Pleosporomycetidae, Dothideomycetes) from a Freshwater Habitat in Thailand

Mycological Progress (2020) 19:1031–1042 https://doi.org/10.1007/s11557-020-01609-0 ORIGINAL ARTICLE Mycoenterolobium aquadictyosporium sp. nov. (Pleosporomycetidae, Dothideomycetes) from a freshwater habitat in Thailand Mark S. Calabon1,2 & Kevin D. Hyde1,3 & E. B. Gareth Jones4 & Mingkwan Doilom5,6 & Chun-Fang Liao5,6 & Saranyaphat Boonmee1,2 Received: 25 May 2020 /Revised: 25 July 2020 /Accepted: 28 July 2020 # German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract A study of freshwater fungi in Thailand led to the discovery of Mycoenterolobium aquadictyosporium sp. nov. Evidence for the novelty and placement in Mycoenterolobium is based on comparison of morphological data. The new species differs from the type species, M. platysporum, in having shorter and wider conidia, and from M. flabelliforme in having much longer and wider conidia. The hyphomycetous genus Mycoenterolobium is similar to Cancellidium but differs in the arrangement of conidial rows of cells at the attachment point to the conidiophores. The conidia of the former are made up of rows of cells, radiating in a linear pattern from a single cell attached to the conidiophore, while in Cancellidium, adherent rows of septate branches radiate from the conidiophore. Cancellidium conidia also contain branched chains of blastic monilioid cells arising from the conidia, while these are lacking in Mycoenterolobium.AtmaturityinMycoenterolobium, the two conidial lobes unite and are closely appressed. Phylogenetic analyses based on a combined LSU, SSU, ITS, TEF1-α,andRPB2 loci sequence data support the placement of Mycoenterolobium aquadictyosporium close to the family Testudinaceae within Pleosporomycetidae, Dothideomycetes. The novel species Mycoenterolobium aquadictyosporium is described and illustrated and is compared with other morphologically similar taxa. Keywords 1 new taxon . Freshwater hyphomycete . Asian mycology . Multi-locus analysis . Thailand Introduction Section Editor: Gerhard Rambold The incorporation of molecular data in phylogenetic and evo- * Saranyaphat Boonmee [email protected] lutionary studies has revolutionized the current knowledge of fungal systematics (Tanabe et al. 2005; Shenoy et al. 2006; 1 Center of Excellence in Fungal Research, Mae Fah Luang Taylor and Berbee 2006; Choi and Kim 2017; Liu et al. 2017; University, Chiang Rai 57100, Thailand Tedersoo et al. 2018; Naranjo-Ortiz and Gabaldón 2019; 2 School of Science, Mae Fah Luang University, Chiang Rai 57100, Samarakoon et al. 2019). Natural taxonomic placements of Thailand taxa and development of backbone trees based on molecular 3 Innovative Institute for Plant Health, Zhongkai University of phylogeny have been possible (Ebersberger et al. 2012;He Agriculture and Engineering, Guangzhou 510225, Guangdong et al. 2019; Luo et al. 2019; Hongsanan et al. 2020;Hydeetal. ’ Province, People s Republic of China 2020a; Wijayawardene et al. 2020). The knowledge of fungal 4 Department of Botany and Microbiology, College of Science, King taxonomy and classification are however limited as only ap- Saud University, P. O Box 2455, Riyadh 11451, Kingdom of Saudi proximately 8% (120,000 described taxa) of the 2.2 to 3.8 Arabia million estimated global fungal species are described 5 CAS Key Laboratory for Plant Diversity and Biogeography of East (Hawksworth and Lücking 2017) and even fewer have been Asia, Kunming Institute of Botany, Chinese Academy of Science, Kunming 650201, Yunnan, People’s Republic of China sequenced. Several authors have suggested areas as to where to find the “missing fungi,” such as understudied countries 6 Honghe Innovation Center for Mountain Futures, Kunming Institute of Botany, Honghe County 654400, Yunnan, People’sRepublicof and hosts, particularly in tropical regions and biodiversity China hot spots, little explored habitats or niches, and cryptic species 1032 Mycol Progress (2020) 19:1031–1042 hidden under well-established names [see Hyde 2001;Jones samples were incubated for 5 days and observed using stereo- 2011; Hawksworth and Lücking 2017;Hydeetal.2018]. microscope for the presence of fruiting bodies. Macro- The taxonomy and classification of Dothideomycetes have morphological features were photographed using a Motic been comprehensively studied, and considerable changes have SMZ 168 Series dissection microscope for fungal structures occurred over the past decade (Schoch et al. 2009; Hyde et al. on the woody substrate, while microscopic characters were 2013; Hongsanan et al. 2020). Hyde et al. (2013) listed 105 documented using Nikon Eclipse 80i microscope. Single families under Dothideomycetes. Several published works spore isolation was used to obtain pure cultures and colonial have also focused on different ecological groups of characteristics described. Herbarium-type specimen was de- Dothideomycetes that include terrestrial (Ariyawansa et al. posited in Mae Fah Luang University (MFLU). Ex-type living 2013, 2014; Dai et al. 2014; Thambugala et al. 2014a, b; culture was deposited at Mae Fah Luang University Culture Jayasiri et al. 2018; Pem et al. 2019), rock-dwelling (Ruibal Collection (MFLUCC). The new species was registered in et al. 2009), freshwater (Shearer et al. 2009; Shearer et al. 2014; Faces of Fungi (http://www.facesoffungi.org; Jayasiri et al. Dong et al. 2020), and marine habitats (Suetrong et al. 2009; 2015) and MycoBank database (http://www.mycobank.org/). Jones et al. 2012). Divergence time estimates using molecular data and fossil records have helped to confirm the establish- ment of higher-level taxa, such as families, orders, and classes. DNA extraction, PCR amplification, and sequencing Pleosporales is the largest order of Dothideomycetes (Kirk et al. 2008; Zhang et al. 2009, 2012; Hyde et al. 2013; Fungal mycelia from pure cultures grown in malt extract agar Hongsanan et al. 2020) and comprises ascomycetes with (MEA) for 30 days were scraped off using sterilized scalpel bitunicate asci and ceolomycetous or hyphomycetous asexual and kept in a 1.5 ml microcentrifuge tube. Genomic DNA was morphs (Wijayawardene et al. 2016;Jayasirietal.2018;Pem extracted using the Biospin Fungus Genomic DNA Extraction et al. 2019;Lietal.2020). Twenty-eight families and 175 kit (BioFlux®, China) following the manufacturer’sprotocol. genera (with 12 genera listed as genera incertae sedis)were Polymerase chain reaction (PCR) was used to amplify five included in the Pleosporales by Lumbsch and Huhndorf genes: the large subunit (28SrDNA), small subunit (2010). Wijayawardene et al. (2018) updated the outline of (18SrDNA), internal transcribed spacers (ITS1-5.8S-ITS2), Lumbsch and Huhndorf and included 75 families and 52 gen- translation elongation factor 1-alpha (TEF1-α), and DNA- era in Pleosporales genera incertae sedis.IntheOutline of dependent RNA polymerase II second largest subunit Fungi and fungus-like taxa, Wijayawardene et al. (2020)in- (RPB2). The 28SrDNA was amplified using the primers cluded 87 families and 533 genera, with 49 genera listed as LROR and LR5 (Vilgalys and Hester 1990), while genera incertae sedis in Pleosporales. 18SrDNA was amplified using the primers NS1 and NS4 The asexual genus Mycoenterolobium was introduced by (White et al. 1990). For ITS, primers ITS5 and ITS4 were Goos (1970)withMycoenterolobium platysporum as the type used (White et al. 1990). The TEF1-α was amplified using species growing on decaying wood of Araucaria from primers EF1–983F and EF1–2218R (Rehner and Buckley Hawaii. Subsequently, Karandikar et al. (2015) introduced 2005), while RPB2 was amplified using primers fRPB2-5F/ Mycoenterolobium flabelliforme from dead bark of Tectona fRPB2-7cR (Liu et al. 1999). Polymerase chain reaction was grandis collected from Toranmal region of Maharashtra State, performed in a volume of 25 μl, which contained 12.5 μlof India. There is no sequence data for these two species; there- 2× Power Taq PCR MasterMix (Bioteke Co., China), 1 μlof fore, taxonomic placement of the genus was not possible until each primer (10 μM), 1 μl genomic DNA, and 9.5 μldeion- the current study. ized water. The PCR thermal cycle programs for LSU, SSU, In our survey of freshwater fungi in northern Thailand and TEF1-α amplification were as follows: initial denaturing (Hyde et al. 2016), we observed and isolated a saprobic hy- step of 94 °C for 3 min, followed by 40 cycles of denaturation phomycete from submerged wood. Phylogenetic analysis of at 94 °C for 45 s, annealing at 56 °C for 50 s, elongation at combined LSU, SSU, ITS, TEF1-α,andRPB2 shows that the 72 °C for 1 min, and final extension at 72 °C for 10 min. The genus Mycoenterolobium groups close to Testudinaceae. PCR thermal cycle program for the RPB2 was initial denatur- ingstepof95°Cfor5min,followedby40cyclesofdena- turation at 95 °C for 1 min, annealing at 52 °C for 2 min, Materials and methods elongation at 72 °C for 90 s, and final extension at 72 °C for 10 min. Agarose gel electrophoresis was done to confirm the Sample collection, morphological observation, and presence of amplicons at the expected molecular weight. PCR fungal isolation products were purified and sequenced with the primers men- tioned above at a commercial sequencing provider (BGI, Samples of submerged decayed wood were collected from a Shenzhen, China). A BLAST search of the newly generated freshwater stream in Phitsanulok Province, Thailand. The sequences was carried out to exclude contamination and to Mycol Progress (2020) 19:1031–1042 1033 search for related taxa in GenBank database

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