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Aquapeziza: a New Genus from Freshwater and Its Morphological and Phylogenetic Relationships to Pezizaceae

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Aquapeziza: a New Genus from Freshwater and Its Morphological and Phylogenetic Relationships to Pezizaceae Mycologia, 104(2), 2012, pp. 540–546. DOI: 10.3852/11-123 # 2012 by The Mycological Society of America, Lawrence, KS 66044-8897 Aquapeziza: a new genus from freshwater and its morphological and phylogenetic relationships to Pezizaceae Dian-Ming Hu Key words: Aquapeziza globispora, ascomycetes, International Fungal Research & Development Centre, fungi, phylogenetics, taxonomy The Research Institute of Resource Insects, Chinese Academy of Forestry, Bailongsi, Kunming 650224, P.R.China, and School of Chemistry and Life Science, INTRODUCTION Gannan Normal University, Ganzhou 341000, Freshwater fungi comprise a distinct ecological P.R.China assemblage of fungi and to date some 548 ascomycete Hang Chen species have been reported from this milieu (Raja International Fungal Research & Development Centre, et al. 2009). This ecological group however is still The Research Institute of Resource Insects, Chinese poorly studied (Wong et al. 1998, Raja et al. 2009). We Academy of Forestry, Bailongsi, Kunming 650224, have investigated the diversity of freshwater fungi for P.R.China more than 20 y (Hyde 1992, 1995; Cai et al. 2003b; Lei Cai1 Zhang et al. 2009; Reblova et al. 2010), most recently State Key Laboratory of Mycology, Institute of concentrating on southwestern China (Cai et al. 2002; Microbiology, Chinese Academy of Sciences, Beijing Luo et al. 2004; Cai and Hyde 2007; Hu et al. 2007, 100101, P.R.China 2010b) and Thailand (Hu et al. 2010a). During this Ali Hassan Bahkali study we collected an interesting discomycete that Botany and Microbiology Department, College of cannot be accommodated in any described genus, Science, King Saud University, Riyadh, Saudi Arabia based on its distinct morphological characters and 2 sequence data (28S and ITS rDNA sequences). The Kevin D. Hyde taxon is characterized morphologically by its com- International Fungal Research & Development Centre, bined characters of epigenous, white ascomata, ovoid, the Research Institute of Resource Insects, Chinese Academy of Forestry, Bailongsi, Kunming 650224, P.R. amyloid asci, and multi-guttulate, single-celled, smooth, China, School of Science, Mae Fah Luang University, globose ascospores. In this paper we introduce a new Chiang Rai, Thailand, and Botany and Microbiology species and genus to accommodate the taxon and Department, College of Science, King Saud University, provide a description and illustrations. Analyses of 28S Riyadh, Saudi Arabia and ITS rDNA sequences from the new taxon with reference sequences from GenBank were used to infer its phylogenetic relationships to Pezizaceae. Abstract: An investigation of freshwater fungi on submerged wood in southwestern China led to the discovery of a new discomycete species from a small MATERIALS AND METHODS stream in Yunnan Province. The taxon is character- Sample collection and specimen examination.—Fifty samples ized morphologically by its combined characters of were collected randomly from Yulu Stream, Yunnan epigenous, white ascomata, ovoid, amyloid asci and Province, China, 7 Feb 2010, returned to the laboratory multi-guttulate, single-celled, smooth, globose asco- and incubated following the method described by Cai et al. spores. Because the taxon cannot be accommodated (2006). The fungal specimens were deposited as herbarium in any known genus based on morphological charac- specimens in IFRDC. Samples were examined for fungal ters and molecular data (28S and ITS rDNA sequenc- fruiting bodies under a dissecting microscope (Leica MZ16A) during incubation. Observations and photographs es) a new genus and species Aquapeziza globispora is were prepared from material mounted in water under a proposed to accommodate it. The new genus is compound microscope (Nikon E800); Melzer’s reagent was circumscribed and a description and illustrations used to test the amyloid reaction. The single-spore isolation of the new species are provided. Relationships of method outlined by Choi et al. (1999) was used to attempt Aquapeziza in Pezizaceae are inferred from 28S and to isolate the fungus but was not successful. ITS rDNA sequence analyses. DNA extraction, amplification and sequencing.—Total DNA was extracted from the ascomata. A single ascoma was picked from the substrate with caution taken to ensure that Submitted 17 Apr 2011; accepted for publication 17 Aug 2011. no other fungal spores or hyphae adhered to the ascoma. 1 E-mail: [email protected] The ascoma was placed in sterile water and washed 3–5 2 Corresponding author. E-mail: [email protected] times. Total DNA was extracted with a Biospin Fungus 540 HUETAL.: A NEW FRESHWATER GENUS AQUAPEZIZA 541 Genomic DNA Extraction Kit (BioFluxH) according to the (Roberge ex Desm.) Ces. & de Not. (Sordariomy- manufacturer’s protocol. cetes) as the outgroup taxon. The final dataset Fragments of the partial large subunit (LSU) and internal comprised 849 characters after alignment without transcribed spacer (ITS) rDNA were amplified by the ambiguous regions. Parsimony analysis resulted in polymerase chain reaction (PCR). Primers LROR & LR6 four trees without significant difference. One of the (Vilgalys and Hester 1990, Rehner and Samuels 1995) and most parsimonious trees (TL 5 1339, CI 5 0.419, RI ITS4 & ITS5 (White et al. 1990) were used respectively for PCR amplification of LSU and ITS rDNA sequences. The 5 0.682, RC 5 0.286, HI 5 0.581) is illustrated here amplification was performed in a 50 mM reaction volume (FIG. 1). In the tree all Pezizaceae taxa formed a (buffer, MgCl2 1.5 mM, dNTP 0.2 mM, 0.8 mM of each distinct lineage with phylogenetic distance from primer and 1 U Taq DNA Polymerase). Thermal-cycling Pyronemataceae and Ascobolaceae; Aquapeziza globis- parameters included an initial denaturation of 95 C for pora nested in the clade of Pezizaceae with a close 2 min, followed by 35 cycles consisting of denaturation at relationship to Boudiera Cooke. 94 C for 1 min, annealing at 50 C for 1 min and extension The ITS rDNA dataset included sequences from 25 of 72 C for 1 min. A final extension at 72 C for 10 min was strains representing 11 genera of the Pezizaceae, one included at the end of the thermal cycling. The PCR Pyronemataceae (Pyronema domesticum)andone products were purified and sequenced with LROR and LR6 and ITS4 and ITS5 primers respectively (ABI-PRISM3730) Ascobolaceae (Ascobolus crenulatus), with Sordaria at Sangon Biotech Co. Ltd., Shanghai. fimicola (Sordariomycetes) as the outgroup taxon. The final dataset comprised 866 characters after Sequence alignment and phylogenetic analyses.—Sequences alignment without ambiguous regions. Parsimony were aligned with BioEdit (Hall 1999). Two novel sequences analysis resulted in two trees without significant (GenBank accession numbers HM996916, JF432065) from difference. One of the most parsimonious trees (TL the new taxon, together with reference sequences obtained 5 2145, CI 5 0.567, RI 5 0.615, RC 5 0.349, HI 5 from GenBank, were aligned with Clustal X (Thomson et al. 1997). Alignment was manually adjusted to allow maximum 0.433) is illustrated here (FIG. 2). In the tree all alignment and minimize gaps. Pezizaceae taxa formed a distinct lineage and showed Phylogenetic analyses were performed with maximum phylogenetic distance from Pyronemataceae and parsimony as implemented in PAUP* 4.0b10 (Swofford Ascobolaceae; Aquapeziza globispora nests in the clade 2002). Characters were equally weighted and gaps were of Pezizaceae, with a close relationship to Pachyph- treated as missing data. Trees were inferred with the loeus Tul. & C. Tul. heuristic search option with TBR branch swapping and 1000 random sequence additions. MAXTREES were unlimit- TAXONOMY ed, branches of zero length were collapsed and all parsimonious trees were saved. Clade stability was assessed Aquapeziza D.M. Hu, L. Cai & K.D. Hyde, gen. nov. with a bootstrap (BT) analyses with 1000 replicates, each MycoBank MB561613. with 10 replicates of random stepwise of taxa. Kishino- Etymology. Aquapeziza refers to the aquatic habitat of Hasegawa tests (KH Test) (Kishino and Hasegawa 1989) this fungus. were performed to determine whether trees were signifi- Apothecia dispersa, epigenous, sessilia, turbinata, alba; cantly different. Trees were drawn with TreeView (Page excipulum ectale et excipulum medullare exhibentes. 1996). Paraphyses cylindracei, hyalinae. Asci late cylindracei vel The model of evolution was estimated with MrModeltest ovoidei, apice rotundati, amyloid, 8-ascosporae. Ascosporae 2.2. Posterior probabilities (PP) (Rannala and Yang 1996, globosae, guttulatae, leavae, hyalinae. Zhaxybayeva and Gogarten 2002) were determined by Markov chain Monte Carlo sampling (BMCMC) in MrBayes Typus. Aquapeziza globispora D.M. Hu, L. Cai & K.D. 3.0b4 (Huelsenbeck and Ronquist 2001). Six simultaneous Hyde Markov chains were run 1 000 000 generations and trees Apothecia scattered, epigenous, sessile, turbinate, were sampled every 100 generations (resulting 10 000 total white; excipulum composed of an ectal exipulum of trees). The first 2000 trees, which represented the burn-in textura globulosa to textura angularis and medullary phase of the analyses, were discarded and the remaining excipulum of textura intricata. Paraphyses cylindrical, 8000 trees were used for calculating posterior probabilities septate, hyaline. Asci unitunicate, not protruding, (PP) in the majority rule consensus tree. operculum not distinct, at first broad cylindrical becoming ovoid at maturity, with a rounded apex, RESULTS hyaline, amyloid, eight-ascospored. Ascospores glo- bose, one-celled,
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