Mycologia, 103(6), 2011, pp. 1351–1371. DOI: 10.3852/11-102 # 2011 by The Mycological Society of America, Lawrence, KS 66044-8897 Savoryellales (Hypocreomycetidae, Sordariomycetes): a novel lineage of aquatic ascomycetes inferred from multiple-gene phylogenies of the genera Ascotaiwania, Ascothailandia, and Savoryella Nattawut Boonyuen1 Canalisporium) formed a new lineage that has Mycology Laboratory (BMYC), Bioresources Technology invaded both marine and freshwater habitats, indi- Unit (BTU), National Center for Genetic Engineering 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 order we erect a new Plant 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/Bertia/ Mycology Laboratory (BMYC), Bioresources Technology Melanospora) in the Hypocreomycetidae incertae Unit (BTU), National Center for Genetic Engineering sedis. and Biotechnology (BIOTEC), 113 Thailand Science Key words: ascomycota incertae sedis, fungal Park, Phaholyothin Road, Khlong 1, Khlong Luang, systematics, Hypocreomycetidae, unitunicate ascomy- Pathumthani 12120, Thailand cetes Ka-Lai Pang Institute of Marine Biology, 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 species has been widely (Jones and Hyde 1992), while S. appendiculata and S. debated, and the genus 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 ascospores. We performed two com- cetes, Sordariomycetidae (Vijaykrishna et al. 2006). bined phylogenetic analyses of different genes: (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 Hypocreales, Halosphaer- tower run on brackish water. Savoryella species are iales (now Microascales), Sordariales and Xylariales, 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- spores, 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. palmicola (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 morphology and sporulation submerged grasses and A. mauritiana (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-spore 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 mycelium 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