Mycologia, 104(3), 2012, pp. 000–000. DOI: 10.3852/11-238 # 2012 by The Mycological Society of America, Lawrence, KS 66044-8897

Annulatascus aquatorba sp. nov., a lignicolous freshwater ascomycete from Sirindhorn Peat Swamp Forest, Narathiwat, Thailand

Nattawut Boonyuen1 species were Acrogenospora sphaerocephala, Annulatas- Mycology Laboratory (BMYC), Bioresources Technology cus sp.1, A. velatisporus, Cancellidium applanatum, Unit (BTU), National Center for Genetic Engineering Chaetopsina sp.2, Cryptophialoidea unilateralis, Dictyo- and Biotechnology (BIOTEC), 113 Thailand Science chaeta sp.3, Helicomyces roseus, Hypocrea sp.1, Jahnula Park, Phaholyothin Road, Khlong 1, Khlong Luang, Pathumthani 12120, Thailand, and appendiculata, Monodictys sp.1, Monodictys sp.2, Tricho- Department of Plant Pathology, Faculty of Agriculture, derma sp.1, Verticillium sp.1 and Xylomyces chlamydos- Kasetsart University, 50 Phaholyothin Road, poris. The site is situated in an area subject to mon- Chatuchak, Bangkok 10900, Thailand soonal seasonality, with high relative humidity and Veera Sri-indrasutdhi rainfall nearly year round (2560 mm on average). It Satinee Suetrong offers an unusual habitat for tropical forests with its Somsak Sivichai acidic water (with pH approximately 4.5–6.0) and E.B. Gareth Jones relative humidity, low input of nutrients and lack of Mycology Laboratory (BMYC), Bioresources Technology soil or firm ground for the growth of plants (Anon Unit (BTU), National Center for Genetic Engineering 1994, 1996). Test blocks were recovered at intervals over and Biotechnology (BIOTEC), 113 Thailand Science an 8 y period and examined for fungi that resulted in Park, Phaholyothin Road, Khlong 1, Khlong Luang, the discovery of this new taxon. Numerous new species Pathumthani 12120, Thailand have been described from this site (Pinnoi et al. 2003a, b; Pinruan et al. 2004a, b; Sivichai and Boonyuen 2010). Hyde (1992) described the (type Abstract: As part of a long term study of fungi col- species Annulatascus velatispora) from submerged onizing submerged wood in freshwater streams a new dead wood collected in northern Queensland, Austra- Annulatascus species, A. aquatorba, is described and lia. The genus is characterized by perithecioid, im- illustrated from Erythrophleum teysmannii test blocks mersed or superficial, black ascomata with long necks; from Sirindhorn Peat Swamp Forest, southern Thai- wide, tapering paraphyses; cylindrical, unitunicate asci land. It differs from other species in the genus in with a relatively large, refractive apical ring; fusiform, ascospore measurements, thickness of the cell wall, 1– septate, hyaline ascospores with appendages or sheaths. 3-septate, fusoid to lunate shape, with central brown Currently 16 Annulatascus species (, cells and subhyaline end cells and without a mucilag- , , Pezizomycotina, inous sheath. Asci are cylindrical, pedicellate, with a ) have been described on submerged sub- distinct, wedge-shaped and non-amyloid apical ring. strata (decaying wood, stems of Phragmites australis, Phylogenetic relationships of this species, based on dead petiole of Licuala ramsayi, bamboo) from the combined partial 18S and 28S rDNA, place it freshwater habitats (TABLE I). Annulatascus is a cosmo- in the same clade as A. velatisporus (type species), A. politan genus with four species described from Queens- hongkongensis and A. nilensis. land, Australia and nine species reported from tropical Key words: Annulatascaceae, freshwater, peat southeastern Asia (TABLE I). Two species have been de- swamp, phylogeny, scribed from Africa, whereas only one species has been reported from South America (TABLE I). Tsui et al. INTRODUCTION (2002) provided a key to species and a synoptic table of In an ongoing long-term survey of fungi colonizing 12 Annulatascus species based on morphological char- nine timber species submerged in aquatic habitats in acteristics, while A. apiculatus (Barbosa et al. 2008), A. Thailand a referable to the genus Annulatas- bipolaris (Hyde 1992) and A. liputii (Cai et al. 2002) cus K.D. Hyde was collected on Erythrophleum were described subsequently. teysmannii Craib test blocks in Sirindhorn Peat At the molecular level, based on 28S rDNA sequenc- Swamp Forest, Narathiwat Province. This fungus was es, Abdel-Wahab et al. (2011) showed that A. nilensis collected along with 22 other species (17 mitosporic grouped with A. velatisporus and A. hongkongensis in fungi and five ascomycetes). The most common the same clade, but A. biatriisporus did not group with them, suggesting Annulatascus might be polyphyletic. Submitted 17 Jul 2011; accepted for publication 26 Oct 2011. In this paper we describe and illustrate a new 1 Corresponding author. E-mail: [email protected] Annulatascus species with morphological and molecular

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TABLE I. Comparative table of the morphological features of Annulatascus species modified from Tsui et al. 2002 (all species listed alphabetically and measured in micrometers)

Apical ring Fungi Ascomata (high 3 diam) Neck Asci (high 3 diam)

A. aquatorba 310–350 3 390–500 100–120 3 400–480 102.5–152.5 3 6.25–8.75 5.0–7.5 3 2.5–3

A. apiculatus 400–5503 240–410 100–250 3 100–180 175–250 3 10–13 6–7.2 3 1.8–2.4

A. aquaticus 220–600 3 240–600 400–800 3 60–80 150–175 3 10–12 3.5–4 3 4.5–5

A. biatriisporus 195–325 3 390–520 No data 210–260 3 12–17 No data

A. bipolaris 155–235 diam No data 174–205 3 9–10.5 4 3 6

A. citriosporus 224–310 3 350–485 No data 137.5–178.8 3 10–12 2.75–3.25 35–5.5

A. fusiformis 170–220 diam ca. 210–230 in high 141–235 3 7.5–10.5 4–5 3 3–4

A. hongkongensis 210–250 3 250–280 140–150 3 35–40 250–275 3 25–30 3.5–4.5 3 4.7–6.0

A. joannae 180–200 3 150–200 150–200 3 40–60 150–200 3 10–13 2.5–4 3 5–6.5

A. lacteus 140–200 3 100–130 50 3 40–50 130–170 3 9–10.5 3–4 3 4–5

A. licualae 77.5–95 3 202.5–460 56–82 diam 88.8–125 3 5.8–7.8 1.5–2 3 2.5–2.75

A. liputii 200–300 3 180–260 250–400 3 45–70 130–187.5 3 8.5–10 2 3 3.5

A. nilensis 400–600 3 220–280 240–360 3 96–112 260–400 3 12–14 3–4 3 5–6

A. palmietensis 150–440 diam 200 3 70 98–142 3 7–10.5 3–4 3 4–4.5

A. tropicalis 250–260 3 250–270 100–50 3 30–50 190–255 3 12–18 3–5 3 3–5

A. velatisporus 450 3 260–410 384 3 140 220–290 3 12–18 7–8 3 4–5

Annulusmagnus 222–353 diam 110 in high 140–218 3 9–14 3 3 5.5 (Annulatascus) triseptatus evidence (two loci of 18S and 28S rDNA sequences) Valeton, Erythrophleum teysmannii Craib, Melaleuca cajuputi from submerged E. teysmannii test blocks in the peat Powell, Shorea obtusa Wall, S. roxburghii G. Don, S. siamensis swamp forest. Miq, Wrightia tomentosa Roem. & Schult, Xylia xylocarpa (Roxb.) W. Theob, and Zollingeria dongnaiensis Pierre) MATERIALS AND METHODS were submerged in Sirindhorn Peat Swamp Forest, Sungai Kolok, in Narathiwat, Thailand (6.00295N, 101.9571889E). Specimen collection, fungal isolates, identification and growth.— After 2 y (22 Feb 2003) four sets of nine timber species Nine timber species (Azadirachta indica var. siamensis (15 3 2.5 3 2.5 cm3) were recovered and returned to the

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TABLE I. Extended

Known Ascospores Septate/aseptate Substrate distribution Reference

15–22.5 3 5–7.5, fusoid to lunate, lacking (1–)3, constricted Erythrophleum Narathiwat, This study appendages or a sheath at the septa teysmannii Thailand (test blocks) 23–36.5 3 8.8–10, uniseriate, fusiform, 0–3-septate, not Submerged Brazil Barbosa et al. with a narrow mucilaginous sheath constricted wood (2008 at septum 19–24 3 6–7, uniseriate, hyaline, ellipsoidal, Aseptate Submerged Hong Kong, Ho et al. (1999a) with a mucilaginous sheath wood China 40–58 3 8–10, fusiform, with a mucilaginous Aseptate Submerged Queensland, Hyde (1995) sheath wood Australia 21–30 3 6.5–8.5, uniseriate, hyaline, Aseptate Submerged Queensland, Hyde (1992) fusiform, with a polar appendage and a wood Australia mucilaginous sheath 22.5–30.5 3 6.4–8.5, fusiform, with a thin Aseptate Dead petiole of Brunei Fro¨hlich and mucilaginous sheath Licuala sp. Darussalam Hyde (2000) (plam) 16.5–25.5 3 6–9, uniseriate, hyaline, 1–5-septate Submerged The Philippines Hyde and Wong fusiform, with a bipolar mucilaginous wood (2000) pad–like appendage 35–37.5 3 12.5–15, uniseriate, hyaline, Three-septate Submerged Hong Kong, Ho et al. (1999b) ellipsoidal, with a mucilaginous sheath wood China 20–28 3 9–12, uniseriate, hyaline, Aseptate Submerged Hong Kong, Tsui et al. (2002) ellipsoidal to fusiform, with a thin wood China mucilaginous sheath 24–28 3 6–8, uniseriate, hyaline, fusiform, Aseptate Submerged Hong Kong, Tsui et al. (2002) lacking appendages or a sheath wood China 15–17.8 3 3.8–5, overlapping –uniseriate, Aseptate Dead petiole of Queensland, Fro¨hlich and fusiform–rhomboid, with a thin Licuala ramsayi Australia Hyde (2000) mucilaginous sheath (plam) 15–22.5 3 6.5–7.5, , overlapping – Two-septate Submerged The Philippines Cai et al. (2002) uniseriate, hyaline, fusiform, with a thin bamboo Islands mucilaginous sheath 32–52 3 7–10, uniseriate –overlapping 5–11-septate, Submerged Sohag, Egypt Abdel-Wahab uniseriate, hyaline, fusoid, with bipolar constricted stems of et al. (2011) mucilaginous pad–like appendage and at the septa Phragmites sheath australis 20–26 3 6–7, seriate, hyaline fusiform with Three-septate Submerged KwaZulu–Natal, Hyde et al. (1998) blunt ends, lacking a mucilaginous sheath wood South Africa 42.5–52.5 3 7.5–10, uniseriate, hyaline, 1–3-septate Submerged Hong Kong, Tsui et al. (2002) fusiform, lacking appendages or a sheath wood China 26–42 3 9–12, fusiform, with mucilaginous Aseptate Submerged Queensland, Hyde (1992) sheath wood Australia 18–33 3 6–12, uniseriate, hyaline, fusiform, Three-septate, Submerged Brunei Campbell and with a thin mucilaginous sheath constricted wood Darussalam Shearer (2004); at the septa Wong et al. (1999)

laboratory. Each set of test blocks were separated and cornmeal agar (CMA). Plates were incubated at 20 C in a placed in pre-sterilized plastic boxes with tissue papers cabinet with cool, white fluorescent light and examined layered at the base. They were incubated at 20 C in a daily for germinating ascospores. Six to eight germinated cabinet with cool white fluorescent light and examined at ascospores were transferred to new plates and incubated in intervals of 1 wk, 1, 2 and 3 mo for the presence of spor- the same cabinet. A culture was deposited in the BIOTEC ulating fungi. culture collection (BCC) as BCC16735 and dried material Assessment procedures were as described by Sivichai in the BIOTEC Bangkok Herbarium (BBH), as BBH et al. (2002). Single-ascospore isolations were made on 29936.

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DNA extraction, polymerase chain reactions (PCR), sequencing partitions as in the searches for the optimal trees using fast and phylogenetic analyses.—DNA was extracted from fresh bootstrapping 1000 pseudoreplicates (Stamatakis et al. mycelium as described by Sri-indrasutdhi et al. (2010) with 2006). extraction buffer. To amplify the partial 18S ribosomal DNA (18S rDNA) the sequencing primers are NS1 and NS4 RESULTS (White et al. 1990). To amplify the partial 28S ribosomal DNA (28S rDNA) the sequencing primers are LROR and Molecular phylogeny of combined 18S and 28S LR7 (Bunyard et al. 1994, Landvik 1996). PCR reactions and rDNA sequence.—The combined 18S rDNA and 28S amplification cycles were carried out according to Boo- nyuen et al. (2011). Purified PCR products were sent to rDNA dataset including sequences from the new Macrogen Inc. (Korea) with the above mentioned primers species (highlighted in FIG.1andTABLE II) was aligned for DNA sequencing. Newly generated sequences from with 84 representative taxa from the subclass Hypo- isolates were blasted in GenBank to exclude the possibility creomycetidae (Savoryellales, , Hypo- of contamination, assembled in Bioedit 7.5.03 (Hall 2006) creales and Hypocreomycetidae Incertae sedis), Sordar- to get consensus sequence and deposited in GenBank iomycetidae (, Chaetosphaeriales, (TABLE II). A phylogenetic tree was generated. Boliniales, Diaporthales, Ophiostomatales, Sordariales, The assembled sequences (18S rDNA and 28S rDNA) Sordariomycetidae incertae sedis), were aligned with related sequences obtained from Gen- and Xylariales (TABLE II). Two representative taxa of Bank (Campbell and Shearer 2004, Zhang et al. 2006, the order Pezizales (Morchella escuenta and Disciotis Abdel-Wahab et al. 2011, Boonyuen et al. 2011, Sakayaroj venosa)wereusedasoutgrouptaxa. et al. 2011) with Clustal W 1.6 (Thompson et al. 1994) incorporated in Bioedit 7.5.03 (Hall 2006) and improved in The final aligned dataset comprised 2317 characters, MUSCLE 3.6 (Edgar 2004) with ambiguously aligned out of which 762 were parsimony informative, 302 regions excluded from phylogenetic analyses. Missing data parsimony uninformative and 1253 constant characters. at the 59– and 39– ends of the partial sequences were The result of Kishino-Hasegawa maximum likelihood excluded from the analysis. Representatives of the order tests of the combined 18S rDNA and 28S rDNA from Pezizales were selected as an outgroup. unweight parsimony analysis yielded three most parsi- Maximum parsimony (MP) analyses were performed with monious trees with a tree length of 4030 (CI 5 0.405; RI PAUP 4.0b10 (Swofford 2002) with Windows and Home 5 0.687; RC 5 0.278; HI 5 0.595; 2ln likelihood 5 Mac OS 9.2 (Apple Studio, Mac OS X 10.4.10) as heuristic 23 519.13633; Difference ln L 5 the best tree and a searches with 1000 random addition replicates and tree significant difference at P , 0.05). One of the three bisection-reconnection (TBR) branch swapping to obtain the most parsimonious trees. Gaps were given equal weight and most parsimonious trees, inferred from the combined treated as missing data. To estimate the best tree topology two-locus dataset, was the best tree (FIG. 1). Three trees the Kishino-Hasegawa test was used (Kishino and Hasegawa in the interordinal relationship within the subclasses 1989). The final alignments (18S rDNA and 28S rDNA Hypocreomycetidae and Sordariomycetidae formed a dataset) and the trees obtained were deposited in TreeBASE stable branching pattern but with minor branch (http://www.treebase.org) as accession number 11739. swapping of Annulatascus biatriisporus and branch Clade stability for the branching topologies was evaluated length in Cyanoannulus petersenii (results not shown). by bootstrap analysis (BSMP) derived from 1000 replicates A comparison of the combined 18S rDNA and 28S with 10 random addition replicates each of a full heuristic rDNA dataset with the individual 28S rDNA dataset search (Felsenstein 1985). Descriptive tree statistics tree using Kishino-Hasegawa maximum likelihood tests length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) (tree not shown) showed the topology of the were calculated for each tree and phylogenetic trees were combined two-locus of 18S rDNA and 28S rDNA displayed with Treeview (Page 1996). dataset was almost identical to that of the individual Bayesian phylogenetic analysis was performed in MrBayes 28S rDNA dataset (TL 5 3166, CI 5 0.365; RI 5 3.0b4 with a uniform GRT + I + G model as the best 0.650; RC 5 0.237; HI 5 0.635, total character 5 nucleotide substitution model (Huelsenbeck and Ronquist 1149; constant character 5 447; parsimony informa- 2001). This model was chosen with MrModeltest 2.2 tive character 5 486 and parsimony uninformative (Nylander 2004). Chains were analyzed with random starting variable characters 5 216) (data not shown). trees for 2 000 000 generations and sampled every 100 Bayesian analysis provided a topology similar to generations. Trees collected before the stable likelihood the combined 18S rDNA and 28S rDNA analyses (data value points were discarded as burn-in. The Metropolis- not shown). Although minor differences in the coupled Markov chain Monte Carlo (MCMC) sampling approach after the exclusion of the initial set of 2000 burn- position of Papulosa amerospora, Ascobrunneispora in trees were used to calculate posterior probabilities (BYPP). aquatica and Mirannulata samuelsii were noted, this RAxML bootstrap values (BSML) were conducted in does not affect the overall topology of the tree in RAxML 7.0.3 with the same models in MrBayes 3.0b4 (GRT maximum parsimony analyses and the conclusions + I + G model of nucleotide substitution) and data drawn.

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TABLE II. DNA sequences in Sordariomycetes used in the phylogenetic analyses, their specimen voucher and GenBank accession numbers

GenBank accession numbers Classification Original codes SSU LSU

Annulatascaceae incertae sedis (Sordariomycetidae) - Annulatascus aquatorba* SS 2424 JN226106 JN226107 - Annulatascus nilensis* MF 808 N/A HQ616536 - Annulatascus hongkongensis* HKUCC 3702 N/A AF132319 - Annulatascus velatisporus A 70–18a N/A AY316354 - Annulatascus velatisporus R 047a N/A AY316355 - Annulatascus velatisporus* HKUCC3701 N/A AF132320 - Annulatascus biatriisporus A 464–3a N/A AY316352 - Pseudoproboscispora caudae–suis A 336–2Db N/A AY094192 - aquatica A 95–1Bb N/A AY094193 - Submersisphaeria aquatica A 354–14b N/A AY094194 - triseptatus A 325–1Db N/A AY590288 - Annulusmagnus triseptatus A 353–1Fb N/A AY590289 - Annulusmagnus triseptatus A 413–6b N/A AY590285 - Annulusmagnus triseptatus A 54–10Ab N/A AY590286 - Annulusmagnus triseptatus A 54-10Eb N/A AY590287 - Ascitendus austriacus* A 44–28Ab N/A AY590292 - Ascitendus austriacus* A 324–1Bb N/A AY590293 - Ascitendus austriacus* A 324–1Fb N/A AY590294 - Ascitendus austriacus CBS 102665 N/A AF261067 Sordariomycetidae Incertae sedis - hyalomura R 038b N/A AY590291 - Aquaticola ellipsoidea A 411–3a N/A AY316356 - Aquaticola hongkongenis* HKUCC 3703 N/A AF132321 - Cyanoannulus petersenii R 044aa N/A AY316358 - Fusoidispora aquatica* HKU(M) 17484c N/A AY780365 - Ascocollumdensa aquatica* HKUCC 3707 N/A AF132325 - Vertexicola caudatus* HKUCC 3715 N/A AF132331 - Cataractispora receptaculorum HKUCC 3710 N/A AF132327 - Rhamphoria delicatula MR 1396/98 N/A AF261068 Papulosaceae Incertae Sedis (Sordariomycetidae) - Papulosa amerospora AFTOL 748 DQ470998 DQ470950 - Ascobrunneispora aquatica HKUCC 3708 N/A AF132326 Order Ophiostomatales - Endomyces scopularum NRRL Y–17633d (CBS N/A AF268488 131.86) - Ophiostoma piliferum AFTOL 910 DQ471003 DQ470955 - Ophiostoma stenoceras AFTOL 1038 DQ836897 DQ836904 - Ophiostoma ulmi* ATCC 32437 M83261 DQ368627 Order Boliniales - Camarops microspora AFTOL 1361 DQ471036 AY083821 - Camarops ustulinoides AFTOL 72 DQ470989 DQ470941 - Cornipulvina ellipsoides* SMH 1378 N/A DQ231441 Order Chaetosphaeriales - Chaetosphaeria innumera SMH 2748 N/A AY017375 - Menispora tortuosa AFTOL 278 AY544723 AY544682 - Striatosphaeria codinaeophora SMH 1524 N/A AF466088 Order Coniochaetales - Barrina polyspora AWR 9560A N/A AY346261 - Coniochaeta ligniaria F 3331e N/A AF353583

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TABLE II. Continued

GenBank accession numbers

Classification Original codes SSU LSU - Lecythophora lignicola* CBS 267.33T AJ496246 AF353601 - Poroconiochaeta discoidea SANK 12878 N/A AY346297 Order Diaporthales - Apiognomonia errabunda AFTOL 2120 DQ862045 AF408334 - Diaporthe eres AFTOL 935 DQ471015 AF408350 - Gnomonia gnomon AFTOL 952 DQ471019 AF408361 - Melanconis marginalis AFTOL 2128 DQ862053 AF408373 - Melanconis stilbostoma AFTOL 2129 DQ862054 AF408374 - Valsa ambiens AFTOL 2131 DQ862056 AF362564 - Valsa nivea AFTOL 2125 DQ862050 AF362558 Order Sordariales - Gelasinospora tetrasperma AFTOL 1287 DQ471032 DQ470980 - Immersiella caudata CBS 606.72 DQ368659 AY999113 - Lasiosphaeria ovina AFTOL 17 DQ836894 AY436413 - Neurospora crassa* CBS 709.71 X04971 AF286411 - Sordaria fimicola Carolina Biological Supply AY545724 AY545728 Company 15–6291 - Sordaria macrospora ATCC 36709/Buck s.n. AY641007 AY346301 Order Savoryellales - Ascotaiwania lignicola NIL 00006 HQ446285 HQ446365 - Ascotaiwania mitriformis* HKUCC 3706 N/A AF132324 - Ascotaiwania sawadae SS 00051 HQ446283 HQ446363 - Ascothailandia grenadoidia* SS 03615 GQ390252 GQ390267 - Canalisporium caribense SS 03839 GQ390253 GQ390268 - Savoryella lignicola NTOU 791 HQ446299 HQ446377 Sordariomycetidae Incertae sedis - Conioscyphascus varius* CBS 113653 AY484511 AY484512 - Carpoligna pleurothecii AFTOL 281 AY544689 AY544685 Order Microascales - Kochiella crispa* BCC 33502 N/A HQ111020 - Morakotiella salina BCC 12781 N/A AY864844 - Nimbospora effusa JK 5104A U46877 U46892 - Nohea umiumi JK 5103F U46878 U46893 - Ocostaspora apilongissima LP 53 N/A HQ111005 - Remispora maritima BBH 28309 HQ111002 HQ111012 - Sablecola chinensis BCC 22809 N/A HQ111024 Order Hypocreales - Calonectria colombiensis* CBS 112221 N/A GQ280689 - Cordyceps militaris NRRL 28021 AF049146 AF327374 - Cylindrocladium floridanum ATCC 22677 N/A U17408 - Melanopsamma pomiformis* ATCC 18873 AY489677 AY489709 - Nectria cinnabarina GJS 89–107 U32412 U00748 - Sedecimiella taiwanensis* CY 5100 HM451495 HM451496 - Stachybotrys chartarum ATCC 66238 AY489680 AY489712 Sordariomycetes Incertae sedis - Mirannulata samuelsii* smh 1880 N/A AY578353 Order Trichosphaeriales - Trichosphaeria pilosa 089319bb N/A AY590297 Order Xylariales

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TABLE II. Continued

GenBank accession numbers

Classification Original codes SSU LSU

- Daldinia concentrica* ATCC 36659 U32402 U47828 - Xylaria acuta AFTOL 63 AY544719 AY544676 - Xylaria hypoxylon AFTOL 51 NG013136 NG027599 Order Pezizales - Disciotis venosa AFTOL 179 AY544711 AY544667 - Morchella esculenta AFTOL 60 AY544708 AY544664

Bold: new sequence generated for this study; Not applicable with the prefix N/A, information in a certainfield on a table is not provided; Asterisks: ex-holotype strains; AFTOL: Assembling the Fungal Tree of Life; ATCC: The American Type Culture Collection, Manassas, VA; BCC: BIOTEC Culture Collection, Thailand; BBH: BIOTEC Bangkok Herbarium, Thailand; CBS: Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; CY: City University of Hong Kong Culture Collection, Hong Kong; HKUCC: University of Hong Kong Culture Collection, Hong Kong; MF: Marine-Earth Science and Technology (JAMSTEC), Japan; NRRL: Northern Regional Research Center (NRRL), USDA, Peoria, IL, USA; Culture and specimen abbreviations: a H.A. Raja, J. Campbell and C.A. Shearer; b J. Campbell and C.A. Shearer; c D. Vijaykrishna, R. Jeewon, and K.D. Hyde; d S.-O. Suh and M, Blackwell; e E. Weber; AWR: A.W. Ramaley; GJS: G.J. Samuels; LP: J.Sakayaroj; JK: J. Kohlmeyer; MR: M. Reblovi; NIL; K-L Pang; SANK: J. C. Krug; SMH, smh: S.M. Huhndorf; SS: S. Sivichai.

A. aquatorba (FIG. 1) placed in the Annulatascaceae Paraphysis hyalinus, septa, filiformis, simplex clade with a BSMP of 66%,BSMLof59% and a BYPP versus rariflorus ramus, usque ad 2.5 mm latus. Asci of 0.98. It forms a sister clade to A. velatisporus, A. 102.5–152.5 3 6.25–8.75 mm(x 5 114 mm, n 5 30) hongkongensis and A. nilensis that are grouped in the cylindriculus, 8-spora, pedicellus, unitunicatata, dis- same clade but without significant support (BSML of tinctus, cuneatus, non–amyloideus annulusio apica- 55%). Other taxa in the Annulatascaceae clade includ- lis, 5.0–7.5 mm latus. Ascospora 15–22.5 3 5–7.5 mm ed the freshwater genera Annulusmagnus J. Campbell & (x 5 19.2 3 6.5 mm, n 5 30), uniseriata versus Shearer, Ascitendus J. Campbell & Shearer, Pseudopro- biseriata imbricate, in ascus, fusoidus, 3-septata, boscispora Punith and Submersisphaeria K.D. Hyde. aliquando leviter constrictus, strictus versus aliquan- Annulatascus biatriisporus grouped in a distal clade tum curvus, laevis, centrale cella brunnea, extremum with Fusoidispora aquatica and Ascocollumdensa aqua- cella rotundus cum subhyalinus. Ascospora destitutus tica (Sordariomycetidae Incertae sedis)withlow appendiculatus vel vagina. bootstrap support (BYPP 0.99) and showed no af- Holotype: BBH 29936. finities with other Annulatascus species (A. velatis- Etymology: aqua: water and torba: peat swamp, porus, A. aquatorba, A. hongkongensis and A. nilensis). belonging to the aquatic habitat of the fungus. Ascomata 390–500 mm high (x 5 445 mm, n 5 4), 310–350 mm(x 5 312 mm, n 5 4) diam, semi– TAXONOMY immersed or superficial, globose, brown to dark Annulatascus aquatorba Boonyuen & Sri-indrasutdhi brown, ostiolate, periphysate, solitary (FIG. 2). Neck sp. nov. FIGS. 2–15 400–480 3 100–120 mm(x 5 450 3 110 mm, n 5 8), MycoBank MB519473 cylindrical, bending perpendicularly or becoming Anamorph. Not observed. erect. Peridium thick-walled, brown, pseudoparenchy- Ascomata 390–500 mm alta (x 5 445 mm, n 5 4), matous cells in the outer layers and inner layer 310–350 mm(x 5 312 mm, n 5 4) diam, semi-immersa compressed (FIG. 3). Hamathecium: Paraphyses hya- vel superficialia, globosa, brunea usque atra, ostiolata, line, septate, filiform, simple to rarely branched, up to periphysis, solitaria. Collo 400–480 3 100–120 mm(x 2.5 mm wide (FIG. 4). Asci 102.5–152.5 3 6.25–8.75 mm 5 450 3 110 mm, n 5 8), cylindriculus, flexus per- (x 5 114 mm, n 5 30) cylindrical, eight-spored, pendicular versus erectusescens. Peridium crassus pedicellate, unitunicate with a distinct, wedge-shaped, paries, brunea, pseudoparenchymatous cella in exte- nonamyloid apical ring, 5.0–7.5 mm wide (FIGS. 5–10). rior stratum et compressus. Hamathecium: Ascospores 15–22.5 3 5–7.5 mm(x 5 19.2 3 6.5 mm, n

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FIG. 1. Phylogenetic analyses of Annulatascus aquatorba and related species based on combined partial 18S and 28S rDNA sequences. Bootstrap values higher than 50% from maximum parsimony analysis (BSMP) (top left) and from RAxML (BSML) (top right) are given above the nodes respectively. Bayesian posterior probabilities greater than 0.95 are indicated below the nodes (BYPP). The internodes that are highly supported by all bootstrap proportions (100%) and posterior probabilities (1.00) are shown as a thicker line. Asterisks are indicated as ex-holotype.

5 30), uniseriate to overlapping biseriate in the ascus, apices, subhyaline (FIGS. 11–15). Ascospores lack fusoid to lunate, straight to somewhat curved, three- appendages or a sheath. septate, slightly constricted at the septa, smooth- Culture characteristics: Colonies diffuse, no aerial walled, central cells brown, end cells with rounded mycelium, hyaline, becoming pale brown to dark

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FIGS. 2–15. Annulatascus aquatorba. (Holotype SS2424.01). Light microscope micrographs. 2. Ascoma with long neck on the test blocks (arrowed). 3. Section of ascomata with thick peridial wall. 4. Asci and hyaline paraphyses. 5. Apical ring (arrowed). 6–10. Asci at various stages of development with ascospores uniseriate to overlapping biseriate. 11–15. Ascospores three-septate, fusoid to lunate, end cells subhyaline. Bars: 2 5 1000 mm, 3 5 150 mm, 4 5 30 mm, 8–15 5 15 mm, 5–7 5 10 mm.

Mycologia myco-104-03-13.3d 25/4/12 17:04:38 9 Cust # 11-238R1 0MYCOLOGIA brown. Cultures grew moderately well, reaching 16 mm and Submersisphaeria aquatica K.D. Hyde (Campbell in 24 d at 25–28 C in normal daylight conditions. et al. 2003, Campbell and Shearer 2004). However Annulatascus aquatorba was isolated on CMA, with Submersisphaeria aquatica differs from A. aquatorba in ascospores germinating within 24–48 h on CMA. having 1–2-septate ascospores with cap-like append- Appressorial-like pegs developed from each terminal ages while Ascitendus austriacus differs from A. cell and usually in synchrony (BCC16735). aquatorba in having ascospores not constricted at Specimen examined: Thailand, Narathiwat Province, Sir- the septa and a roughened spore wall. indhorn Peat Swamp Forest on submerged wood test block Taxa grouping in the Annulatascaceae include of Erythrophleum teysmannii, 22 Feb 2003, collected by N. Annulatascus, Annulusmagnus, Ascitendus, Pseudopro- Boonyuen and V. Sri-indrasutdhi (BBH 29936). boscispora and Submersisphaeria, all freshwater ascomy- Habitat: On submerged test blocks of Erythrophleum cetes growing on submerged wood. Within the teysmannii, in a peat swamp forest. Annulatascaceae clade three subclades are discerned Mode of life: Saprobic. (FIG. 1). Subclade A includes the type species A. Distribution: Thailand. velatisporus, which forms a sister group to A. hongkon- gensis,withA. nilensis and A. aquatorba as basal taxa DISCUSSION with low bootstrap support. Subclade B includes various strains of Annulusmagnus triseptatus and Based on morphological features (FIGS. 2–15), A. Ascitendus austriacus. Two strains of Submersisphaeria aquatorba is referred to the genus Annulatascus aquatica group in subclade C with high bootstrap because it is characterized by dark, immersed to support. Pseudoproboscispora forms a basal subclade superficial ascomata with tapering paraphyses. Asci with moderate support (FIG.1). Annulatascus aqua- are cylindrical with a relatively large refractive apical torba therefore can be distinguished from other taxa in nonamyloid apical ring with a plug to the ascus and the Annulatascaceae based on morphological and versicolorous, thick-walled ascospores, slightly con- molecular evidence. stricted at the septa (Hyde 1992, Tsui et al. 2002). Annulatascus species have been reported from Campbell and Shearer (2004) opined that taxa various substrata and geographical locations, with assigned to Annulatascus were not monophyletic as most growing on submerged wood in freshwater currently circumscribed based on molecular data, and streams. Annulatascus aquatorba was collected on a this is confirmed by Abdel-Wahab et al. (2011) and submerged test block of Erythrophleum teysmannii in this study. Annulatascus species differ in morphology, a peat swamp, while the remaining species (n 5 13) dimensions of the ascomata, asci and ascospores and are reported from unknown submerged timbers, the substrata they naturally colonize. The most dead palm petioles and stems of Phragmites pronounced differences are in size of the ascus and australis. apical ring, ascospore septation and the presence of a Annulatascus aquatorba occurred only once on the mucilaginous sheath (Tsui et al. 2002). Eight test block that had been exposed 2 y and was not Annulatascus species (TABLE I) have ascospores that found on the other eight timber species submerged at are aseptate (A. aquaticus, A. biatriisporus, A. bipolaris, the peat swamp (on Azadirachta indica var. siamensis, A. citriosporus, A. joannae, A. lacteus, A. licualae and A. Melaleuca cajuputi, Shorea obtusa, S. roxburghii, S. velatispora), while the remainder are (1–)-3 septate (A. siamensis, Wrightia tomentosa, Xylia xylocarpa and aquatorba, A. apiculatus, A. hongkongensis, A. palmie- Zollingeria dongnaiensis). In addition it has not been tensis, A. triseptatus and A. tropicalis), 1–5-septate (A. collected on natural submerged wood and test blocks fusiformis), 5–11-septate (A. nilensis) and two-septate at other test sites in Thailand (e.g. Doi Inthanon (A. liputii). National Park, Khao Yai National Park, Kaeng Annulatascus apiculatus, A. hongkongensis and An- Krachang National Park, Mo Ko Chang National nulusmagnus triseptatus have ascospores that are larger Park, Tai Rom Yen National Park, Ton Nga Chang than A. aquatorba and have a mucilaginous sheath Wildlife Sanctuary and Hala Bala Wildlife Sanctuary) (TABLE I). Annulatascus fusiformis has ascospores of a in the past 8 y, indicating it might be a rare species similar size to A. aquatorba but differs in having bipolar and adapted to the acidic waters of the peat swamp mucilaginous pad–like appendages (TABLE I). forest. Annulatascus aquatorba differs from A. palmietensis and A. tropicalis because A. palmietensis has larger ascomata and ascospores (TABLE I), while A. tropicalis ACKNOWLEDGMENTS has larger asci and larger ascospores (TABLE I). This study was supported by the TRF/BIOTEC special Annulatascus aquatorba resembles two other fresh- Program for Biodiversity Research and Training grants BRT water ascomycetes: Ascitendus J. Campbell & Shearer R_145006, BRT R_252057 and R_251009. Prof Morakot

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Tanticheroen, Drs Kanyawim Kirtikara and Lily Eurwilai- Ho WH, Hyde KD, Hodgkiss IJ. 1999a. Ultrastructure of chitr at BIOTEC are thanked for their continued support of Annulatascus aquaticus sp. nov., a freshwater ascomy- the freshwater work in Thailand. Our thanks to Sunthorn cete on submerged wood from Hong Kong. Fungal Taodam for support with the field work at Sirindhorn Peat Divers 2:119–128. Swamp, Jureerat Ueapattanakit for DNA extraction and ———, Ranghoo VM, Hyde KD, Hodgkiss IJ. 1999b. Ascal PCR of Annulatascus aquatorba, Juntima Chanprasert for ultrastructural study on Annulatascus hongkongensis sp. running the RAxML analysis, Prof Mohamed A. Abdel- nov., a freshwater ascomycete. Mycologia 91:885–892, Wahab for the sequence of A. nilensis and Dr Clement K.M. doi:10.2307/3761542 Tsui for invaluable comments on Annulatascus taxonomy. Huelsenbeck JP, Ronquist F. 2001. MrBayes: Bayesian We are also grateful for the help of two anonymous inference of phylogeny. 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