well the generic concept of Diaporthe (Wehmeyer 1933), except for the absence of a stroma. In the phylogenetic tree (FIG. 1) based on ITS rdna sequences, the two sequences of D. aquatica clustered in the Diaporthe/Phomopsis clade. Diaporthe aquatica therefore is placed in Diaporthe. In the tree D. aquatica is phylogenetically closely related to D. strumella var. longispora Wehm. (FIG 1). Morphologically D. aquatica differs from D. strumella var. longispora in having shorter ascospores (10 12 µm vs µm) (Wehmeyer 1936). D. aquatica is unusual among Diaporthe species in that it lacks a stroma and is rare in freshwater habitats. Most Diaporthe species have been recorded as plant pathogens or endophytes (Crous et al. 2011, Hodges 1980, Sommer and Beraha 1975, Wehmeyer 1933), while some other species are saprobes (Wong and Hyde 2001). Diaporthe beckhausii Nitschke, the only species previously reported from freshwater habitats (Ho et al. 2001), morphologically resembles D. aquatica (Wehmeyer 1933) but differs in having irregularly pustulate-effuse entostromata. Furthermore, D. beckhausii was known normally as a parasite on various plants (Wehmeyer 1933) with a solely report from freshwater (Ho et al. 2001) (i.e. D. beckhausii is an opportunistic freshwater species). To date D. aquatica is the only Diaporthe species restricted to freshwater habitats. We assume that D. aquatica reduced its stroma structure during its adaptation in freshwater habitats. More investigation on fungal diversity in freshwater habitats and adjacent environment may shed light on the adaption mechanism and speciation of freshwater fungi. 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, features that fit well with the generic concept of Ophioceras (Saccardo 1883). The phylogenetic trees (FIGS. 3, 4) inferred from 28S

11 and 18S sequences also show that O. aquaticus clusters in the Ophioceras clade. O. aquaticus morphologically resembles O. commune Shearer, J.L. Crane & W. Chen and O. hongkongense K.M. Tsui, H.Y.M. Leung, K.D. Hyde & Hodgkiss. However, the ascospores of O. aquaticus are broader and shorter when compared to O. commune ( µm vs µm) (Shearer et al. 1999). The ascospores of O. aquaticus are shorter when compared to O. hongkongense ( µm vs µm). Furthermore, the ascospores of O. aquaticus are falcate or sigmoid, compared to the falcate and non-sigmoid ascospores of O. hongkongense (Tsui et al. 2001). Moreover, the 18S and 28S trees (FIGS. 3, 4) show phylogenetic distance between O. aquaticus, O. commune and O. hongkongense. The ITS rdna of O. aquaticus was sequenced and deposited in GenBank (accession number JQ797440). Because there was no reference sequence in GenBank, we could not construct ITS phylogenetic tree of Ophioceras. Ophioceras species were recorded frequently in freshwater habitats (Hu et al. 2010b, Tsui et al. 2001). Including the new species introduced in this paper, 10 Ophioceras species have been recorded from freshwater habitats. Mostert et al. (2006) studied the taxonomy and pathology of Togninia and its Phaeoacremonium anamorphs and accepted 10 species in the genus. They also provided keys to the genus and to the anamorphs. Three species of Togninia have been described since (Damm et al. 2008, Réblová and Mostert 2007). Togninia aquatica is characterized by globose ascomata with long necks, clavate and truncate asci clustered on distinct ascogenous hyphae, and small, reniform, hyaline ascospores. The morphological characters of T. aquatica fit well with the concept of the genus (Mostert et al. 2006). T. aquatica is morphologically similar to T. austroafricana L. Mostert, W. Gams & Crous in having long ascomatal necks (up to 1450 µm) and

12 reniform ascospores (Mostert et al. 2006). However, the ascospores of T. aquatica are narrower when compared to T. austroafricana (1 1.5 µm vs µm), and the peridia of the former is much thicker when compared to the latter (35 µm vs µm). The result of ITS sequences analysis (FIG 2) shows that T. aquatica appears as a distinct lineage and clustered together with the representatives of Togninia. In the tree T. aquatica is closely related phylogenetically to T. parasitica L. Mostert, W. Gams & Crous (FIG. 1). Morphologically, T. aquatica differs from T. parasitica in having longer ascomatal necks (1450 µm vs µm) and reniform ascospores as compared to the allantoid ascospores of T. parasitica (Mostert et al. 2006). Furthermore, T. aquatica is unique among species of Togninia in its freshwater habitat because most Togninia species are plant pathogens (Damm et al. 2008, Mostert et al. 2006). ACKNOWLEDGMENTS Financial support for research was provided by the Grant for Essential Scientific Research of the National Nonprofit Institute (No. CAFYBB ). The authors also express deep thanks to Prof Xiaoming Chen, Ma Tao, Hang Chen, HaiXia Wu and YanMei Li (The Research Institute of Resource Insects, Chinese Academy of Forestry) for their valuable help. Staff of the International Fungal Research and Development Centre at The Research Institute of Resource Insects, Chinese Academy of Forestry, also are thanked for providing help. Further financial support was provided by NSFC Dian-Ming Hu thanks Fang Liu for collecting samples in Beijing. LITERATURE CITED Cai L, Guo XY, Hyde KD Morphological and molecular characterisation of a new anamorphic genus Cheirosporium, from freshwater in China. Persoonia 20: DOI: / X314732, Hyde KD New species of Clohiesia and Paraniesslia collected from freshwater habitats

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16 Réblová M, Mostert L Romellia is congeneric with Togninia, and description of Conidiotheca gen. nov. for one species of this genus with polysporous asci. Mycol Res 111: DOI: /j.mycres Rehner SA, Samuels GJ Molecular systematics of the Hypocreales: a teleomorph gene phylogeny and the status of their anamorphs. Can J Bot 73: Saccardo PA Sylloge Pyrenomycetum. Vol. II. Sylloge Fungorum 2: Santos JM, Phillips AJL Resolving the complex of Diaporthe (Phomopsis) species occurring on Foeniculum vulgare in Portugal. Fungal Divers 34: Shearer CA, Crane JL, Chen W Freshwater ascomycetes: Ophioceras species. Mycologia 91: DOI: / , Descals E, Kohlmeyer B, Kohlmeyer J, Marvanova L, Padgett D, Porter D, Raja HA, Schmit JP, Thorton HA et al Fungal biodiversity in aquatic habitats. Biodivers Conserv 16: DOI: /s z, Raja HA, Miller AN, Nelson P, Tanaka K, Hirayama K, Marvanova L, Hyde KD, Zhang Y The molecular phylogeny of freshwater Dothideomycetes. Stud Mycol 64: DOI: /sim , Freshwater Ascomycetes Database: (Accessed 14 Mar 2012). Sivanesan A, Shaw DE Gnomonia papuana sp. nov. with a Sesquicillium conidial state. Trans Br Mycol Soc 68:85 90.

17 Sommer NF, Beraha L Diaporthe actinidiae, a new species causing stem-end rot of Chinese gooseberry fruits. Mycologia 67: Swofford DL PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4b10. Sunderland, Massachusetts: Sinauer Associates. Teng SJ Notes on Sphaeriales from China. Sinensia 4: Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG The Clustal X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: DOI: /nar/ Thongkantha S, Jeewon R, Vijaykrishna D, Lumyong S, McKenzie EHC, Hyde KD Molecular phylogeny of Magnaporthaceae (Sordariomycetes) with a new species, Ophioceras chiangdaoense from Dracaena loureiroi in Thailand. Fungal Divers 34: Tsui CKM, Leung YM, Hyde KD, Hodgkiss IJ Three new Ophioceras species (Ascomycetes) from the tropics. Mycoscience 42: DOI: /BF Udayanga D, Liu X, McKenzie EHC, Chukeatirote E, Bahkali AHA, Hyde KD The genus Phomopsis: biology, applications, species concepts and names of common phytopathogens. Fungal Divers 50:1 37. DOI: /s Vilgalys R, Hester M Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172: Walker J Gaeumannomyces, Linocarpon, Ophiobolus and several other genera of scolecospored ascomycetes and Phialophora conidial states, with a note on hyphopodia. Mycotaxon 11: Wehmeyer LE The genus Diaporthe Nitschke and its segregates. Univ Michigan Stud, Sci Ser

18 9: Cultural studies of three new pyrenomycetes. Mycologia 28: White TJ, Bruns T, Lee S, Taylor JW Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White YJ, eds. PCR protocols: a guide to methods and applications. San Diego, California: Academic Press. p Wong MKM, Hyde KD Fungi on grasses: new species of Ascotaiwania, Diaporthe and Oxydothis (Ascomycetes). Cryptogamie Mycol 22: DOI: /S (01) Zhaxybayeva O, Gogarten JP Bootstrap, Bayesian probability and maximum likelihood mapping: exploring new tools for comparative genome analyses. BMC Genomics 3:4. DOI: / LEGENDS FIG 1. Maximum parsimony phylogram inferred from ITS rdna sequence data, showing the phylogenetic relationship between Diaporthe aquatica and the other Diaporthe/Phomopsis species. Data analyzed with random addition sequence, unweighted parsimony and treating gaps as missing data. Values above the branches are parsimony bootstrap (equal or above 50%). Sequences derived from type cultures are marked with an asterisk. FIG 2. Maximum parsimony phylogram inferred from ITS rdna sequence data, showing the phylogenetic relationship between Togninia aquatica and the other Togninia/Phaeoacremonium species. Data analyzed with random addition sequence, unweighted parsimony and treating gaps as missing data. Values above the branches are parsimony bootstrap (equal or above 50%). Sequences derived from type cultures are marked with an asterisk. FIG 3. Maximum parsimony phylogram inferred from 28S rdna sequence data, showing the phylogenetic relationship among the Magnaporthaceae species. Data analyzed with random addition sequence, unweighted parsimony and treating gaps as missing data. Values above the branches are parsimony bootstrap (equal or above 50%). Branches with Bayesian posterior probabilities equal or

19 above 90% are thickened. The type species of the genera are marked with an asterisk. FIG 4. Maximum parsimony phylogram inferred from 18S rdna sequence data, showing the phylogenetic relationship among the Magnaporthaceae species. Data analyzed with random addition sequence, unweighted parsimony and treating gaps as missing data. Values above the branches are parsimony bootstrap (equal or above 50%). Branches with Bayesian posterior probabilities equal or above 90% are thickened. The type species of the genera are marked with an asterisk. FIG 5. Diaporthe aquatica. a. Ascoma on submerged wood. b. Section of an ascoma. c, d. Peridium. e. Paraphyses. f h. Asci. i k. Ascospores. Bars: a = 200 µm, b = 50 µm, c d = 20 µm, e k = 5 µm. FIG 6. Ophiocera aquaticus. a. Ascomata on submerged wood. b. Paraphyses. c e. Asci. f. Upper part of an ascus, showing the apical ring. g j. Ascospores. k. A germinated ascospore. Bars: a = 500 µm, b = 5 µm, c e = 20 µm, f k = 5 µm. FIG 7. Togninia aquatica. a. Ascomata on submerged wood. b. Section of an ascoma. c. Peridium. d. Squash of an ascoma. e. Paraphyses. f. Ascogenous hyphae. g i. Asci. j n. Ascospores. Bars: a = 200 µm, b = 30 µm, c = 5 µm, d = 50 µm, e n = 5 µm. FOOTNOTES Submitted 26 Dec 2011; accepted for publication 24 May Corresponding authors. mrcailei@gmail.com; kdhyde3@gmail.com

20 TABLE I. Sources and accession numbers of the isolates generated in this study Taxa Source GenBank accession numbers ITS rdna 28S rdna 18S rdna Diaporthe aquatica IFRDCC 3051 JQ Diaporthe aquatica IFRDCC 3015 JQ Togninia aquatica IFRDCC 3035 JQ Ophioceras aquaticus IFRDCC 3091 JQ JQ JQ Pseudohalonectria lignicola IFRDCC 3030 JQ Pseudohalonectria lignicola IFRDCC 3110 JQ797436

21

22

23

24

25

26

27

28 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 , China International Fungal Research & Development Center, the Research Institute of Resource Insects, Chinese Academy of Forestry, Bailongsi, Kunming , China Lei Cai 1 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China Kevin D. Hyde 1 International Fungal Research & Development Center, the Research Institute of Resource Insects, Chinese Academy of Forestry, Bailongsi, Kunming , 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

29 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.

30 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,

31 1.5 mm MgCl 2, 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 generations, and trees were sampled every 100th generation (resulting 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

32 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. The final dataset comprised 872 characters after alignment without ambiguous regions. Parsimony analysis resulted in two trees without significant difference. One of the most parsimonious trees (TL = 337, CI = 0.721, RI = 0.844, RC = 0.608, HI = 0.279) is illustrated (FIG. 3). In the tree Ophioceras aquaticus clustered in the Ophioceras (Magnaporthaceae) clade and is related most closely to Ophioceras commune Shearer, J.L. Crane & W. Chen. The 18S rdna dataset included sequences from 23 Magnaporthaceae strains representing three genera, with Ophiostoma piliferum (Fr.) Syd. & P. Syd.

33 (Ophiostomatales, Ophiostomataceae) as an outgroup taxon. The final dataset comprised 1263 characters after alignment without ambiguous regions. Parsimony analysis resulted in 15 trees without significant difference. One of the most parsimonious trees (TL = 130, CI = 0.877, RI = 0.933, RC = 0.818, HI = 0.123) is illustrated (FIG. 4). In the tree Ophioceras aquaticus clustered in the Ophioceras (Magnaporthaceae) clade and is most closely related to Ophioceras tenuisporum Shearer, J.L. Crane & W. Chen. TAXONOMY Diaporthe aquatica D.M. Hu, L. Cai & K.D. Hyde, sp. nov. FIG. 5 MycoBank MB Etymology: aquatica, referring to the aquatic habitat of the fungus. Stroma absent. Ascomata μm high, μm diam, globose to subglobose, black, coriaceous, immersed to semi-immersed, single to clustered. Neck μm, cylindrical, black. Peridium μm thick, comprising compressed cells of textura angularis, outer layers composed of black brown, thickwalled cells, inner layers composed of pale brown to hyaline thin-walled cells. Paraphyses ca. 6 μm diam, longer than asci, hyaline, cylindrical, septate. Asci μm, eight-spored, unitunicate, thin-walled, apedicellate, broad cylindrical to obclavate, with a minute apical ring, ca. 1 μm high and 2 μm diam. Ascospores μm (x = μm, n = 30), overlapping biseriate, ellipsoidal to fusiform, hyaline, one-septate, slightly constricted at the septum, thin-walled, smooth-walled, containing four small globules. Specimens examined: CHINA. GUIZHOU PROVINCE: Guiyang City, Tianhetan Park, on wood submerged in a small ditch, 22 May 2009, D.M. Hu (IFRD , HOLOTYPE), ex-type living culure: IFRDCC 3051; ibid (IFRD ), related living culture: IFRDCC Ophioceras aquaticus D.M. Hu, L. Cai & K.D. Hyde, sp. nov. FIG. 6

34 MycoBank MB Etymology: aquaticus referring to the aquatic habitat. Ascomata μm diam, globose, superficial to submerged, solitary to gregarious, black, coriaceous; beak ca μm long, 60 μm diam, cylindrical, black, fragile, periphysate. Peridium thick, blackened, composed of large cells of textura angularis. Paraphyses μm long, μm wide at the base, hypha-like, hyaline, septate, numerous, broad at the base, tapering distally, apically free, not embedded in a gelatinous matrix. Asci μm, eight-spored, cylindrical, apedicellate, unitunicate, persistent, with a J-, thimble-shaped, subapical ring, ca. 2 μm high, 1.5 μm diam, asci becoming detached from the ascogenous hyphae and lying free in the ascomatal cavity. Ascospores μm (x = μm, n = 50), fasciculate, filiform, slightly acute at each end, falcate or sigmoid, 3 5-septate, not constricted at the septa, hyaline, thin-walled, smooth-walled, guttulate. Specimen examined: CHINA. YUNNAN PROVINCE: Mengla County, Dashaba Reservoir (N21 36, E ), on submerged wood, 24 Mar 2010, D.M. Hu ML20 (IFRD , HOLOTYPE), ex-type culture: IFRDCC 3091; ibid DSB26. A KEY TO THE SPECIES OF FRESHWATER OPHIOCERAS 1. Periphyses and paraphyses absent O. leptosporum 1. Periphyses and paraphyses present 2 2. Ascospores heavily guttulate, μm O. guttulatum 2. Ascospores not heavily guttulate 3 3. Ascospores consistently narrow, μm wide O. tenuisporum 3. Ascospores mostly wider than 1.5 μm 4 4. Ascospores μm long 5 4. Ascospores mostly over 110 μm long 8 5. Ascospores 3 6-septate 6

35 5. Ascospores up to seven-septate 8 6. Ascospores μm O. fusiforme 6. Ascospores mostly wider than 3 μm 7 7. Ascospores μm O. hongkongense 7. Ascospores μm O. aquaticus 8. Ascospores consistently 2 μm wide O. commune 8. Ascospores μm wide O. dolichostomum 9. Ascospores 2 4 μm wide, 5(4 6) septate O. vennezuelense 9. Ascospores 4 7 μm wide, 5 12 septate O. arcuatisporum Togninia aquatica D.M. Hu, L. Cai & K.D. Hyde, sp. nov. FIG. 7 MycoBank MB Etymology: aquatica, referring to the aquatic habitat of the fungus. Ascomata ca μm diam, scattered, submerged, globose, black, coriaceous. Neck up to 1450 μm long, ca. 35 μm diam, one per ascoma, erect or lying on substrata, straight or curved, cylindrical, black. Peridium μm thick, comprising 8 10 layers cells of textura angularis, cells of outer layers black-brown to pale brown, inner layers hyaline. Paraphyses hyaline, septate, hypha-like, cylindrical, narrowing toward the tip, thread-like at the apex, longer than asci. Asci μm, eight-spored, unitunicate, clavate, apex truncate, appearing spicate when mature, apedicellate, with truncate bases. Ascogenous hyphae hyaline, septate, simple, smooth-walled, 2 3 μm at the base. Ascospores μm (x = μm, n = 30), biseriate, reniform with rounded ends, unicellular, hyaline, thin-walled, smooth-walled, often containing small guttules at the ends. Specimens examined: CHINA. YUNNAN PROVINCE: Mengla County, in a small stream, on submerged wood, 3 Apr 2009, D.M. Hu (IFRD , HOLOTYPE), ex-type living culture IFRDCC 3035; Beijing, Yanqin County, Songshan Forestry Park, on wood submerged in stream, 8 Jul 2011, F. Liu YQ05.

36 DISCUSSION Diaporthe was established by Nitschke (1870) to accommodate a group of species with stromatic ascomata, ellipsoid to fusiform spores and enclosed unilocular pycnidia anamorphs (Wehmeyer 1933). Wehmeyer (1933) revised the genus and segregated the species into five genera (i.e. Apioporthe, the species with unequally two-celled ascospores; Diaporthe, the species with blackened zones present in the substratum and equally two-celled ascospores; Diaporthopsis, the species with one-celled ascospores; Diaporthella, the species with strongly developed, widely erumpent I-like disks, lacking blackened zones in the substratum and equally two-celled ascospores; and Cryptodiaporthe, the species without strongly developed disks, lacking blackened zones in the substratum and equally two-celled ascospores). The anamorph is in Phomopsis, which has more than 900 recorded names with few linked to the teleomorphs (Udayanga et al. 2011). To date 829 names are listed in Diaporthe (Index Fungorum: thus resolving the phylogenetic relationships among all the species of the genus is challenging and has not been attempted. Some reports have focused on resolving the phylogenetic relationship within the species complex on certain hosts (Diogo et al. 2010, Santos and Phillips 2009) and the anamorphic genus Phomopsis (Udayanga et al. 2011). The anamorphic state is important in the systematics of Diaporthe (Diogo et al. 2010, Santos and Phillips 2009), but we have not observed conidial formation in pure culture after 3 mo. Diaporthe aquatica is characterized by globose to subglobose, black ascomata with long necks, broad cylindrical to obclavate asci, and hyaline, small, one-septate, ellipsoidal to fusiform ascospores. The morphological characters of D. aquatica fit

37 well the generic concept of Diaporthe (Wehmeyer 1933), except for the absence of a stroma. In the phylogenetic tree (FIG. 1) based on ITS rdna sequences, the two sequences of D. aquatica clustered in the Diaporthe/Phomopsis clade. Diaporthe aquatica therefore is placed in Diaporthe. In the tree D. aquatica is phylogenetically closely related to D. strumella var. longispora Wehm. (FIG 1). Morphologically D. aquatica differs from D. strumella var. longispora in having shorter ascospores (10 12 μm vs μm) (Wehmeyer 1936). D. aquatica is unusual among Diaporthe species in that it lacks a stroma and is rare in freshwater habitats. Most Diaporthe species have been recorded as plant pathogens or endophytes (Crous et al. 2011, Hodges 1980, Sommer and Beraha 1975, Wehmeyer 1933), while some other species are saprobes (Wong and Hyde 2001). Diaporthe beckhausii Nitschke, the only species previously reported from freshwater habitats (Ho et al. 2001), morphologically resembles D. aquatica (Wehmeyer 1933) but differs in having irregularly pustulate-effuse entostromata. Furthermore, D. beckhausii was known normally as a parasite on various plants (Wehmeyer 1933) with a solely report from freshwater (Ho et al. 2001) (i.e. D. beckhausii is an opportunistic freshwater species). To date D. aquatica is the only Diaporthe species restricted to freshwater habitats. We assume that D. aquatica reduced its stroma structure during its adaptation in freshwater habitats. More investigation on fungal diversity in freshwater habitats and adjacent environment may shed light on the adaption mechanism and speciation of freshwater fungi. 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, features that fit well with the generic concept of Ophioceras (Saccardo 1883). The phylogenetic trees (FIGS. 3, 4) inferred from 28S

38 and 18S sequences also show that O. aquaticus clusters in the Ophioceras clade. O. aquaticus morphologically resembles O. commune Shearer, J.L. Crane & W. Chen and O. hongkongense K.M. Tsui, H.Y.M. Leung, K.D. Hyde & Hodgkiss. However, the ascospores of O. aquaticus are broader and shorter when compared to O. commune ( μm vs μm) (Shearer et al. 1999). The ascospores of O. aquaticus are shorter when compared to O. hongkongense ( μm vs μm). Furthermore, the ascospores of O. aquaticus are falcate or sigmoid, compared to the falcate and non-sigmoid ascospores of O. hongkongense (Tsui et al. 2001). Moreover, the 18S and 28S trees (FIGS. 3, 4) show phylogenetic distance between O. aquaticus, O. commune and O. hongkongense. The ITS rdna of O. aquaticus was sequenced and deposited in GenBank (accession number JQ797440). Because there was no reference sequence in GenBank, we could not construct ITS phylogenetic tree of Ophioceras. Ophioceras species were recorded frequently in freshwater habitats (Hu et al. 2010b, Tsui et al. 2001). Including the new species introduced in this paper, 10 Ophioceras species have been recorded from freshwater habitats. Mostert et al. (2006) studied the taxonomy and pathology of Togninia and its Phaeoacremonium anamorphs and accepted 10 species in the genus. They also provided keys to the genus and to the anamorphs. Three species of Togninia have been described since (Damm et al. 2008, Réblová and Mostert 2007). Togninia aquatica is characterized by globose ascomata with long necks, clavate and truncate asci clustered on distinct ascogenous hyphae, and small, reniform, hyaline ascospores. The morphological characters of T. aquatica fit well with the concept of the genus (Mostert et al. 2006). T. aquatica is morphologically similar to T. austroafricana L. Mostert, W. Gams & Crous in having long ascomatal necks (up to 1450 μm) and

39 reniform ascospores (Mostert et al. 2006). However, the ascospores of T. aquatica are narrower when compared to T. austroafricana (1 1.5 μm vs μm), and the peridia of the former is much thicker when compared to the latter (35 μm vs μm). The result of ITS sequences analysis (FIG 2) shows that T. aquatica appears as a distinct lineage and clustered together with the representatives of Togninia. In the tree T. aquatica is closely related phylogenetically to T. parasitica L. Mostert, W. Gams & Crous (FIG. 1). Morphologically, T. aquatica differs from T. parasitica in having longer ascomatal necks (1450 μm vs μm) and reniform ascospores as compared to the allantoid ascospores of T. parasitica (Mostert et al. 2006). Furthermore, T. aquatica is unique among species of Togninia in its freshwater habitat because most Togninia species are plant pathogens (Damm et al. 2008, Mostert et al. 2006). ACKNOWLEDGMENTS Financial support for research was provided by the Grant for Essential Scientific Research of the National Nonprofit Institute (No. CAFYBB ). The authors also express deep thanks to Prof Xiaoming Chen, Ma Tao, Hang Chen, HaiXia Wu and YanMei Li (The Research Institute of Resource Insects, Chinese Academy of Forestry) for their valuable help. Staff of the International Fungal Research and Development Centre at The Research Institute of Resource Insects, Chinese Academy of Forestry, also are thanked for providing help. Further financial support was provided by NSFC Dian-Ming Hu thanks Fang Liu for collecting samples in Beijing. LITERATURE CITED Cai L, Guo XY, Hyde KD Morphological and molecular characterisation of a new anamorphic genus Cheirosporium, from freshwater in China. Persoonia 20: DOI: / X314732, Hyde KD New species of Clohiesia and Paraniesslia collected from freshwater habitats

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42 Mostert L, Groenewald JZ, Summerbell RC, Gams W, Crous PW Taxonomy and pathology of Togninia (Diaporthales) and its Phaeoacremonium anamorphs. Stud Mycol 54: Müller E, Dennis RWG Fungi venezuelani VIII. Plectascales, Sphaeriales, Loculoascomycetes. Kew Bull 19: Nitschke T Pyrenomycetes Germanici 2: Page RDM TreeVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12: Pinruan U, Sakayaroj J, Jones EBG, Hyde KD Aquatic fungi from peat swamp palms: Phruensis brunneispora gen. et sp. nov. and its hyphomycete anamorph. Mycologia 96: DOI: / Raja HA, Miller AN, Shearer CA Freshwater Ascomycetes: Hyalorostratum brunneisporum, a new genus and species in the Diaporthales (Sordariomycetidae, Sordariomycetes) from North America. Mycosphere 1: , Schmit JP, Shearer CA Latitudinal, habitat and substrate distribution patterns of freshwater ascomycetes in the Florida Peninsula. Biodivers Conserv 18: DOI: /s Ranghoo VM, Tsui CKM, Hyde KD Brunneosporella aquatica gen. et sp. nov., Aqualignicola hyalina gen. et sp. nov., Jobellisia viridifusca sp. nov. and Porosphaerellopsis bipolaris sp. nov.(ascomycetes) from submerged wood in freshwater habitats. Mycol Res 105: DOI: /S Rannala B, Yang Z Probability distribution of molecular evolutionary trees: a new method of phylogenetic inference. J Mol Evol 43: DOI: /BF

43 Réblová M, Mostert L Romellia is congeneric with Togninia, and description of Conidiotheca gen. nov. for one species of this genus with polysporous asci. Mycol Res 111: DOI: /j.mycres Rehner SA, Samuels GJ Molecular systematics of the Hypocreales: a teleomorph gene phylogeny and the status of their anamorphs. Can J Bot 73: Saccardo PA Sylloge Pyrenomycetum. Vol. II. Sylloge Fungorum 2: Santos JM, Phillips AJL Resolving the complex of Diaporthe (Phomopsis) species occurring on Foeniculum vulgare in Portugal. Fungal Divers 34: Shearer CA, Crane JL, Chen W Freshwater ascomycetes: Ophioceras species. Mycologia 91: DOI: / , Descals E, Kohlmeyer B, Kohlmeyer J, Marvanova L, Padgett D, Porter D, Raja HA, Schmit JP, Thorton HA et al Fungal biodiversity in aquatic habitats. Biodivers Conserv 16: DOI: /s z, Raja HA, Miller AN, Nelson P, Tanaka K, Hirayama K, Marvanova L, Hyde KD, Zhang Y The molecular phylogeny of freshwater Dothideomycetes. Stud Mycol 64: DOI: /sim , Freshwater Ascomycetes Database: (Accessed 14 Mar 2012). Sivanesan A, Shaw DE Gnomonia papuana sp. nov. with a Sesquicillium conidial state. Trans Br Mycol Soc 68:85 90.

44 Sommer NF, Beraha L Diaporthe actinidiae, a new species causing stem-end rot of Chinese gooseberry fruits. Mycologia 67: Swofford DL PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4b10. Sunderland, Massachusetts: Sinauer Associates. Teng SJ Notes on Sphaeriales from China. Sinensia 4: Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG The Clustal X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: DOI: /nar/ Thongkantha S, Jeewon R, Vijaykrishna D, Lumyong S, McKenzie EHC, Hyde KD Molecular phylogeny of Magnaporthaceae (Sordariomycetes) with a new species, Ophioceras chiangdaoense from Dracaena loureiroi in Thailand. Fungal Divers 34: Tsui CKM, Leung YM, Hyde KD, Hodgkiss IJ Three new Ophioceras species (Ascomycetes) from the tropics. Mycoscience 42: DOI: /BF Udayanga D, Liu X, McKenzie EHC, Chukeatirote E, Bahkali AHA, Hyde KD The genus Phomopsis: biology, applications, species concepts and names of common phytopathogens. Fungal Divers 50:1 37. DOI: /s Vilgalys R, Hester M Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172: Walker J Gaeumannomyces, Linocarpon, Ophiobolus and several other genera of scolecospored ascomycetes and Phialophora conidial states, with a note on hyphopodia. Mycotaxon 11: Wehmeyer LE The genus Diaporthe Nitschke and its segregates. Univ Michigan Stud, Sci Ser

45 9: Cultural studies of three new pyrenomycetes. Mycologia 28: White TJ, Bruns T, Lee S, Taylor JW Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White YJ, eds. PCR protocols: a guide to methods and applications. San Diego, California: Academic Press. p Wong MKM, Hyde KD Fungi on grasses: new species of Ascotaiwania, Diaporthe and Oxydothis (Ascomycetes). Cryptogamie Mycol 22: DOI: /S (01) Zhaxybayeva O, Gogarten JP Bootstrap, Bayesian probability and maximum likelihood mapping: exploring new tools for comparative genome analyses. BMC Genomics 3:4. DOI: / LEGENDS FIG 1. Maximum parsimony phylogram inferred from ITS rdna sequence data, showing the phylogenetic relationship between Diaporthe aquatica and the other Diaporthe/Phomopsis species. Data analyzed with random addition sequence, unweighted parsimony and treating gaps as missing data. Values above the branches are parsimony bootstrap (equal or above 50%). Sequences derived from type cultures are marked with an asterisk. FIG 2. Maximum parsimony phylogram inferred from ITS rdna sequence data, showing the phylogenetic relationship between Togninia aquatica and the other Togninia/Phaeoacremonium species. Data analyzed with random addition sequence, unweighted parsimony and treating gaps as missing data. Values above the branches are parsimony bootstrap (equal or above 50%). Sequences derived from type cultures are marked with an asterisk. FIG 3. Maximum parsimony phylogram inferred from 28S rdna sequence data, showing the phylogenetic relationship among the Magnaporthaceae species. Data analyzed with random addition sequence, unweighted parsimony and treating gaps as missing data. Values above the branches are parsimony bootstrap (equal or above 50%). Branches with Bayesian posterior probabilities equal or

46 above 90% are thickened. The type species of the genera are marked with an asterisk. FIG 4. Maximum parsimony phylogram inferred from 18S rdna sequence data, showing the phylogenetic relationship among the Magnaporthaceae species. Data analyzed with random addition sequence, unweighted parsimony and treating gaps as missing data. Values above the branches are parsimony bootstrap (equal or above 50%). Branches with Bayesian posterior probabilities equal or above 90% are thickened. The type species of the genera are marked with an asterisk. FIG 5. Diaporthe aquatica. a. Ascoma on submerged wood. b. Section of an ascoma. c, d. Peridium. e. Paraphyses. f h. Asci. i k. Ascospores. Bars: a = 200 μm, b = 50 μm, c d = 20 μm, e k = 5 μm. FIG 6. Ophiocera aquaticus. a. Ascomata on submerged wood. b. Paraphyses. c e. Asci. f. Upper part of an ascus, showing the apical ring. g j. Ascospores. k. A germinated ascospore. Bars: a = 500 μm, b = 5 μm, c e = 20 μm, f k = 5 μm. FIG 7. Togninia aquatica. a. Ascomata on submerged wood. b. Section of an ascoma. c. Peridium. d. Squash of an ascoma. e. Paraphyses. f. Ascogenous hyphae. g i. Asci. j n. Ascospores. Bars: a = 200 μm, b = 30 μm, c = 5 μm, d = 50 μm, e n = 5 μm. FOOTNOTES Submitted 26 Dec 2011; accepted for publication 24 May Corresponding authors. mrcailei@gmail.com; kdhyde3@gmail.com

47 TABLE I. Sources and accession numbers of the isolates generated in this study Taxa Source GenBank accession numbers ITS rdna 28S rdna 18S rdna Diaporthe aquatica IFRDCC 3051 JQ Diaporthe aquatica IFRDCC 3015 JQ Togninia aquatica IFRDCC 3035 JQ Ophioceras aquaticus IFRDCC 3091 JQ JQ JQ Pseudohalonectria lignicola IFRDCC 3030 JQ Pseudohalonectria lignicola IFRDCC 3110 JQ797436

48

49

50

51

52