Mycologia, 104(5), 2012, pp. 1178–1186. DOI: 10.3852/11-379 # 2012 by The Mycological Society of America, Lawrence, KS 66044-8897

Conlarium duplumascospora gen. et. sp. nov. and Jobellisia guangdongensis sp. nov. from freshwater habitats in China

Fang Liu partial nuclear large subunit ribosomal DNA (LSU Dian-Ming Hu rDNA) sequences was conducted to establish the sys- Lei Cai1 tematic placement of this . Our data showed State Key Laboratory of Mycology, Institute of that this is a new that presents sufficient Microbiology, Chinese Academy of Sciences, Beijing distinctions to other related genera. It is described 100101, P.R. China herein as Conlarium duplumascospora gen. et. sp. nov. Jobellisia M.E. Barr originally was placed in the Clypeosphaeriaceae of Xylariales based on the mor- Abstract: Conlarium duplumascospora gen. et. sp. nov. phological characteristics (Barr 1993). However, phy- and Jobellisia guangdongensis sp. nov. are described logenetic studies indicated that Jobellisia does not and illustrated from submerged wood collected from belong to Xylariales and a new family Jobellisiaceae Guangdong Province, China. Conlarium duplumascos- was established to accommodate this genus (Re´blova´ pora is characterized by gregarious, coriaceous and 2008). Jobellisiaceae appears basal to Diaporthales beaked ascomata; cylindrical, unitunicate asci with a and Calosphaeriales in the phylogenetic tree but its bipartite apical ring; biseriate, fusiform, hyaline, 0–5- ordinal placement awaits further investigation (Re´- septate ascospores with or without appendages; and blova´ 2008). Jobellisia (Barr 1993) is characterized by anamorph with muriform conidia. Jobellisia guang- large ascomata with a papilla or a long, stout neck; dongensis is characterized by globose to subglobose, two- or three-layered perithecial wall in longitudinal gregarious and papillate ascomata; three-layered pe- section; and brown, aseptate ascospores with one or ridium; cylindrical, unitunicate asci with a refractive two polar germ pores. Our recent investigation of apical ring; and one-septate, fusiform, greenish brown freshwater fungi yielded an undescribed Jobellisia ascospores. Sequence analyses of partial nuclear large species from Guangdong Province of China. The new subunit ribosomal DNA (LSU rDNA) were performed species is described, illustrated and compared with to infer the phylogenetic affinities of these new taxa. other species in genus Jobellisia. Phylogenetic rela- A key to species of Jobellisia is provided. tionships are inferred with partial LSU rDNA se- Key words: Ascomycetes, freshwater fungi, system- quences data. atics,

MATERIALS AND METHODS INTRODUCTION Sample collection.—Submerged woody samples were col- The S.W. Wong, K.D. Hyde & E.B.G. lected from streams at Dinghu Mountain Nature Reserve in Jones is a family of freshwater ascomycetes charac- Zhao Qing, Guangdong Province, China, in Dec 2010. terized by beaked, dark brown or black ascomata; Samples were returned to the laboratory and incubated in cylindrical, unitunicate asci with a relatively large re- sealed plastic bags with moist tissues to maintain humid fractive apical ring; and hyaline ascospores mostly conditions. They were incubated at room temperature (20– with appendages or surrounded by a mucilaginous 30 C) under normal light. After 2 wk, the woody substrates sheath (Wong et al. 1998). Recent phylogenetic studies were examined for the presence of fungi periodically have revealed that Annulatascaceae is polyphyletic during the incubation period (usually 3 mo). The fruiting (Abdel-wahab et al. 2011, Campbell and Shearer 2001, bodies were picked off with a sterilized needle, and single- Campbell and Shearer 2004, Raja et al. 2003). Species spore isolations were made on water agar (WA) with anti- with relative large refractive apical ring were found biotics following the method described by Choi et al. (1999). Single germinated spores were picked up with a to be scattered in several clades of pyrenomycetes sterilized needle and transferred onto new potato dextrose (Vijaykrishna et al. 2005). During our investigation agar (PDA) plate and incubated at 25 C under normal light of freshwater fungi in China, a fungus with typical for further study. We made morphological observation of characteristics of Annulatascaceae was collected. Mor- material mounted in distilled water. Microphotographs of phological comparisons were made with genera in fungi were taken from material mounted in water using a Annulatascaceae, and phylogenetic analysis using Nikon 80i microscope. Fifty conidia or ascospores of each fungus were measured. The range between minimum and Submitted 16 Nov 2011; accepted for publication 27 Mar 2012. maximum values are included, with extreme value in pa- 1 Corresponding author. E-mail: [email protected] rentheses. The specimens are deposited in the herbarium of

1178 LIU ET AL.: NEW FRESHWATER ASCOMYCETES 1179

Microbiology, Academia Sinica (HMAS), while living were used to calculate posterior probabilities (PP) in the cultures deposited in China General Microbiological majority rule consensus tree. Culture Collection Center (CGMCC).

DNA extraction.—Total genomic DNA was extracted from RESULTS ascomata or axenic cultures with modified CTAB protocol described in Guo et al. (2000). For DNA extraction, All sequences obtained, including the ones not used ascomata were picked from the substrate with sterile forcep in phylogenetic analyses, were submitted in GenBank with a fine tip. The ascomata were washed in sterile distilled with accession numbers JN936987–JN936997. The water before DNA extraction to ensure no other fungal alignments were deposited in TreeBASE (S12514). material adhered to the ascomata. A BLAST query in NCBI showed that the LSU se- quence of Conlarium duplumascospora was most sim- PCR and sequencing.—Partial small subunit (SSU), large ilar to Papulosa amerospora Kohlm. & Volkm. -Kohlm. subunit (LSU) and internal transcribed spacer (ITS) rDNA were amplified with fungal specific primers NS1/NS4, (DQ470950, 95%) (Spatafora et al. 2006), Tectonidula LROR/LR5 and ITS1/ITS4 (Vilgalys and Hester 1990, hippocrepida Re´blova´ (FJ617557, 95%) (Re´blova´ and White et al. 1990). PCR was performed in a 25 mL reaction Stepa´nek 2009), Calosphaeria barbirostris (Fr.) Ellis & containing 17.3 mL double distilled water, 2.5 mL103 PCR Everh. (EF577059, 94%) (Re´blova´ 2007), Cryptadel- buffer, 2 mL dNTP mix (2.5 mM), 1 mL each primer (10 mM), phia groenendalensis (Sacc., E. Bommer & M. Rous- 1 mL DNA template, 0.2 mL Taq DNA polymerase. Thermal- seau) Re´blova´ & Seifert (AY281104, 94%) (Re´blova´ cycling parameters included an initial denaturation of 95 C and Seifert 2004), cuspidata (Fr.) for 5 min, followed by 35 cycles consisting of denaturing at Re´blova´ (FJ617558, 94%) (Re´blova´ and Stepa´nek 95 C for 30 s, annealing at 54 C for 30 s and extension of 2009) and Dactylaria parvispora (Preuss) de Hoog & 72 C for 1 min, followed by 10 min at 72 C for extension. von Arx (EU107296, 94%) (Bhilabutra et al. unpubl). The PCR products were purified and sequenced with NS1/ NS4, LROR/LR5 and ITS1/ITS4 primers mentioned above The LSU rDNA dataset consisted of 35 sequences at SinoGenoMax Co. Ltd. representing Annulatascaseae sensu lato and other members of representative groups, such as Tricho- Sequence data analysis.—The SSU, LSU and ITS rDNA sphaeriales, Thyridiaceae, Papulosaceae, Orbiliales sequences were obtained. However, SSU and ITS sequences and Magnaporthales. Phaeosphaeria typharum (Desm.) failed to give sufficient information when we analyzed the L. Holm (Phaeosphaeriaceae, Pleosporales) was used phylogenetic affinity of our isolates with related taxa, be- as an outgroup. The final dataset comprised 836 cause most freshwater fungi lack available SSU and ITS sequences in GenBank. LSU rDNA sequences together with characters after alignment without ambiguous regions. reference sequences from GenBank were aligned by Clustal Parsimony analysis resulted in 10 equally parsimonious X (Thompson et al. 1997). trees. One of the trees (TL 5 1194, CI 5 0.559, RI 5 Alignments were optimized manually with BioEdit (Hall 0.580, RC 5 0.324, HI 5 0.441) is illustrated (FIG. 1). 1999). Phylogenetic analyses were performed with PAUP* In the phylogenetic tree, the three C. duplumascospora 4.0b10 (Swofford 2002). Ambiguously aligned regions were strains form a distinct lineage and have affinity with excluded from all analyses. Unweighted parsimony (UP) several members in Annulatascaceae sensu lato. analysis was performed. Trees were inferred with the The LSU sequence of Jobellisia guangdongensis is heuristic search option with TBR branch swapping and most similar to J. luteola (Ellis & Everh.) M.E. Barr 1000 random sequence additions. MAXTREES were unlimit- (AY346286, 95%) and J. fraterna Huhndorf, Lodge & ed, branches of zero length were collapsed and all multiple % parsimonious trees were saved. Clade stability was assessed F.A. Ferna´ndez (AY346285, 94 ) (Huhndorf et al. in a bootstrap analysis with 1000 replicates, each with 10 2004) in NCBI through a BLAST query. The LSU replicates of random stepwise addition of taxa. Shimodaira- rDNA dataset consisted of 26 sequences represent- Hasegawa test (SH test) (Shimodaira and Hasegawa 1999) ing members of Sordariales, Boliniales, Coniochae- were performed to determine whether trees were signifi- tales, Diaporthales, Calosphaeriales, Jobellisiaceae, cantly different. Trees were visualized in TreeVIEW (Page Xylariales and Hypocreales, with Pleospora herbarum 1996). (Pers.) Rabenh. and Setosphaeria monocera Alcorn Model of evolution was estimated with MrModeltest 2.2 as out group. The final dataset comprised 840 char- (Nylander 2004). Posterior probabilities (PP) (Rannala and acters after alignment without ambiguous regions. Yang 1996, Zhaxybayeva and Gogarten 2002) were deter- Parsimony analysis resulted in two equally parsimo- mined by Markov chain Monte Carlo sampling (BMCMC) in nious trees. One of the trees (TL 5 888, CI 5 0.528, MrBayes 3.0b4 (Huelsenbeck and Ronquist 2001), using the 5 5 5 estimated model of evolution. Six simultaneous Markov RI 0.673, RC 0.356, HI 0.472) is illustrated chains were run 1 000 000 generations, and trees were sam- (FIG. 2). In the tree, Jobellisia fraterna, J. luteola and pled every 100th generation (resulting 10 000 total trees). J. guangdongensis form a strongly supported group The first 2000 trees, which represented the burn-in phase of (100%) basal to the clade with members of Dia- the analyses, were discarded, and the remaining 8000 trees porthales and Calosphaeriales. 1180 MYCOLOGIA

FIG. 1. Maximum parsimony phylogram showing phylogenetic relationships of Conlarium duplumascospora with closely related taxa based on LSU rDNA. Bootstrap support values above 50% are above the branches. Thickened branches represent significant Bayesian posterior probability ($ 95%). The tree is rooted with Phaeosphaeria typharum.

TAXONOMY 175–250 mm high, with cylindrical neck, straight or slightly flexuous, 125–175 mm long, 50–75 mm wide. Conlarium F. Liu & L. Cai, gen. nov. Peridium comprising several layers of textura angu- MycoBank MB564382 laris cells, which are usually darker externally and pale Etymology. Conlarium, in latin means ‘‘together’’, internally, 15.5–28.5 mm thick. Paraphyses 4 mm wide, referring to the gregarious ascomata. hyaline, branched, septate, not embedded in a ma- Ascomata perithecioid, superficial, or partially im- trix. Asci eight-spored, unitunicate, short pedicellate, mersed, dark brown to black, gregarious, smooth, glo- cylindrical, 126–151.5 3 11–14 mm, with a large, bi- bose to subglobose; neck elongated, cylindrical, straight partite apical ring, 5 mm wide, 4 mm high. Ascospores or slightly curved. Paraphyses hyaline, branched, sep- biseriate, fusiform, straight or cymbiform, 0–5-septate, tate, not embedded in a matrix. Asci eight-spored, hyaline, guttulate, 29–32 3 5–7 mm, with or without unitunicate, short pedicellate, cylindrical, with bipartite globose or papillary appendages at one or each end of apical ring. Ascospores biseriate, fusiform, straight or slightly curved, aseptate to multiseptate, hyaline, guttu- the ascospores. late, with or without appendages at one or each end of Cultures and anamorph.—Colonies reach 8 mm diam the ascospores. after 2 mo, dark brown to black, planar, smooth. Co- nidiophores 18.5–34.0 3 2.0–5.4 mm, micronematous Conlarium duplumascospora F. Liu & L. Cai, sp. nov. or semi-macronematous, mononematous, septate or FIG.3 aseptate, unbranched or irregularly branched, straight MycoBank MB564383 or flexuous, hyaline, becoming brown with age. Etymology. Duplumascospora, referring to the biseri- Conidiogenous cells determinate, doliiform, cylindrical, ate arrangement of ascospores. 5.0–10.0 3 3.0–6.0 mm. Conidia brown, muriform, Ascomata perithecioid, superficial, or partially im- irregularly globose or subglobose, constricted at the mersed, coriaceous, dark brown to black, gregarious, septa, 15.5–35.0 3 11–26.5 mm. Chlamydospores not smooth, globose to subglobose, 200–250 mm diam, observed. LIU ET AL.: NEW FRESHWATER ASCOMYCETES 1181

FIG. 2. Maximum parsimony phylogram showing phylogenetic relationships of Jobellisia guangdongensis with closely related taxa based on LSU rDNA sequences. Bootstrap support values above 50% are above the branches. Thickened branches represent significant Bayesian posterior probability ($ 95%). The tree is rooted with Pleospora herbarum and Setosphaeria monoceras.

Habitat. Saprobic on submerged wood. slightly constricted and thick-walled at the septum, Distribution. China, collected in Guangdong greenish-brown. Province. Habitat. Saprobic on submerged wood. Holotype. CHINA. GUANGDONG PROVINCE: Zhaoqing Distribution. China, collected in Guangdong Province. Dinghu Mountain, in a stream. On submerged wood, 29 Holotype. CHINA. GUANGDONG PROVINCE: Zhaoq- Dec 2010, F. Liu, HMAS243129, ex-type living culture ing Dinghu Mountain, in a stream. On submerged wood, 29 CGMCC3.14938; ibid GD34-5-1, living culture CGMCC3.14939, Dec 2010, F. Liu, HMAS251240. No culture was obtained ibid GD34-5-2, living culture CGMCC3.14940. despite several attempts.

Jobellisia guangdongensis F. Liu & L. Cai, sp. nov. KEY TO ACCEPTED SPECIES OF JOBELLISA FIG.4 MycoBank MB564385 1. Asci with large refractive apical ring, ascospores one- Etymology. Guangdongensis, referring to the prov- septate, with darker band of pigment at septum . . . 2 ince where the type was collected. 19. Asci with delicate ring-like structures, ascospores Ascomata 200–400 mm high, 175–400 mm diam, glo- one-septate or one-celled without darker band of bose to subglobose, superficial, dark brown to black, pigment at septum ...... J. nicaraguensis 2. Ascospores ellipsoidal and reddish-brown . . . 3 gregarious, papillate, ostiolate. Peridium 60 mm wide, 29. Ascospores not as above ...... 5 three-layered in longitudinal section. Paraphyses 2.5– 3. Ascomata luteous, orange to yellow-brown, collaps- 5 um wide, numerous, filamentous, septate, constrict- ing, ascospores 11–14 3 4.5–5.5 mm..... J. luteola ed at the septa, not embedded in a gelatinous ma- 39. Ascomata dark brown, black or dark blue-green trix. Asci 76.5–97 3 5–8 mm, eight-spored, cylindrical, iridescence, not collapsing ...... 4 short pedicellate, unitunicate, with a refractive api- 4. Ascoma wall smooth, black to dark blue-green cal apparatus, 3.5–4.5 mmindiam.Ascospores iridescence, easily shed exposing bright orange 8.0–10.5 3 (3.0–)3.5–4.5 mm, overlapping uniseriate, middle wall; ascospores 9–11.5(–11.8) 3 3– fusoid to fusiform, with acute ends, one-septate, 4.5 mm ...... J. barrii 1182 MYCOLOGIA

FIG.3. Conlarium duplumascospora (from holotype). a. Ascomata on natural substratum. b. Peridium. c. Paraphyses. d–f. Asci. Note the bipartite refractive apical ring. g–h. Upper and reverse colony after 2 mo cultivation. i–j. Ascospores. Note the polar appendages (arrowed). k. Conidiophore bearing mature (left) and immature (right) conidium. l–m. Mature conidia. Bars: a 5 100 mm; b–f, i–m 5 10 mm; g–h 5 10 mm. LIU ET AL.: NEW FRESHWATER ASCOMYCETES 1183

FIG.4. Jobellisia guangdongensis (from holotype). a. Ascomata on natural substratum. b. Section of ascoma. c–d. Asci. Note the refractive apical ring. e. Paraphyses. f–h. Ascospores. Bars: a 5 200 mm, b–h 5 10 mm. 1184 MYCOLOGIA

49. Ascoma wall roughened, dark brown, not cells. With only a few exceptions, such as Chaetoros- easily shed, without bright orange middle trum quincemilensis Zelski, Raja, A.N. Mill & Shearer wall; ascospores (9.5–)11–14.5(–15) 3 4.25– (Zelski et al. 2011), most of the members in 5 mm ...... J. fraterna Annulatascaceae sensu lato do not have reported 5. Asci cylindrical, 135–150 3 7–8 mm; ascospores ob- anamorphs. long, (16–)18–19(–20) 3 5–6 mm... J. saliciluticola Jobellisia was established to accommodate taxa that 59. Ascospores shorter than 16 mm ...... 6 produce immersed or superficial ascomata surround- 6. Asci 76.5–97 3 5–8 mm; ascospores 8–10.5 3 (3–)3.5–4.5 mm ...... J. guangdongensis ed by stromatic tissues, and brown, ellipsoid, one- 69. Asci 96–105.5 3 6–7 mm; ascospores 11–13.5 3 celled or one-septate ascospores (Barr 1993, 1994). 3.5–5 mm ...... J. viridifusca Jobellisia guangdongensis fits this morphological cir- cumscription well. It is similar morphologically to J. viridifusca K.M. Tsui & K.D. Hyde in having superfi- DISCUSSION cial, gregarious ascomata, bright orange middle wall layer, cylindrical asci with relatively large refractive The genus Conlarium is established for C. duplumas- apical rings and fusiform, greenish brown ascospores cospora based on the morphological characters and (Ranghoo et al. 2001). By re-examination of the type molecular phylogeny inferred from LSU rDNA specimen of J. viridifusca (IFRD 8771, holotype), we sequences. Conlarium is unique among genera in concluded that J. guangdongensis differs from J. Annulatascaceae sensu lato because of its unusual viridifusca in the size of asci (76.5–97 3 5–8 mm vs. combination of several morphological characters (i.e. 96–105.5 3 6–7 mm) and ascospores (8–10.5 3 [3–] gregarious, dark brown to black ascomata with long 3.5–4.5 mm vs. 11–13.5 3 3.5–5 mm). The dimensions neck; biseriate ascospores with or without globose we obtained upon re-examination of the holotype or papillary appendages at one or each end, and material were concordant with the original descrip- anamorph with muriform conidia). In Annulatasca- tion of J. viridifusca (Ranghoo et al. 2001). Unfortu- ceae sensu lato, Conlarium differs from Aqualignicola nately we failed to obtain genomic DNA from the Ranghoo, K.M. Tsui & K.D. Hyde, Fryar & K.D. holotype of J. viridifusca. J. guangdongensis grouped Hyde and Brunneosporella Ranghoo & K.D. Hyde in with J. fraterna and J. luteola in a strongly supported producing coriaceous ascomata, while the latter three distinct clade (FIG. 2). As reported by Re´blova´ (2008), have membranous ascomata. Moreover, Aqualignicola Jobellisiaceae appears distant from Xylariales but is characterized by short neck and unicellular bears a close relationship to Diaporthales and Calo- ascospores, and Brunneosporella species produce sphaeriales (FIG. 2). Six species currently are recog- uniseptate ascospores. Torrentispora K.D. Hyde, nized in Jobellisia, namely J. luteola (Ellis & Everh.) W.H. Ho, E.B.G. Jones, K.M. Tsui & S.W. Wong is M.E. Barr, J. nicaraguensis (Ellis & Everh.) M.E. Barr, different from Conlarium in producing solitary J. barrii Huhndorf, Lodge & F.A. Ferna´ndez, J. ascomata and uniseriate, one-celled ascospores. Clo- fraterna Huhndorf, Lodge & F.A. Ferna´ndez, J. hiesia K.D. Hyde and K.D. Hyde are distinct saliciluticola P. Leroy and J. viridifusca (Barr 1993, from Conlarium in producing clypeate ascomata. Huhndorf et al. 1999, Leroy 2006, Ranghoo et al. Conlarium also is comparable morphologically to 2001). Jobellisia rhynchostoma (Ho¨hn.) M.E. Barr has Diluviocola K.D. Hyde, S.W. Wong & E.B.G. Jones been transferred to Bellojisia (Lasiosphaeriaceae, and Fluminicola S.W. Wong, K.D. Hyde & E.B.G. Sordariales) based on phylogenetic analyses of partial Jones; Conlarium however differs from them in having LSU rDNA sequence data (Re´blova´ 2008), which is polar appendages. Results from our phylogenetic supported by our molecular analysis (FIG. 2). analysis indicated that the Annulatascaceae sensu lato Freshwater fungi, such as the taxa described herein, is polyphyletic as previously reported (Abdel-wahab colonize submerged organic matter (e.g. leaves, twigs, et al. 2011, Campbell and Shearer 2001, Campbell branches) (Cai et al. 2003, Ranghoo et al. 2001, and Shearer 2004, Raja et al. 2003), and Conlarium Vijaykrishna and Hyde 2006) and play an important has affinities with members in Annulatascaceae sensu role in degrading woody debris and leaves in lato but could not be included in any existing genera freshwater environments (Cai et al. 2006, Pascoal (FIG.1).Theanamorphicstageof Conlarium is et al. 2005, Simonis et al. 2008, Suberkropp 1997). comparable to Monodictys at first glance but differs They also are known to produce various enzymes and in having constrictions at the septa. It also is similar antifungal metabolites (Bucher et al. 2004, Simonis morphologically to Canalisporium in producing muri- et al. 2008, Yuen et al. 1999). In contrast with ter- form conidia. The conidia of Canalisporium however restrial environments, wood-inhabiting fungi in the are complanate and have ordered rows of cells. While freshwater environment in mainland China have in Conlarium conidia are composed of anomalistic received relatively less attention. According to our LIU ET AL.: NEW FRESHWATER ASCOMYCETES 1185 statistics, 894 freshwater species have been recorded ———, Miller AN, Ferna´ndez FA. 2004. Molecular system- from China, and it has also been estimated that many atics of the Sordariales: the order and the family new species remain to be discovered (Cai et al. 2012). Lasiosphaeriaceae redefined. Mycologia 96:368–387, doi:10.2307/3762068 Leroy P. 2006. 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