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Mycologia, 103(6), 2011, pp. 1351–1371. DOI: 10.3852/11-102 # 2011 by The Mycological Society of America, Lawrence, KS 66044-8897

Savoryellales (, ): a novel lineage of aquatic ascomycetes inferred from multiple- phylogenies of the genera , Ascothailandia, and Savoryella

Nattawut Boonyuen1 Canalisporium) formed a new lineage that has Laboratory (BMYC), Bioresources Technology invaded both marine and freshwater habitats, indi- Unit (BTU), National Center for cating that these genera share a common ancestor and Biotechnology (BIOTEC), 113 Thailand Science and are closely related. Because they show no clear Park, Phaholyothin Road, Khlong 1, Khlong Luang, Pathumthani 12120, Thailand, and Department of relationship with any named we erect a new Pathology, Faculty of Agriculture, Kasetsart order Savoryellales in the subclass Hypocreomyceti- University, 50 Phaholyothin Road, Chatuchak, dae, Sordariomycetes. The genera Savoryella and Bangkok 10900, Thailand Ascothailandia are monophyletic, while the position Charuwan Chuaseeharonnachai of Ascotaiwania is unresolved. All three genera are Satinee Suetrong phylogenetically related and form a distinct clade Veera Sri-indrasutdhi similar to the unclassified group of marine ascomy- Somsak Sivichai cetes comprising the genera Swampomyces, Torpedos- E.B. Gareth Jones pora and Juncigera (TBM clade: Torpedospora// Mycology Laboratory (BMYC), Bioresources Technology ) in the Hypocreomycetidae incertae Unit (BTU), National Center for Genetic Engineering sedis. and Biotechnology (BIOTEC), 113 Thailand Science Key words: , fungal Park, Phaholyothin Road, Khlong 1, Khlong Luang, systematics, Hypocreomycetidae, unitunicate ascomy- Pathumthani 12120, Thailand cetes Ka-Lai Pang Institute of Marine , National Taiwan Ocean INTRODUCTION University, Pei-Ning Road, Keelung 20224, Taiwan (ROC) Savoryella is one of the most commonly reported unitunicate ascomycetous genera from submerged wood in rivers and streams (Sivichai et al. 2002, Abstract: The taxonomic placement of freshwater Sivichai and Jones 2003) and the marine environment and marine Savoryella has been widely (Jones and Hyde 1992), while S. appendiculata and S. debated, and the has been tentatively assigned melanospora have been recovered from wood in to various orders in the Sordariomycetes. The genus is contact with sand (Jones and Hyde 1992, Abdel- characterized as possessing paraphyses that deli- Wahab and Jones 2000). The phylogenetic position of quesce early, elongate, clavate to cylindrical asci with the genus is unresolved, and it has been referred to a a poorly developed apical ring and versicolored, number of orders and families in the Sordariomy- three-septate . We performed two com- cetes, (Vijaykrishna et al. 2006). bined phylogenetic analyses of different : (i) Eleven species are recognized in Savoryella: S. partial small subunit rRNA (SSU), large subunit rRNA appendiculata, S. aquatica, S. curvispora, S. fusiformis, (LSU), DNA-dependent RNA polymerase II largest S. grandispora, S. lignicola, S. limnetica, S. longispora, subunit (rpb2) dataset and (ii) SSU rDNA, LSU rDNA, S. melanospora, S. paucispora and S. verrucosa.Of DNA-dependent RNA polymerase II largest subunit these species five are found in marine and six in (rpb1 and rpb2), translation elongation factor 1-alpha freshwater habitats (Cai et al. 2006). (tef1), the 5.8S ribosomal DNA (5.8S rDNA) dataset. Jones and Eaton (1969) established the genus Our results indicate that Savoryella species formed a Savoryella with S. lignicola as the type species, which monophyletic group within the Sordariomycetes but was isolated from wooden slats in a water-cooling showed no affinity to the , Halosphaer- tower run on brackish water. Savoryella species are iales (now ), and , characterized by dark brown to black ascomata, despite earlier assignments to these orders. Savoryella, clavate to cylindrical asci with a comparatively Ascotaiwania and Ascothailandia (and its anamorph, flattened apical ring and versicolorous septate asco- , brown central cells and hyaline end cells. No Submitted 4 Jan 2011; accepted for publication 4 Apr 2011. anamorph has been reported for Savoryella (Tsui and 1 Corresponding author. E-mail: [email protected] Hyde 2003). The genus has been referred to the

1351 1352 MYCOLOGIA

Sphaeriales incertae sedis (Kohlmeyer and Kohl- teristics suggested an affinity to Ascothailandia with meyer 1979), ascomycetes incertae sedis (Kohlmeyer the Hypocreales (subclass Hypocreomycetidae, Sor- 1986, Eriksson and Hawksworth 1986), Amphisphaer- dariomycetes), and it was referred to the Ascomycota iaceae (Eriksson and Hawksworth 1987) and Sordar- incertae sedis (Sri-indrasutdhi et al. 2010). iales (Jones and Hyde 1992). Barr (1990) and Read et To better understand the relationships and ordinal al. (1993) thought that Savoryella was best referred to placements of Savoryella, Ascotaiwania and Ascothai- the Halosphaeriales (now Microascales) based respec- landia (and the anamorphic genus Canalisporium)we tively on morphological features (catenophyses-like conducted a phylogenetic study with multiple genes. paraphyses) and ultrastructural observations. Based The objectives of this study were (i) to determine the on large subunit (LSU) rDNA data, Vijaykrishna et al. taxonomic placement of Savoryella with a multiple- (2006) and Cai et al. (2006) referred S. elongata and gene approach, (ii) to elucidate the phylogeny of S. longispora to the order Hypocreales within the Savoryella and morphologically similar genera includ- subclass Hypocreomycetidae, but with weak statistical ing Ascotaiwania and Ascothailandia and (iii) to use support. molecular data to examine the higher order position The type species of Ascotaiwania, A. lignicola, was of the above freshwater and marine genera. described by Sivanesan and Chang (1992) from driftwood collected in a freshwater stream at Wulae, MATERIALS AND METHODS Taiwan. Currently 12 species have been described from senescent palm material, submerged wood, Specimen collection.—Fungi were isolated from substrata leaves and dead wood collected from freshwater and collected from freshwater and marine locations in Thailand terrestrial habitats. Seven Ascotaiwania species have and Taiwan (Pinruan et al. 2002, Sivichai and Boonyene been reported from submerged wood (A. hsilio, 2004, Sakayaroj et al. 2004). Isolates were maintained on TM Chang et al. 1998; A. hughesii, Fallah et al. 1999; A. cornmeal agar (CMA, from Criterion , Santa Maria, California), seawater cornmeal agar (SCMA), potato dex- mitriformis, Ranghoo and Hyde 1998; A. pallida, Hyde trose agar (PDA, from DifcoTM, Becton Dickinson, Sparks, and Goh 1999; A. persoonii, Fallah et al. 1999; A. Maryland) and seawater potato dextrose agar (SPDA) media sawada, Chang et al. 1998; A. wulai, Chang et al. and prepared with either seawater or freshwater. 1998) while A. licualae (Fro¨hlich and Hyde 2000) and A. (Hyde 1995) were described from palms, Fungal isolates, identification and growth.—Fungal strains A. pennisetorum (Wong and Hyde 2001) from were identified based on their and sporulation submerged grasses and A. (Dulymamode on naturally occurring substrata. Sporulating material was mounted in lactophenol-cotton blue or freshwater or et al. 2001) from dead leaves of Pandanus palustris. seawater, depending on their natural habitat, and observed Ascotaiwania (Sivanesan and Chang 1992, Ho et al. under Olympus compound and stereo microscopes (CH40, 1997, Chang et al. 1998) morphologically resembles CX31, CH-2). Single- isolates were made and grown on Savoryella in having versicolorous ascospores and long CMA,SCMA,PDAandSPDAmedia.Cultureswere pale ascomatal necks. However Ascotaiwania differs in deposited and maintained in the BIOTEC Culture Collec- having cylindrical asci with a relatively massive, non- tion (BCC), and dried material deposited in the BIOTEC amyloid apical ring and ascospores that are three- to Bangkok Herbarium (BBH). Taxa used in this study are seven-septate. Additionally Ascotaiwania anamorphs listed (TABLE I). All cultures were grown on appropriate have been reported belonging to Monotosporella (A. media at 25 C for 4–16 weeks, depending on the growth rate sawadae, A. mitriformis) and Helicoon farinosum (A. of each species. hughesii) (Sivichai et al. 1998, Cai et al. 2006). Genomic extraction and PCR amplification.—Actively grow- Molecular studies have failed to resolve the taxonom- ing was scraped from the surface of a culture and ic position of Ascotaiwania (Ranghoo et al. 1999, transferred to microcentrifuge tubes. The biomass was Campbell and Shearer 2004), while Cai et al. (2006) lyophilized at 280 C for 2 d before DNA extraction, which referred it to the Sordariales incertae sedis. followed under conditions modified from Tigano-Milani et In our ongoing research on Thai freshwater fungi al. (1995) and described by Sri-indrasutdhi et al. (2010). (Pang et al. 2002; Sivichai et al. 2002; Pinruan et al. The sequencing primers used for different regions are 2002, 2004a, b; Sivichai and Jones 2003; Pinnoi et al. NS1, NS3, NS4, NS5 and NS6 (White et al. 1990) for the 2003) we have recovered several Canalisporium partial SSU ribosomal DNA; JS1, JS8, LROR, LR5 and LR7 (Bunyard et al. 1994, Landvik 1996 ) for the partial LSU species from submerged or trapped wood (Sivichai ribosomal DNA; RPB2-5F1, RPB2-5F2, RPB2-7CR and RPB2- and Boonyene 2004). Recently a new ascomycete 7R (Liu et al. 1999) for rpb2 region; RPB1-CRPB1 and RPB1- genus, Ascothailandia, was described that morpholog- CR (Castlebury et al. 2004) for rpb1 region; EF1-983F, EF1- ically resembles Savoryella and Ascotaiwania and has a 2218R, EF1-CEFF2 and EF1-CEFR2 (Rehner 2001) for tef1 Canalisporium anamorph (Sri-indrasutdhi et al. region; ITS1, ITS4 and ITS5 (White et al. 1990) for 5.8S 2010). Phylogenetic data and morphological charac- ribosomal DNA. PCR reactions were carried out in a total TABLE I. Sequences used in this study representing the Savoryellales clade with taxa, culture numbers, GenBank sequence numbers, substrate origins and collecting sites

GenBank accession numbers Culture no SSU LSU rpb2 rpb1 tef1 5.8S rDNA Substrate origin Colleting site Ascotaiwania SS00051 HQ446283a HQ446363a HQ446418a HQ446383a HQ446306a HQ446340a Submerged Road marker at km 29.2, Khao sawadae hardwood Yai National Park, Nakhon Ratchasima, Thailand Ascotaiwania HKUCC3705 N/A AF132323b N/A N/A N/A AF177148b N/A N/A sawadae Ascotaiwania P2-6 N/A AY316357b N/A N/A N/A N/A Submerged N/A hughesii wood Ascotaiwania HKUCC3706 N/A AF132324b N/A N/A N/A N/A N/A Hong Kong mitriformis Ascotaiwania NIL00005 HQ446284a HQ446364a HQ446419a N/A HQ446307a HQ446341a Submerged Wulai, Taiwan

lignicola natural wood B Ascotaiwania NIL00006 HQ446285a HQ446365a N/A N/A HQ446308a HQ446342a Submerged Wulai, Taiwan AL ET OONYUEN lignicola natural wood Ascothailandia SS03615 GQ390252c GQ390267c HQ446420a HQ446384a HQ446309a GQ390282c Wrightia Khlong Ai-kading, Hala Bala grenadoidia tomentosa Wildlife Sanctuary, Narathiwat, Thailand Canalisporium SS03839 GQ390253c GQ390268c HQ446421a HQ446385a N/A GQ390283c Submerged Khlong Ai-kading, Hala Bala :S .: caribense natural wood Wildlife Sanctuary, Narathiwat, Thailand AVORYELLALES Canalisporium SS03683 GQ390254c GQ390269c HQ446422a HQ446386a N/A GQ390284c Submerged Wang Kan Lueang Waterfall, caribense natural wood Wang Kan Lueang Arboretum, Lop Buri, Thailand Canalisporium SS00523 GQ390255c GQ390270c HQ446423a HQ446387a HQ446310a GQ390285c Xylia Road marker at km 29.2, Khao elegans dolabriformis Yai National Park, Nakhon Ratchasima, Thailand Canalisporium SS00877 HQ446286a HQ446366a HQ446424a HQ446388a HQ446311a HQ446343a Submerged Kaeng Krachan National Park, elegans natural wood Prachuap Khiri Khan, Thailand Canalisporium SS00895 GQ390256c GQ390271c HQ446425a HQ446389a HQ446312a GQ390286c Stereospermum Road marker at km 29.2, Khao elegans neuranthum Yai National Park, Nakhon Ratchasima, Thailand Canalisporium SS03491 GQ390257c GQ390272c HQ446426a HQ446390a HQ446313a GQ390287c Submerged Kaeng Krachan National Park, jinghongensis natural wood Prachuap Khiri Khan, Thailand Canalisporium SS03483 GQ390258c GQ390273c HQ446427a HQ446391a HQ446314a GQ390288c Submerged Bo Khlueng Hot-spring, jinghongensis natural wood Ratchaburi, Thailand Canalisporium SS03819 GQ390259c GQ390274c HQ446428a HQ446392a HQ446315a GQ390289c Submerged Khao Pra - Bang Khram Wildlife pulchrum natural wood Sanctuary, Krabi, Thailand Canalisporium SS03823 GQ390260c GQ390275c HQ446429a HQ446393a HQ446316a GQ390290c Submerged Khao Pra - Bang Khram Wildlife

pulchrum natural wood Sanctuary, Krabi, Thailand 1353 34M 1354 TABLE I. Continued

GenBank accession numbers

Taxon Culture no SSU LSU rpb2 rpb1 tef1 5.8S rDNA Substrate origin Colleting site Canalisporium SS00170 GQ390261c GQ390276c HQ446430a HQ446394a HQ446317a GQ390291c Alstonia Road marker at km 29.2, Khao pulchrum scholaris Yai National Park, Nakhon Ratchasima, Thailand Canalisporium SS03982 GQ390262c GQ390277c HQ446431a HQ446395a HQ446318a GQ390292c Submerged Heo Narok Waterfall, Khao Yai pulchrum natural wood National Park, Nakhon Nayok, Thailand Canalisporium SS03773 GQ390263c GQ390278c HQ446432a HQ446396a HQ446319a GQ390293c Submerged Khlong Ai-kading, Hala Bala pulchrum natural leaf Wildlife Sanctuary, Narathiwat, Thailand Canalisporium SS03788 HQ446287a HQ446367a HQ446433a HQ446397a HQ446320a HQ446344a Submerged Than Bok Khorani National Park, pulchrum natural wood Krabi, Thailand Canalisporium SS00091 GQ390264c GQ390279c HQ446434a HQ446398a HQ446321a GQ390294c Alstonia Road marker at km 29.2, Khao pallidum scholaris Yai National Park, Nakhon Ratchasima, Thailand Canalisporium SS00498 GQ390265c GQ390280c HQ446435a HQ446399a HQ446322a GQ390295c Xylia Road marker at km 29.2, Khao

pallidum dolabriformis Yai National Park, Nakhon YCOLOGIA Ratchasima, Thailand Canalisporium SS00809 GQ390266c GQ390281c HQ446436a HQ446400a N/A GQ390296c Submerged Khao Soi Dao Wildlife Sanctuary, exiguum natural wood Chanthaburi, Thailand Canalisporium SS03732 HQ446288a HQ446368a HQ446437a HQ446401a N/A HQ446345a Submerged Kaeng Krachan National Park, sp. natural wood Prachuap Khiri Khan, Thailand Monotosporella HKUCC3713 N/A AF132334b N/A N/A N/A N/A N/A N/A setosa Savoryella SS00096 HQ446289a HQ446369a HQ446438a HQ446402a HQ446323a HQ446346a Anisoptera Road marker at km 29.2, Khao aquatica oblonga Yai National Park, Nakhon Ratchasima, Thailand Savoryella SS00359 HQ446290a HQ446370a HQ446439a HQ446403a HQ446324a HQ446347a Alstonia Tad Tha Phu, Khao Yai National aquatica scholaris Park, Nakhon Ratchasima, Thailand Savoryella SS00583 HQ446291a HQ446371a HQ446440a HQ446404a HQ446325a HQ446348a Xylia Tad Tha Phu, Khao Yai National aquatica dolabriformis Park, Nakhon Ratchasima, Thailand Savoryella SS03801 HQ446292a HQ446372a HQ446441a HQ446405a HQ446326a HQ446349a Submerged Khao Pra - Bang Khram Wildlife aquatica wood Sanctuary, Krabi, Thailand Savoryella NF00206 HQ446293a N/A HQ446442a HQ446406a HQ446327a HQ446350a Submerged Hat Mae Ramphueng, Khao appendiculata Nypa fruticans Laem Ya - Mu Ko Samet National Park Rayong, Thailand TABLE I. Continued

GenBank accession numbers Taxon Culture no SSU LSU rpb2 rpb1 tef1 5.8S rDNA Substrate origin Colleting site Savoryella SS00783 HQ446294a N/A HQ446443a HQ446407a HQ446328a HQ446351a Submerged Chet Kot Waterfall, Khao Yai fusiformis natural wood National Park, Nakhon Ratchasima, Thailand Savoryella SS00042 HQ446295a HQ446373a HQ446444a HQ446408a HQ446329a HQ446352a Submerged Khao Yai National Park, Nakhon verrucosa Elephant grass Ratchasima, Thailand Savoryella SS00052 HQ446296 HQ446374a HQ446445a HQ446409a HQ446330a HQ446353a Submerged twig Khao Yai National Park, Nakhon verrucosa Ratchasima, Thailand a a a a a

Savoryella SS00582 HQ446297 HQ446375 HQ446446 HQ446410 HQ446331 HQ446354 Xylia Tad Tha Phu, Khao Yai National B verrucosa dolabriformis Park, Nakhon Ratchasima, AL ET OONYUEN Thailand Savoryella SS03331 HQ446298a HQ446376a HQ446447a HQ446411a HQ446332a HQ446355a Stereospermum Tad Tha Phu, Khao Yai National verrucosa neuranthum Park, Nakhon Ratchasima, Thailand Savoryella NTOU791 HQ446299a HQ446377a HQ446448a HQ446412a HQ446333a HQ446356a Unidentified Morib, Malaysia lignicola mangrove S .:

wood AVORYELLALES Savoryella NF00204 HQ446300a HQ446378a N/A HQ446413a HQ446334a HQ446357a Submerged Nakluea , Phra Samut Chedi, lignicola Nypa fruticans Samut Prakan, Thailand Savoryella SAT00320 HQ446301a HQ446379a HQ446449a HQ446414a HQ446335a HQ446358a Mangrove wood Tammarang Pier, Satun, longispora Thailand Savoryella SAT00322 HQ446302a HQ446380a HQ446450a HQ446415a HQ446336a HQ446359a Mangrove wood Tammarang Pier, Satun, longispora Thailand Savoryella SAT00866 HQ446303a HQ446381a HQ446451a HQ446416a HQ446337a HQ446360a Mangrove wood Laem TaLum Phuk, paucispora Nakhonsithammarat, Thailand Savoryella SAT00867 HQ446304a HQ446382a HQ446452a HQ446417a HQ446338a HQ446361a Mangrove wood Laem TaLum Phuk, paucispora Nakhonsithammarat, Thailand Savoryella sp. NF00205 HQ446305a N/A N/A N/A HQ446339a HQ446362a Submerged Ban Ko Sanoe, Surat Thani, Nypa fruticans Thailand

a This paper; b Ranghoo et al. (1999), Campbell and Shearer (2004). c Sri-indrasutdhi et al. (2010). Isolates with the prefix SS, NF and SAT, NIL are from the BIOTEC Culture Collection (BCC); Isolates with the prefix NTOU is from National Taiwan Ocean University; Not applicable with the prefix N/A, information not provided. 1355 1356 MYCOLOGIA volume of 50 mL containing 1 mL 10–50 ng DNA template, Swofford 2002). A partition homogeneity test indicated 5 mL103 PCR buffer, 5 mL 25 mM MgCl2,1mL10mM the two datasets, P value . 0.05, could be combined and dNTPs, 1 mL10mM each primer, 5 mL 4 M enhancer, 30.8 mL showed that none of the individual datasets resulted in sterile distilled water and 0.2 mL 0.2U of Taq Polymerase significantly incongruent trees (data not shown). In DNA Polymerase Kit, from FERMENTAS (Burlington, addition an independent analyses were run for each of Canada). the sequenced gene regions to check for possible conflicts Amplification cycles were performed with MJ Research in the topology by comparison of the bootstrap supports of DNA Engine ALD1244 thermal cycler according to the the nodes of individual trees (data not shown). procedure of Sri-indrasutdhi et al. (2010) and Tang et al. Bootstrapping analyses (Felsenstein 1985) were per- (2007). PCR products were purified with the NucleoSpinH formed with 1000 replicates of a full heuristic search (each Extract Kit (Macherey-Nagel, Germany) following the consisting of 10 replicates of the random-swapping algo- manufacturer’s instructions. The quantity and quality of rithm). The tree length (TL), consistency index (CI), each purified product was verified by electrophoresis on a rescaled consistency index (RC), retention index (RI) and 1% agarose gel. Finally purified PCR products were used homoplasy index (HI) were calculated for each tree. directly for DNA sequencing. Representative sequences for each order within the Sordariomycetes were retrieved from GenBank (TABLE II) DNA sequencing.—PCR products were sequenced by Macro- and added to the alignment. gen Inc. (Korea) in an Applied Biosystem 373XL DNA Bayesian phylogenetic inference was calculated with Analyzer with the same forward and reverse primers used MrBayes 3.0b4 with a general time reversible (GTR) model for amplification (White et al. 1990, Bunyard et al. 1994, of DNA substitution as the best fit and a gamma distribution Landvik 1996, Liu et al. 1999, Rehner 2001, Castlebury et al. rate variation across sites (Huelsenbeck and Ronquist 2004). Each sequence was checked for ambiguous bases and 2001). This model was chosen as the result from a pretest assembled with BioEdit 7.5.03 (Hall 2006). All sequences with MrModeltest 2.2 (Nylander 2004). After this was were deposited in GenBank and the accession numbers are determined GTR + I + G model as the best nucleotide listed (TABLE I). substitution model were used for the combined three-gene Sequence alignment and phylogenetic analyses.—The SSU dataset and the combined six-gene dataset. Four Markov rDNA, LSU rDNA, rpb2, rpb1, tef1 and 5.8S rDNA sequences chains were run from random starting trees for 2 000 000 were aligned with related sequences obtained from Gen- generations and sampled every 100 generations. The first Bank (Campbell and Shearer 2004, Zhang et al. 2006, 2000 generations were discarded as burn-in. A majority rule Hibbett et al. 2007, Schoch et al. 2007, Spatafora et al. 2006, consensus tree of the remaining 18 000 trees as well as the Tang et al. 2007, Sri-indrasutdhi et al. 2010) with Clustal W posterior probabilities (BYPP) was calculated with the 1.6 (Thompson et al. 1994). Alignments were further Metropolis-coupled Markov chain Monte Carlo (MCMC) adjusted manually with BioEdit 7.5.0.3 (Hall 2006) and sampling approach after the exclusion of the initial set of improved in MUSCLE 3.6 (Edgar 2004) with ambiguously 2000 burn-in trees. Parsimony bootstrap values (BSMP) aligned regions excluded from phylogenetic analyses. greater than 50%, and Bayesian posterior probabilities Missing data at the 59– and 39 –ends of the partial sequences (BYPP) greater than 0.95 are given above and below each were excluded from the analysis. Representatives of the clade respectively. order were chosen as the outgroup taxa for all In addition in the combined six locus dataset of the analyses. partial SSU rDNA + LSU rDNA + rpb2 + rpb1 + tef1 + 5.8S Aligned datasets were analyzed with maximum parsimony sequences, maximum likelihood (ML) analysis and their (MP) in PAUP 4.0b10 (Swofford 2002) on Windows and bootstrapping values were performed with RAxML 7.0.3 Home Mac OS 9.2 ( Studio, Mac OS X 10.4.10). (Stamatakis et al. 2006). A general time reversible (GTR) Phylogenetic trees were displayed with Treeview (Page with a discrete gamma distribution and four rate classes was 1996). Phylogenetic analyses of two datasets were performed applied to each partition. A tree was obtained by simulta- with maximum parsimony (MP), maximum likelihood neously running with fast bootstrapping using 1000 (ML) and Bayesian algorithms. pseudoreplicates, followed by searching the most likely tree To obtain the most parsimonious trees (MPTs) we and bootstrapping (BSML). The program also was used with performed heuristic searches with tree bisection reconnec- 100 successive searches in RAxML under the GTR model of tion (TBR) branch swapping and 1000 replicates of random nucleotide substitution with gamma rate distribution and stepwise sequence addition. Gaps were treated as missing starting on each searching from a randomized tree. RAxML data and were given equal weight. The Kishino-Hasegawa bootstrap values (BSML) greater than 50% are shown to the (K-H) test was used to estimate the best tree topology top left of each branch. (Kishino and Hasegawa 1989) and the alignments in Two datasets were analyzed as follows: (i) an alignment of TreeBASE (www.treebase.org). Sequence alignments and the SSU + LSU + rpb2 sequences for the major orders of the trees of the SSU + LSU + rpb2 and the SSU + LSU + rpb2 + Sordariomycetes, including representative taxa within rpb1 + tef1 + 5.8SrDNA were deposited in TreeBASE with Savoryella, Ascotaiwania and Canalisporium; and (ii) an accession number 11113. The combinability of the three alignment of the SSU + LSU + rpb2 + rpb1 + tef1 + 5.8S loci and six loci datasets was tested with the partition rDNA sequences for an expanded set of taxa for homogeneity test implemented with PAUP using 1000 Ascotaiwania for which only nrLSU sequence was available random repartitions for each test (Farris et al. 1995, and used to determine the closet relative of Savoryella, BOONYUEN ET AL.: SAVORYELLALES 1357

TABLE II. Sequences of fungal taxa obtained from GenBank, with their GenBank accession numbers

GenBank accession no. Taxon Source SSU LSU rpb2 Order microspora AFTOL 1361 DQ471036 AY083821 DQ470937 Camarops ustulinoides AFTOL 72 DQ470989 DQ470941 DQ470882 Order Menispora tortuosa AFTOL 278 AY544723 AY544682 DQ836884 Order ostrea AFTOL 915 DQ471007 DQ470959 DQ470909 Order Bertia moriformis SMH4320 N/A AY695260 AY780151 phaeostroma SMH4585 N/A AY346274 AY780172 Nitschkia grevillei SMH4663 N/A AY346294 N/A Nitschkia menicoidea SMH1523 N/A AY695270 N/A Order errabunda AFTOL 2120 DQ862045 AF408334 DQ862014 parasitica AFTOL 2123 DQ862048 N/A DQ862017 hypodermia AFTOL 2124 DQ862049 DQ862028 DQ862018 eres AFTOL 935 DQ471015 AF408350 N/A destructiva ATCC 76230 AF429718 AF362568 AF277147 gnomon AFTOL 952 DQ471019 AF408361 DQ470922 marginalis AFTOL 2128 DQ862053 AF408373 DQ862022 Melanconis stilbostoma AFTOL 2129 DQ862054 AF408374 DQ862023 ambiens AFTOL 2131 DQ862056 AF362564 DQ862025 Valsa nivea AFTOL 2125 DQ862050 AF362558 DQ862019 Order Hypocreales henningsiana AEG 96-27a AY489683 AY489715 DQ522413 ochroleuca GJS 90-227 AY489684 AY489716 DQ522415 AEG 97-2 AF543765 AF543789 DQ522417 capitata OSC 71233 AY489689 AY489721 DQ522421 Cordyceps cardinalis OSC 93609 AY184973 AY184962 DQ522422 NRRL 28021 AF049146 AF327374 AY545732 Cordyceps ophioglossoides OSC 106405 AY489691 AY489723 DQ522429 Epichloe typhina ATCC 56429 U32405 U17396 DQ522440 haematococca GJS 89-70 AY489697 AY489729 DQ518180 erubescens ATCC 36093 AY545722 AY545726 AY545732 americana AFTOL 52 AY544693 AY544649 DQ522853 Hypocrea lutea ATCC 208838 AF543768 AF543791 DQ522446 cinnabarina GJS 89-107 U32412 U00748 DQ522456 rousseliana AR 2716 AF543767 U17416 DQ522459 rufula CBS 346.85 DQ522561 DQ518776 DQ522461 Sphaerostilbella berkeleyana GJS 82-274 AF543770 U00756 DQ522465 Valetoniellopsis laxa GJS 96-174 AY015624 AY015635 DQ368638 Order Melanospora tiffanii ATCC 15515 AY015619 AY015630 AY015637 Melanospora zamiae ATCC 12340 AY046578 AY046579 AY046580 Order Microascales xylebori AFTOL 1285 DQ471031 DQ470979 N/A chesapeakensis ATCC 32818 U46870 U46882 DQ470896 fimbriata CBS 374.83 U32418 AF221009 DQ368641 halima JK 5473F U47843 U47844 N/A maritima CBS 264.59 U46871 U46884 N/A 1358 MYCOLOGIA

TABLE II. Continued

GenBank accession no.

Taxon Source SSU LSU rpb2 stemonitis AFTOL 1380 DQ836901 DQ863907 N/A Faurelina elongata CBS 126.78 DQ368657 DQ368625 DQ368639 penicillioides AFTOL 1415 DQ471038 AF027384 DQ470938 fibrosa JK 5132C/JK 5166A/ AF352078 U46886 N/A AT2060 appendiculata CBS 197.60 U46872 U46885 N/A laevis JK 5180A U46873 U46890 DQ836886 cirrosus CBS 217.31 M89994 AF275539 N/A Microascus longirostris AFTOL 1237 DQ471026 AF400865 N/A Microascus trigonosporus AFTOL 914 DQ471006 DQ470958 N/A Nimbospora effusa JK 5104A U46877 U46892 DQ836887 umiumi JK 5103F U46878 U46893 N/A setifera AFTOL 956 DQ471020 DQ470969 DQ836883 Varicosporina ramulosa RVG-113 U43846 U44092 DQ836888 Order piliferum AFTOL 910 DQ471003 DQ470955 DQ470905 Ophiostoma stenoceras AFTOL 1038 DQ836897 DQ836904 DQ836891 ATCC 32437/CBS M83261 DQ368627 DQ368645 298.87 Order Pezizales esculenta AFTOL 60 AY544708 AY544664 DQ470880 scutellata AFTOL 62 DQ247814 DQ247806 DQ247796 Order Sordariales elatum IFO 6554 M83257 DQ368628 AF107791 tetrasperma AFTOL 1287 DQ471032 DQ470980 DQ470932 Immersiella caudata CBS 606.72 DQ368659 AY999113 DQ368646 ovina AFTOL 17 DQ836894 AY436413 N/A crassa CBS 709.71 X04971 AF286411 AF107789 fimicola Caroline Biological AY545724 AY545728 N/A Supply company 15-6291 Sordaria ATCC 36709/Buck AY641007 AY346301 AY780195 s.n. TBM clade Incertae Sedis Etheirophora blepharospora JK 5397A EF027717 EF027723 EF027731 Etheirophora unijubata JK 5443B EF027718 EF027725 EF027733 Torpedospora ambispinosa CY 3386 AY858941 AY858946 N/A Torpedospora radiata AFTOL 751 DQ470999 DQ470951 DQ470902 Swampomyces clavatispora LP 83 AY858945 AY858952 N/A Swampomyces aegyptiacus CY 2973 AY858943 AY858950 N/A Swampomyces armeniacus JK 5059C EF027721 EF027728 N/A Swampomyces triseptatus CY 2802 AY858942 AY858953 N/A Juncigena adarca JK 5235A EF027719 EF027726 EF027734 Order Xylariales concentrica ATCC 36659 U32402 U47828 DQ368651 acuta AFTOL 63 AY544719 AY544676 DQ247797 Xylaria AFTOL 51 AY544692 AY544648 DQ470878

Not applicable with the prefix N/A, information in a certain field on a table is not provided AFTOL: Assembling the Fungal Tree of ; ATCC: The American Type Culture Collection, Manassas, VA; BCC: Biotec Culture Collection, Thailand; CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; BOONYUEN ET AL.: SAVORYELLALES 1359

TABLE II. Continued

CY: City University of Hong Kong Culture Collection, Hong Kong; IFO: Institute for (IFO), Osaka, Japan; OSC: Oregon State University Herbarium, U.S.A.; NRRL: Northern Regional Research Center (NRRL), USDA, Peoria, IL, USA; Culture and specimen abbreviations: AEG: H.G. Sung and J.W. Spatafora; AR: Stephen A. Rehner; GJS: G.J. Samuels; LP: J.Sakayaroj; JK: J. Kohlmeyer; RVG: J. Kohlmeyer and B. Volkmann-Kohlmeyer; SMH: S.M. Huhndorf.

Canalisporium and related species. MP, ML and Bayesian The SSU + LSU + rpb2 dataset showed that the analyses datasets on a LSU dataset showed that A. persoonii branches leading to the major orders in the Sordar- (AY590295 and AY094190) is characterized by short-length iomycetes were reasonably stable and highlighted a sequences in the Ascotaiwania clade and was excluded from number of other consistent and well supported clades the dataset. with 100% support and posterior probabilities of 1.00. Within this class the Sordariomycetes can be divided RESULTS into two major subclasses; Hypocreomycetidae com- prised two orders, and the TBM and Savoryellales Combined SSU, LSU and rpb2 phylogeny.—The type clades with a bootstrap support (BSMP) of 84% and a species of the genera Ascothailandia (A. grenadoidia), Bayesian posterior probability (BYPP) less than 0.95, Ascotaiwania (A. lignicola), Savoryella (S. lignicola), and the Sordariomycetidae composed of six orders and Canalisporium (C. caribense) were included in the with a BSMP of 67% and a BYPP of 1.00, in which they 18S rDNA + 28S rDNA + rpb2 dataset to determine formed a monophyletic group. The interordinal their monophyly. Sequences from 41 sequences of relationships within the Hypocreomycetidae showed Ascotaiwania, Ascothailandia, Canalisporium and Sa- a stable branching pattern, but with weak bootstrap voryella (TABLE I) were aligned with 80 sequences % from the GenBank representing the two main support in some minor clades, lower than 50 and subclasses of the Sordariomycetes: Hypocreomyceti- 0.95 BYPP for example Melanosporales and some taxa in the TBM clade (Swampomyces, Etheirophora, Torpe- dae and Sordariomycetidae (TABLE II). As a result of excluding ambiguously aligned dospora, and Juncigena). regions, the combined SSU + LSU + rpb2 dataset The maximum parsimony trees resolved a previ- consisted of 3195 characters and 121 taxa. A total of ously unknown monophyletic grouping in the Hypo- 1275 characters are constant, 1453 of which were creomycetidae comprising three well supported sub- parsimony informative and 467 variable characters clades (A, C, S). The type species of the genera, were parsimony uninformative. An initial analysis of Ascothailandia (A. grenadoidia), with the anamorph, this dataset yielded 18 trees with a tree length of Canalisporium (C. caribense), Ascotaiwania (A. ligni- 14 013 (CI 5 0.245, RI 5 0.695, RC 5 0.170, HI 5 cola) and Savoryella (S. lignicola), are well placed in 0.755). The major branches were stable and varied each subclade in the Hypocreomycetidae. These only with minor differences in the position of results are almost consistent with those of the Savoryella sp. (NF00205) and some taxa of the individual SSU dataset, LSU dataset and combined Microascales and Hypocreales. However these differ- SSU + LSU dataset with high bootstrap support and ences did not affect the overall topology of the tree posterior probabilities (data not shown). (trees not shown). Bayesian inference with likelihood The genera Ascotaiwania (A), Ascothailandia (C) scores for the combined analysis provided mostly and Savoryella (S) including Canalisporium species identical topology to other analyses and the best KH- formed a well supported clade (100% BSMP, 1.00 likelihood scores. Although there is a minor differ- BYPP), that was clearly distinct from the Hypocreales, ence in the position of some genera in the Hypo- Melanosporales and Microascales (Hypocreomyceti- creales and A. hughesii (AY316357) and Monotospor- dae). In this new lineage Ascothailandia and six ella setosa (AF132334), all branches were supported by Canalisporium species (C. caribense, C. elegans, C. posterior probabilities. The best phylogenetic hypoth- exigum, C. jinghongensis, C. pallidum and C. pul- esis for the K-H test (2ln L 5 64 623.84037) is chrum) formed a well supported subclade C as a provided (FIG. 1). monophyletic group, with strong statistical support 30M 1360 YCOLOGIA

FIG. 1. One of 18 most parsimonious trees inferred from the combined three-locus dataset of SSU rDNA + LSU rDNA + rpb2 sequences for the Sordariomycetes. The tree is rooted with members of the order Pezizales as outgroups. The numbers above the branches are the bootstrap supports greater than 50% (1000 replicates) while the numbers below the branches higher than 95% are the Bayesian posterior probabilities. Names of taxonomic orders and clade affiliations are provided to the right of the species names indicated by vertical bars. Thickened branches in boldface indicate bootstrap support 100% and Bayesian posterior probability 1.00 in Savoryellales clade. Isolates with the prefixes M and F are from marine and freshwater habitats respectively. BOONYUEN ET AL.: SAVORYELLALES 1361

FIG. 2. The single most parsimonious trees for the subclass Hypocreomycetidae inferred from the combined six-locus dataset of the partial SSU rDNA + LSU rDNA + rpb2 + rpb1 + tef1 + 5.8S sequences. RAxML bootstrap values (BSML) are displayed to the top left and the maximum parsimony bootstrap values (BSMP) are plotted on the top right of each branch while the Posterior probabilities are given below the corresponding branches. The tree is rooted with and Scutellinia scutellelata as members of the Pezizales as outgroups. Isolates with the prefixes M and F are from marine and freshwater habitats, respectively.

(100% BSMP and 1.00 BYPP); seven Savoryella species prised 5025 characters for 44 taxa, of which 1578 were (S. appendiculata, S. aquatica, S. fusiformis, S. parsimony informative, 638 parsimony uninformative lignicola, S. longispora, S. paucispora and S. verrucosa) and 2812 constant. formed a highly supported monophyletic subclade S The maximum parsimony analysis resulted in one (100% BSMP and 1.00 BYPP); while Ascotaiwania most parsimonious tree (MPT) with TL 5 5462, CI 5 species grouped together in subclade A, with moder- 0.586, RI 5 0.822, RC 5 0.482 and HI 5 0.414, while ate support (69% BSMP and less than 0.95 BYPP). the resultant most likely tree from RAxML was log likelihood 232 178.265234. Combined SSU, LSU, rpb2, rpb1, tef1 and 5.8S rDNA The topology of the Savoryellales clade in the six phylogeny.—This dataset included an expanded set of loci dataset was similar to the phylogeny generated taxa: four Ascotaiwania species, eight Savoryella from the individual datasets (data not shown) and was species, seven Canalisporium species and one As- congruent with the combined datasets of SSU + LSU cothailandia species, comprising 42 sequences with + rpb1 (FIG. 1), except in the position of A. hughesii Morchella hughesii and Scutellinia esculenta as the (and its anamorph M. setosa), showing they are outgroup taxa (FIG. 2). The aligned dataset com- distantly related to the Ascotaiwania subclade. The 1362 MYCOLOGIA resulting consensus tree from the Bayesian analysis Shearer (2004), which used only partial sequences of showed nearly identical topology to the maximum the 28S rDNA while Vijaykrishna et al. (2006) used parsimony analysis, but there were minor differences only partial sequences of the 18S rDNA. in the grouping of the Ascotaiwania subclade (data not shown). The monophyly of Ascotaiwania, Canalisporium, Three subclades of the combined SSU, LSU, rpb2, Savoryella and Ascothailandia.—Both analyses sup- rpb1, tef1 and 5.8S rDNA sequences were identified. port the monophyly of the genera Savoryella and The Savoryella subclade (S) was monophyletic and Ascothailandia (with the anamorphic genus Canalis- supported with a BSMP of 100%, BSML of 100% and porium). These genera have been assigned to several a BYPP of 1.00 and comprised two groups: four orders and families within Sordariomycetes, Sordar- marine species (S. appendiculata, S. lignicola, S. iomycetidae, as outlined (TABLE III). The monophyly longispora and S. paucispora) (M), and three fresh- of the genus Ascotaiwania is uncertain as in the three- water species (S. aquatica, S. fusiformis and S. gene dataset, where the five species sequenced group verrucosa)(F).FreshwaterSavoryella species (S. together but with weak support, confirming Campbell aquatica and S. verrucosa) formed a branch that and Shearer (2004). However in the six-gene dataset statistically was supported strongly (1.00 BYPP, A. hughesii and its anamorph, M. setosa, are distantly 100% BSMP and 100% BSML), with S. fusiformis, related to Ascotaiwania sensu stricto. We excluded A. S. appendiculata (NF00206) and Savoryella sp. persoonii from our analysis due to the short length of (NF00205) as a sister group. Savoryella lignicola, S. the sequence (808 bps: AY590295) although Camp- longispora and S. paucispora formed a well supported bell and Shearer (2004) showed that it grouped in a lineage of marine origin with BSMP of 99% from MP, monophyletic clade, to the Hypocreales and BSML of 98% from RAxML and BYPP of 1.00. Microascales and widely separated from taxa in the Ascothailandia grenadoidia (GQ390252) and seven in the Sordariales. Savoryella Canalisporium species grouped together with high (FIG. 3A–D) and Ascothailandia (FIG. 3G, H) share bootstrap and Bayesian support values (100% BSMP, several features in common with Ascotaiwania, such as 100% BSML and 1.00 BYPP), all isolated from ascomatal morphology and versicolorous ascospores freshwater habitats. Ascotaiwania sawadae (SS (FIG. 4A–H, L–V). However Ascotaiwania has long 00051), A. lignicola (NIL00005 and NIL00006) and cylindrical asci with a relatively large (4–8 mm), non- A. mitriformis (AF132324) formed a weakly supported amyloid apical ring, a hamathecium with numerous subclade A (69% BSMP) but with high RAxML (98% -like paraphyses, tapering apically and four- to BSML) support. In this subclade A. mitriformis was eight-septate ascospores (Chang et al. 1998). Cai et al. closely related to A. sawadae (90% BSMP, 100% (2006) opine that Ascotaiwania as currently circum- BSML and 1.00 BYPP) while A. hughesii and scribed may not be monophyletic, and this is borne Monotosporella setosa were distantly placed from out by data in our study. Hyde and Goh (1999), in subclade A with low bootstrap support. describing the new species Ascotaiwania pallida from submerged wood collected in Chedworth Wood, United , commented that placement in the DISCUSSION genus was speculative. In view of the variation in The higher order taxonomic position of the genera morphology reported for Ascotaiwania species, fur- Ascotaiwania and Savoryella was unresolved despite a ther taxon sampling is required to resolve the number of phylogenetic studies (Ranghoo et al. 1999, monophyly of the genus. Campbell and Shearer 2004). Vijaykrishna et al. While the teleomorphic genera Ascothailandia (2006) included two Savoryella species (S. elongata, (FIG. 3G, H) and Savoryella (FIG. 3A–D) are aquatic an invalid name, and S. longispora; neither one is the and found on submerged wood, Ascotaiwania species type species of the genus) in their study of the are known from aquatic and terrestrial habitats on a , origin and evolution of freshwater asco- variety of substrata: A. licualae on petioles of Licuala mycetes. Based on an 18S rDNA dataset, the two ramsayi; A. mauritania on prop roots of Pandanus Savoryella species grouped with Melanospora zamiae in palustris; A. pennisetorum on Pennisetum purpurea; the Hypocreales with no statistical support. and A. palmicola on dead rachis of Iriartia sp. The current study included two strains of the type Ascotaiwania anamorphs include Helicoon and Mono- species and a wider range of Savoryella species tosporella (Cai et al. 2006). (FIGS. 1, 2, 3A–F, 4A–G, M–S) and the type species Based on the sequencing of both, protein coding of Ascotaiwania (A. lignicola)(FIGS. 1, 2, 4T). A wider and ribosomal nuclear loci let us draw two major range of genes were sequenced thus extending the conclusions. First, the type species of Ascothalandia studies of Ranghoo et al. (1999) and Campbell and clusters with Canalisporium, thus the following species BOONYUEN ET AL.: SAVORYELLALES 1363

FIG. 3. Ascomata morphology of the genera Savoryella and Ascothailandica. A–C 5 Savoryella lignicola. Subglobose or ellipsoidal ascomata, partly immersed (A) or superficial (B, C). D–F 5 Savoryella appendiculata. Section of immersed ascoma. D 5 Flask-shaped ascoma, with dark brown peridial wall. E 5 Immersed ascoma with periphysate neck (arrowed). F 5 Immersed ascoma with one-layered peridium, composed of several layers of elongated thick-walled, brown cells (arrowed). G, H 5 Ascothailandia grenadoidia. Light microscope micrographs of ascomata with long neck (arrowed) on the test blocks. Bars: A–C, H 5 250 mm; D 5 50 mm; E, F 5 10 mm; G 5 1000 mm.

are assigned to Ascothalandia (C. elegans, C. pulchrum, form a unique lineage within the Hypocreomycetidae C. jinghongensis, C. exiguum, C. caribense, C. palladi- and a sister clade to a lineage of marine ascomycetes um), the new gen, in line with current nomenclature (TBM: Torpedospora/Bertia/Melanospora clade), Coro- opinion (FIGS. 1, 2). Second, Savoryella, Canalispor- nophorales and Melanosporales (Schoch et al. 2007), ium/Ascothalandia and some Ascotaiwania species and new order Savoryellales proposed. 1364 MYCOLOGIA

FIG. 4. and morphology of the genera Savoryella, Ascotawiania and Ascothailandia.A5 Savoryella aquatica. Asci at various stages of development. B 5 Savoryella curvispora. Cylindrical asci with mature and immature ascospores. C 5 Savoryella lignicola. Cylindrical asci of various stages and paraphyses. D, E 5 Savoryella paucispora.D5 Young two spored ascus. E 5 Mature and immature asci and paraphyses (arrow). F, G 5 Savoryella verrucosa. Cylindrical asci with BOONYUEN ET AL.: SAVORYELLALES 1365

TABLE III. Taxonomic assignment of the genus Savoryella based on published morphological and molecular studies

Authors and reference Subclass Order Family Comments

Jones and Eaton 1969 — — — Authors did not assign to any family Kohlmeyer and — Sphaeriales incertae —— Kohlmeyer 1979 sedis Kohlmeyer 1986 — Ascomycetes insertae — — Eriksson, Hawksworth 1986 Eriksson and Xylariomycetidae Xylariales — Hawksworth 1987 Barr 1990 Hypocreomycetidae Halosphaeriales — Presence of catenophyses-like paraphyeses Jones and Hyde 1992 Sordariomycetidae Sordariales Tripterosporaceae, Presence of brown ascospores, asci with a refractive apical ring Read et al. 1993 — — — It is difficult to assign Savoryella to any existing group and suggested it was congeneric with Ascotaiwania and has shared features with Aniptodera, which belongs to the Halosphaeriales Ho et al. 1997 Sordariomycetidae Sordariales — — Vijaykrishna 2005 Hypocreomycetidae Hypocreales — Based on molecular analysis (SSU data) This study Hypocreomycetidae —— — incertae sedis

r verrucose ascospores. H 5 Ascothailandia grenadoidia. Cylindrical asci and hyaline paraphyses. I 5 Savoryella longispora. Squash illustrating paraphyses (picture from Jones and Hyde 1992 in Botanica Marina). J 5 Savoryella aquatica. Apical ring (arrow). K 5 Ascothailandia grenadoidia. Apical ring (arrow). L 5 Savoryella appendiculata. Ascospore with appendages (arrowed). M 5 Savoryella aquatica. Ascospore ellipsoidal, central cells dark brown end cells hyaline. N 5 Savoryella curvispora. Ascospore fusiform, curved, three-septate, central cells brown, end cells hyaline to pale brown. O 5 Savoryella fusiformis. Ascospores fusiform, three-septate central cells brown, end cells hyaline. P 5 Savoryella lignicola. Ascospore ellipsoidal three- septate central cells brown, end cells smaller and hyaline. Q 5 Savoryella longispora. Ellipsoidal ascospore with three-septate central cells brown, end cells hyaline. R 5 Savoryella paucispora. Fusoid-ellipsoidal ascospore, slightly constricted at the septa, central cells brown, end cells hyaline. S 5 Savoryella verrucosa. Ascospore ellipsoidal, three-septate, constricted at the septa, central cells brown and distinctly verrucose, end cells hyaline. T 5 Ascotaiwania lignicola. Ascospore fusiform, seven-septate, not constricted at the septa, central cells brown, end cells hyaline. U 5 Ascotaiwania sawadae. Fusoid ascospore, three-septate, straight to curved, central cells brown, end cells hyaline to pale brown. V 5 Ascothailandia grenadoidia. Ovoid to fusoid ascospore, three-septate, straight to curved, central cells brown, end cells hyaline to pale brown. W 5 Ascothailandia (Canalisporium) grenadoidia. Squash mount of a portion of the sporodochium. Conidia pale olive to brown, globose to subglobose, slightly curved, with 3–6 longitudinal septa and 4–6 transverse septa. X 5 Ascothailandia (Canalisporium) caribense. Conidia with a single column of longitudinal septa and 3–6 rows of transverse septa, slightly constricted at the septum, brown to reddish dark brown. Y 5 Ascothailandia (Canalisporium) elegans. Conidia irregular with 4–5 columns of longitudinal septa and 5–8 rows of transverse septa, olivaceous brown to brown. Z 5 Ascothailandia (Canalisporium) exiguum. Conidia with a single column of longitudinal septa and 2–3 rows of transverse septa, slightly constricted at the septum, pale olivaceous brown to pale pinkish brown. AA 5 Ascothailandia (Canalisporium) jinghongensis. Conidia irregularly with 4–5 columns of longitudinal septa and 2–4 rows of transverse septa, brown. AB, AC 5 Ascothailandia (Canalisporium) pallidum. Conidia with a single column of longitudinal septa 3-4 and 2–3 rows of transverse septa, scattered, pale olivaceous with clearly visible septa and canals, septa thin and not banned. AD 5 Ascothailandia (Canalisporium) pullchrum. Conidia irregularly with 4–5 columns of longitudinal septa and 3–9 rows of transverse septa, olivaceous brown to brown. Bars: A– E, L–U, W, X 5 20 mm; F–H, Y–AD 5 25 mm; I–K 5 10 mm, V 5 15 mm. 36M 1366 TABLE IV. Morphological characters of Ascotaiwania , Savoryella, Ascothailandia (Savoryellales clade) and selected orders in Hypocreomycetidae, Sordariomycetes (modified from Sakayaroj et al. 2005, Tang et al. 2007, Sri-indrasutdhi et al. 2010)

Savoryellales clade (In this study) Morphological Microascales characters Ascotaiwania Savoryella Ascothailandia TBM clade (+ Halosphaeriales) Hypocreales

Stromatic tissues Present Present Present Absent in some Absent Present in some genera (clypeus) genera (such as or absent Torpedospora spp.) or Found some species with pseudostroma (such as Swampomyces clavatispora ) Ascomal position Partial to fully Superficial or partly Partial immersed, Superficial or Superficial or Superficial or immersed, oblique immersed, coriaceous, immersed immersed immersed to horizontal, coriaceous, ostiolate, papilate coriaceous pyriform, papillate, solitary or gregarious

Ascomal color Dark brown to black Dark brown to black Dark brown to black Dark brown to black Transparent or white White or yellow or red YCOLOGIA or black to brown/ or brown Ostiolar canal Ostiolate Ostiolate Ostiolate Ostiolate Ostiolate Ostiolate Ascomal size 200–240 mm diam 100–250 mm diam 100–150 mm diam. Approx 100–700 mm 50–500 mm diam 100–150 mm diam diam Peridium Membranous Membranous, thin Membranous Membranous Carbonaceous, Soft, fleshy or membranous, membranouse subcarbonaceous Hamathecium Paraphyses, Paraphyses, hyphal - Paraphyses, Paraphyses or Typically Paraphyses numerous, hypha- like sparse if hypha-like, no absence in some catenophyses, like, tapering present, septate, filiform genera in Periphyses distally deliquescing early; 1–2 mm development rounded at their (Melanospora spp.) apex Ascus shape Cylindrical and long Cylindrical to Cylindrical Clavate, oblong to Clavate or ellipsoid, Clavate to cylindrical clavate, ellipsoidal or globose or ovoid pedicellate, apically cylindrical or rounded, with elongate small discoid apical ring unitunicate Ascus persistence Persistent Persistent Persistent Persistent Persistent or Persistent deliquescent in some genera TABLE IV. Continued

Savoryellales clade (In this study) Morphological Microascales characters Ascotaiwania Savoryella Ascothailandia TBM clade (+ Halosphaeriales) Hypocreales Apical ring Present with non- Absent or with a Present with with an Present in some Present with Present with amyloid massive small discoid apial apical ring species or absent non-amyloid or non-amyloid ring pore/ring J-refractive in some species absent in most genera Ascospore colour Brown to black, mid Brown to black, mid Brown to black, mid Brown to black Mostly hyaline or Hyaline to pale brown brown central cells brown to black brown to black red to brown and smaller hyaline central cells and central cells and to sub-hyaline end hyaline to hyaline to

cells sub-hyaline end sub-hyaline end B cells cells AL ET OONYUEN Ascospore number Octosporus Octosporus, Octosporus Octosporus Octosporus Octosporus occasionally two-spored Ascospore shape Fusiform, Ellipsoidal, 3-septate, Ovoid to fusoid, Cylindrical to Ellipsoid or fusiform Muriform, filiform, 3–7-septate, end cell rounded straight to curved, elongate or or reniform, heart falcate, fusoid, :S .: generally more hyaline, ellipsoid or shaped triangular ellipsoid than 3 septa, end 3-euseptate, fusiform or AVORYELLALES cell pointed smooth-walled, with cylindrical to a large guttule in clavate each cell, surrounded by a thin layer of mucilage Ascospore appendage Absent Absent with the Absent Present or absent in Present or absent in Absent exception of S. some genera, some genera appendiculata species Nutritional mode Saprobic Saprobic Saprobic Saprobic Saprobic Saprobic and pathogenic Anamorph stage Monotosporella Unknown Canalisporium Unknown Found in some Mostly present setosa , Helicoon grenadoidia genera farinosum Habitat Freshwater and Freshwater and Freshwater; on On mangrove and Some genera on Mostly saprobic or terrestrial; marine; on woody submerged wood marine drift wood, marine, driftwood, biotrophic on plant submerged wood substrata bark and roots bark and roots or fungi or palm rachis 1367 1368 MYCOLOGIA

TAXONOMY S. melanospora Abdel-Wahab & E.B.G. Jones, My- coscience 41(4):387 (2000) Savoryellales Boonyuen, Suetrong, S. Sivichai, K.L. S. paucispora (Cribb & J.W. Cribb) J. Koch, Nordic Jl Pang & E.B.G. Jones, ord. nov. Bot. 2(2):169 (1982). (FIG. 4D, E, R) MycoBank MB561070. S. verrucosa Minoura & T. Muroi, Trans. Mycol. Soc. Ab ordinibus ceteris Sordariomycetes, Hypocreomyceti- dae differ origine. Ascomata perithecea, immerse vel Japan 19(2):132 (1978). (FIG. 4F, G, S) superficialia, ostiolata, papillata, coriacea, brunnea vel Ascotaiwania Sivan. & H.S. Chang 1992, Mycol. Res. nigra, ostiolata centro vel lateralia in horizontalia, periph- ysata. Peridium multi-stratum, cellularum ex dermis pro- 96(6):481 (1992) fundus, brunneum, cellularum hyalinum internum. Ha- A. lignicola Sivan. & H.S. Chang, Mycol. Res. mathecium hyphae similum, prolixum, paucum vel creber, 96(6):481 (1992). (Types species). (FIG.4T) septatum, simplex. Asci clavati vel cylindricati, pedicellati, A. sawadae H.S. Chang & S.Y. Hsieh, in Chang, Hsieh, unitunicati, parvi vel magni orbis superficies, J-, persisentes. Jones, Read & , Mycol. Res. 102(6):713 (1998). Ascosporae ellipsoidae, fusiformae, rectae vel curvo pauci, (FIG. 4U) multi-septatae, cellularae medius brunneum, cellularae Synonym: Monotosporella sp. terminus hyalinae, teres, dermae profundus, cum vel sine A. hughesii Fallah, J.L. Crane & Shearer, Can. J. Bot. lentesco appendiculatae terminus. Anamorph dematiac- 77(1):89 (1999) eous. Synonym: Helicoon farinosum A new order in the Sordariomycetes, Hypocreomy- Taxonomic position not confirmed: cetidae. Ascomata perithecioid, immersed or superfi- A. mauritiana Dulym., P.F. Cannon, K.D. Hyde & cial, ostiolate, papillate, coriaceous, periphysate, Peerally, Fungal Diversity 8:87 (2001) brown to black, ostiole central or at one end if A. mitriformis Ranghoo & K.D. Hyde, Mycologia ascomata horizontal. Peridium many layers, thick- 90(6):1059 (1998) walled cells, brown, hyaline inwardly. Hamathecium Synonym: Monotosporella sp. hypha-like, wide, numerous or sparse, septate, simple. A. pallida K.D. Hyde & Goh, Mycol. Res. 103(12):1563 Asci clavate to cylindrical, pedicellate, unitunicate, (1999) small or relatively large apical ring, J-, persistent. A. palmicola K.D. Hyde, 47(2):213 (1995) Ascospores ellipsoid, fusiform, straight or slightly A. pennisetorum M.K.M. Wong & K.D. Hyde [as curved, three to many more euseptate, central cells ‘pennisetarum’], Cryptog. Mycol. 22(1):20 (2001) brown, end cells hyaline to subhyaline, smooth, thick- A. persoonii Fallah, J.L. Crane & Shearer, Can. J. Bot. walled, with or without polar mucilaginous pads or 77(1):87 (1999) appendages. Anamorphs dematiaceous. A. wulai H.S. Chang & S.Y. Hsieh, in Chang, Hsieh, Etymology: in reference to the type genus. Jones, Read & Moss, Mycol. Res. 102(6):711 (1998) Type genus: Savoryella E.B.G. Jones & R.A. Eaton. A. hsilio H.S. Chang & S.Y. Hsieh, in Chang, Hsieh, Taxa included in this order: Jones, Read & Moss, Mycol. Res. 102(6):713 (1998) A. licualae J. Fro¨hl. & K.D. Hyde, Fungal Diversity Res. Savoryella E.B.G. Jones & R.A. Eaton, Trans. Br. Ser. (Hong Kong) 3:327 (2000) mycol. Soc. 52(1):161 (1969) S. appendiculata K.D. Hyde & E.B.G. Jones, Bot. Mar. Ascothailandia Sri-indrasutdhi, Boonyuen, Sivichai & 35(2):89 (1992). (FIG. 3D–F, L) E.B.G. Jones, Mycoscience 51(6):414 (2010) S. aquatica K.D. Hyde, Aust. Syst. Bot. 6(2):162 Ascothailandia grenadoidia Sri-indrasutdhi, Boo- (1993). (FIG. 4A, J, M) nyuen, Sivichai & E.B.G. Jones, Mycoscience S. curvispora W.H. Ho, K.D. Hyde & Hodgkiss, Mycol. 51(6):414 (2010). (FIGS. 3G, H, 4H, K, V) Res. 101(7):804 (1997). (FIG. 4B, M) A. elegans (Nawawi & Kuthub.) Boonyuen, Suetrong, S. fusiformis W.H. Ho, K.D. Hyde & Hodgkiss, Mycol. Sivichai & E.B.G. Jones Res. 101(7):804 (1997). (FIG. 4O) Synonym: Canalisporium elegans Nawawi & Kuthub., S. grandispora K.D. Hyde, Mycoscience 35(1):59 (1994) Mycotaxon 34(2):484 (1989). (FIG. 4Y) S. lignicola E.B.G. Jones & R.A. Eaton, Trans. Br. A. pulchrum (Hol.-Jech. & Mercado)(Nawawi & mycol. Soc. 52(1):161 (1969). (Types species). Kuthub.) Boonyuen, Suetrong, Sivichai & E.B.G. (FIGS. 3A–C, 4C, P) Jones S. limnetica H.S. Chang & S.Y. Hsieh, in Chang, Synonym: C. pulchrum (Hol.-Jech. & Mercado) Hsieh, Jones, Read & Moss, Mycol. Res. 102(6):715 Nawawi & Kuthub., Mycotaxon 34(2):481 (1989). (1998) (FIG. 4A, D) S. longispora E.B.G. Jones & K.D. Hyde, Bot. Mar. A. jinghongensis (L. Cai, K.D. Hyde & McKenzie) 35(2):84 (1992). (FIG. 4G, I) Boonyuen, Suetrong, Sivichai & E.B.G. Jones BOONYUEN ET AL.: SAVORYELLALES 1369

Synonym: C.jinghongensis L. Cai, K.D. Hyde & asci that are persistent, clavate to cylindrical, short McKenzie, in Cai, Zhang, McKenzie, Lumyong & pedunculate, with or without paraphyses and gener- Hyde, Cryptog. Mycol. 24(1):4 (2003). (FIG. 4AA) ally with an apical pore (FIG. 4J, K); ascospores with A. caribense (Hol.-Jech. & Mercado) Boonyuen, brown central cells and hyaline end cells, lacking Suetrong, Sivichai & E.B.G. Jones appendages (with the exception of S. appendiculata). Synonym: C. caribense (Hol.-Jech. & Mercado) Nawawi No anamorphs have been reported for Savoryella, & Kuthub., Mycotaxon 34(2):479 (1989). (Types but several dematiaceous have been species). (FIG. 4X) reported in the genus Ascotaiwania, including Mono- A. pallidum (Goh, W.H. Ho & K.D. Hyde) Boonyuen, tosporella sp. (A. sawadae; Sivichai et al. 1998), M. Suetrong, Sivichai & E.B.G. Jones setosa (A. sawadae; Ranghoo et al. 1999) and Helicoon Synonym: C. palldum Goh, W.H. Ho & K.D. Hyde, in (A. hughesii; Fallah et al. 1999, Tsui and Berbee Goh, Ho, Hyde, Whitton & Umali, Can. J. Bot. 2006). Canalisporium (FIG. 4W) is the anamorph of 76(1):148 (1998). (FIG. 4AB, AC) Ascothailandia grenadoidia (Sri-indrasutdhi et al. A. exiguum (Goh & K.D. Hyde) Boonyuen, Suetrong, 2010). Only one Ascothailandia species is known Sivichai & E.B.G. Jones (described), and therefore the only species se- Synonym: C. exiguum Goh & K.D. Hyde, in Goh, Ho, quenced in this study. Hyde, Whitton & Umali, Can. J. Bot. 76(1):145 (1998). (FIG. 4Z) ACKNOWLEDGMENTS Taxonomic position not confirmed: Canalisporium kenyense Goh, W.H. Ho & K.D. Hyde This research was financially supported by the Biodiversity 1998, in Goh, Ho, Hyde, Whitton & Umali, Can. J. Research and Training Program of Thailand (BRT Bot. 76(1):148 (1998) R_252057 and R_251009) with co-financing from the National Center for Genetic Engineering and Biotechnol- Canalisporium panamense A. Ferrer & Shearer, Myco- ogy (BIOTEC). Dr Shaun R. Pennycook is thanked for taxon 93:180 (2005) taxonomic advice. We thank Professor Morakot Tantichar- Canalisporium variabile Goh & K.D. Hyde, Mycologia oen and Dr Kanyawim Kirtikara at BIOTEC for their 92(3):589 (2000) continued interest and constant support. Apiluk Loilong Hibbett et al. (2007) recognized three subclasses is thanked for providing the cultures of S. appendiculata within the Sordariomycetes: Hypocreomycetidae (NF00206), S. lignicola (NF00204) and Savoryella sp. (with the orders Coronophorales, Hypocreales, Mel- (NF00205). KL Pang acknowledges the financial support anosporales and Microascales); Sordariomycetidae by National Science Council of Taiwan (NSC98-2621-B-019- (the orders Boliniales, Chaetosphaeriales, Coniochae- 002-MY3) and Center of Excellence for Marine Bioenviron- tales, Diaporthales, Ophiostomatales and Sordariales) ment and Biotechnology, National Taiwan Ocean Univer- and Xylariomycetidae (the Xylariales). The orders sity. Juntima Chanprasert is thanked for RAxML. Jiranan Techaprasan is thanked for advice on running the partition- , , and Tricho- homogeneity test. sphaeriales were referred to the Sordariomycetes incertae sedis. However the phylogeny of many taxa remain unresolved, for example Annulatacaceae, LITERATURE CITED , Papulosaceae (Zhang et al. 2006) Abdel-Wahab MA, Jones EBG. 2000. Three new marine and the TBM clade, a lineage of marine ascomycetes ascomycetes from driftwood in Australia sand dunes. (Schoch et al. 2007). Mycoscience 41:379–388, doi:10.1007/BF02463951 The genera studied here (Ascotaiwania, Ascothai- Barr ME. 1990. Prodromus to nonlichenized, pyrenomyce- landia and Savoryella) form a new unique clade tous members of class Hymenoascomycetes. Mycotaxon within the Hypocreomycetidae, and a new order, 39:43–184. Savoryellales, is erected. The Savoryellales clade is Bunyard BA, Nicholson MS, Royse DJ. 1994. A systematic distinct from the orders Microascales and Hypo- assessment of Morchella using RFLP analysis of the 28S creales and genera grouping in the TBM clade, which ribosomal RNA gene. Mycologia 86:762–772, doi:10.2307/ includes members of the Coronophorales and Mela- 3760589 nosporales. Morphological features of genera in the Cai L, Hyde KD, Tsui CKM. 2006. Genera of freshwater fungi. Fungal Divers Res Ser 18:1–261. Savoryellales clade are compared with those of the Campbell J, Shearer CA. 2004. and Hypocreales, Microsporales and TBM (TABLE IV). Ascitendum, two new genera in the Annulatascaceae. Members within the Savoryellales clade share a Mycologia 96:821–832, doi:10.2307/3762115 number features: ascomata that are generally swan- Castlebury LA, Rossman AY, Sung GH, Hyten AS, Spatafora like (rarely with a central neck) (FIG. 3A–D, G, H) JW. 2004. Multigene phylogeny reveals new lineage for and periphysate neck (FIG. 3E); peridium with several chartarum, the indoor air . Mycol layers (FIG. 3F); paraphysate (FIG. 4I), unitunicate Res 108:864–872, doi:10.1017/S0953756204000607 1370 MYCOLOGIA

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