Introducing Melanoctona Tectonae Gen. Et Sp. Nov. and Minimelanolocus Yunnanensis Sp. Nov. (Herpotrichiellaceae, Chaetothyriales
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Cryptogamie, Mycologie, 2016, 37 (4): 477-492 © 2016 Adac. Tous droits réservés Introducing Melanoctona tectonae gen. et sp. nov. and Minimelanolocus yunnanensis sp. nov. (Herpotrichiellaceae,chaetothyriales) Qing TIAN a,b,c,d,Mingkhuan DOILOM c, d,Zong-Long LUO c,d,e, Putarak CHOMNUNTI c,d,Jayarama D. BHAT f, Jian-Chu XU a,b &Kevin D. HYDE a, b, c, d* aKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany,Chinese Academy of Sciences, Kunming 650201, Yunnan, People’sRepublic of China bWorld Agroforestry Centre, East and Central Asia, Kunming 650201, Yunnan, People’sRepublic of China cCenter of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100, Thailand dSchool of Science, Mae Fah Luang University,Chiang Rai, 57100, Thailand eCollege of Basic Medicine, Dali University,Dali, Yunnan 671000, China fFormerly at Department of Botany,Goa University,Goa 403 206, India Abstract – Herpotrichiellaceae is an interesting, but confused family in Chaetothyriales; the latter has been considered to represent anatural and well-defined group. Anew genus Melanoctona was collected on decaying wood of Tectona grandis in Chiang Rai Province, Thailand and is introducedinHerpotrichiellaceae.Ithas aunique combination of morphological and phylogenetic characters. Phylogenetic analyses of combined ITS, LSU and SSU sequence data place Melanoctona in adistinct lineage in Herpotrichiellaceae. Melanoctona is distinguished from other genera in Herpotrichiellaceae by its hyphomycetous formation and muriform, ellipsoidal to ovoid, brown to dark brown conidia. Minimelanolocus yunnanensis sp. nov.isalso introduced. This species inhabits decaying wood in freshwater streams and rivers in Yunnan Province, China. Maximum likelihood, maximum parsimony and Bayesian analyses of combined ITS, LSU and SSU sequence data, as well as adistinct morphology provide evidence for this new species. Comprehensive descriptions and illustrations of Melanoctona tectonae gen. et sp. nov.and Minimelanolocus yunnanensis sp. nov.are provided with notes on their taxonomy and phylogeny. Minimelanolocus yunnanensis is compared with morphologically similar taxa. Black moulds /Eurotiomycetes /Fresh water /Phylogeny /Taxonomy *Corresponding author: Kevin D. Hyde, email address: [email protected] doi/10.7872/crym/v37.iss4.2016.477 478 Q. Tian et al. IntroductIon The class Eurotiomycetes is amonophyletic group comprising three major subclasses, Eurotiomycetidae Geiser &Lutzoni, Chaetothyriomycetidae Doweld, and Mycocaliciomycetidae Tibell. Chaetothyriomycetidae accommodates the order Chaetothyriales, which produce ostiolateperithecial ascomata with bitunicate asci, similar to those produced in Dothideomycetes (Geiser et al.,2006). The order Chaetothyriales, presently comprises six families, viz. Chaeto- thyriaceae, Cyphellophoraceae, Epibryaceae, Herpotrichiellaceae, Strelitzianaceae and Trichomeriaceae (Geiser et al.,2006, Kirk et al.,2008, Chomnunti et al.,2012, 2014; Réblová et al.,2013; Feng et al.,2014b; Gueidan et al.,2014; Crous et al., 2015). Most taxa in these families are saprobes, such as species in Herportrichiellaceae which grow on decaying wood and mushrooms(Barr,1987; Untereiner &Naveau, 1999; Untereiner,2000). The asexual morphs of several Herpotrichiellaceae species are black yeasts and are animal or human pathogens or occur on rocks (de Hoog et al., 2000; Prenafeta-Boldú et al.,2006; Réblová et al.,2013; Feng et al.,2014a; Vicente et al.,2014; Zeng et al.,2014; Isola et al.,2016). Genera of Herpotrichiellaceae have been related to species of Coenosphaeria and Trichometasphaeria (Munk, 1953) and latertreated as members of Pleosporales and Dothideales(Müller &von Arx, 1962; Bigelow &Barr,1963; von Arx &Müller, 1975). Although the family Herpotrichiellaceae has been considered to represent a natural and well-definedfamily,the morphological characters used in the delimitation of its genera have been confused (Munk, 1957; Müller &von Arx, 1962; Samuels &Müller,1978). The genus Minimelanolocus was revisited by Liu et al. (2015) who introduced four new species and provided new sequence data and abackbone tree for the order Chaetothyriales and showed the genus is adistinct lineage in Chaetothyriales. In this study,weintroduce Melanoctona tectonae gen. et sp. nov.and Minimelanolocus yunnanensis sp. nov.based on the morphological characteristics and phylogenetic analyses. Descriptions and illustrations are provided for both taxa. MAterIAlS And MethodS Fungal isolation and specimen examination. – Decaying wood was collected from aquatic habitats in Dali, Yunnan Province, China and decaying teak was collected from terrestrial habitats in Chiang Rai Province, Thailand and returned to the laboratory in Zip lock plastic bags. The specimens were incubatedinamoist chamber for 1-2 days at 25°Cand examined at regular intervals for sporulating resident fungi. The fungi were examined using an Olympus SZH10 stereo microscope, and mounted in water.Micromorphological characters were determined with a Nikon ECLIPSE 80i compound microscope and images were captured with aCanon EOS 600D digital camera. Differential interference contrast microscopy was used to visualize hyaline structures. Measurementswere made with Tarosoft (R) Image FrameWork version 0.9.7. Photographic plates were prepared in Adobe Photoshop version CS6 (Adobe Systems, The United States). After morphological examination, single spore isolations were made following the method described by Chomnunti Melanoctona tectonae gen. et sp. nov.and Minimelanolocus yunnanensis sp. nov.479 et al.(2014). Germinating spores were transferred aseptically to malt extractagar (MEA) or potato dextrose agar (PDA) platesand incubated at 25°Cinnormal daylight. Colony colour and culture characters were observed and recorded after one week and again after three weeks. Specimens are deposited in the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), Kunming, China and Mae Fah Luang University (MFLU), Chiang Rai, Thailand. Living cultures are deposited in Mae Fah Luang University Culture Collection (MFLUCC), and duplicated in Kunming Institute of Botany,Chinese Academy of Sciences (KUMCC), Kunming, China. Taxa were registered in Index Fungorum (2016) and Facesoffungi (Jayasiri et al.,2015). Fungal DNA extraction, PCR amplification, sequencing and sequence alignment. – Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux®, P.R. China) was used to extract DNA from fresh mycelium grown on MEA at 25°C for four weeks using the instructions of the manufacturer (Hangzhou, P.R. China). The primers ITS5 and ITS4, NS1 and NS4 (White et al.,1990) and LROR (Rehner &Samuels, 1994) and LR5 (Vilgalys &Hester,1990) were used for the amplification of ITS, SSU and LSU respectively.Polymerase chain reaction (PCR) amplification was carried out following the method of Tian et al. (2015). PCR amplification was confirmed on 1% agarose electrophoresis gels stained with ethidium bromide. The amplified PCR fragments were sequenced by BGI, Ltd Shenzhen, P.R. China). Sequence data are deposited in GenBank (Table 1). The alignment and tree were deposited in TreeBASE under accession number 19479. Phylogenetic analyses. – The closest taxa to our strains were determined with standard nucleotide blast searches in NCBI database (http://www.ncbi.nlm.nih. gov/), and sequences of representative species were selected as in Liu et al. (2015). Sequence data from 35 taxa of Herpotrichiellaceae were selected from Crous et al. (2007), Badali et al. (2008) and Liu et al. (2015) (Table 1). Multiple alignments were made by MAFFTv.7.036 (Katoh &Standley, 2013), and adjusted manually using BioEdit v. 7.2 (Hall, 1999) and ClustalX v. 1.83 (Thompson et al.,1997). The tree was rooted using sequence data from Cyphellophora laciniata (CBS 190.61). MODELTEST v. 2.0 (Nylander,2004), following Akaike Information Criterion, was used to determine the best-fitmodel of evolution for each data set for Maximum Likelihood analyses and MrModel test for Bayesian analyses. Maximum-likelihood (ML) analysis was performed in RAxML (Stamatakis, 2008) implemented in raxmlGUI v.0.9b2 (Silvestro &Michalak, 2012). One thousand non-parametric bootstrap iterations were employed with the available models of generalized time reversible (GTR +Gsubstitution model.) and adiscrete gamma distribution (Stamatakis et al.,2008; Liu et al.,2011). The number of replicates was automatically inferred using the stopping criterion (Pattengale et al.,2009). Maximum likelihood bootstrap values equal to or greater than 50% are given as the first set of numbers above the nodes (Fig. 1). Maximum parsimony (MP) analysis was conducted with PAUP v. 4.0b10 (Swofford, 2002) using the heuristic search option with 1,000 random taxa addition and tree bisection reconnection (TBR) as branch-swapping algorithm. Maxtrees were setup unlimited and azero of maximum branch length collapsed, all multiple, equally parsimonious trees were saved. Gaps were treated as missing data. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated. The robustness of the best parsimonious tree was evaluated by abootstrap (BT) value using 1,000 replications 480 Table 1. GenBank and culture collection accession numbers of species treated in the phylogenetic study.Newly generated sequences are shown in bold Culturecollection/ GenBank accession numbers3 Taxon Type2 Source Locality Isolate1 ITS LSU SSU Cladophialophora