MycoKeys 7 | : | 0 II | 8 (2020) A peer-reviewed open-access journal doi: 10.3897/mycokeys.7 1.55493 RESEARCH ARTICLE fe Mycokeys research http://mycokeys.pensoft.net Launched to accelerate biodiversity Multi-gene phylogenetic evidence suggests Dictyoarthrinium belongs in Didymosphaeriaceae (Pleosporales, Dothideomycetes) and Dictyoarthrinium musae sp. nov. on Musa from Thailand Binu C. Samarakoon'*?, Dhanushka N. Wanasinghe***, Milan C. Samarakoon'”, Rungtiwa Phookamsak'**®?, Eric H. C. McKenzie’, Putarak Chomnunti??, Kevin D. Hyde’, Saisamorn Lumyong!*'®, Samantha C. Karunarathna!*>*? | Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand 2. Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand 3 School of Scien- ce, Mae Fah Luang University, Chiang Rai 57100, Thailand 4 CAS Key Laboratory for Plant Biodiversity and Biogeography of East Asia (KLPB), Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, China 5 World Agro Forestry Centre, East and Central Asia, 132 Lanhei Road, Kunming 650201, Yunnan, China 6 Centre for Mountain Futures (CMF), Kunming Institute of Botany, Kunming 650201, Yunnan, China 7 Innovative Institute of Plant Health, Zhongkai University of Agriculture and En- gineering, Guangdong Province, Peoples Republic of China 8 Manaaki Whenua-Landcare Research, Private Bag 92170, Auckland, New Zealand 9 Research Center of Microbial Diversity and Sustainable Utilization, Faculty of Sciences, Chiang Mai University, Chiang Mai 50200, Thailand \0 Academy of Science, The Royal Society of Thailand, Bangkok 10300, Thailand Corresponding authors: S. C. Karunarathna (samanthakarunarathna@gmail.com); S. Lumyong (scboi009@gmail.com) Academic editor: Huzefa Raja | Received 16 June 2020 | Accepted 12 July 2020 | Published 5 August 2020 Citation: Samarakoon BC, Wanasinghe DN, Samarakoon MC, Phookamsak R, McKenzie EHC, Chomnunti P, Hyde KD, Lumyong S, Karunarathna SC (2020) Multi-gene phylogenetic evidence suggests Dictyoarthrinium belongs in Didymosphaeriaceae (Pleosporales, Dothideomycetes) and Dictyoarthrinium musae sp. nov. on Musa from Thailand. MycoKeys 71: 101-118. https://doi.org/10.3897/mycokeys.7 1.55493 Abstract Dead leaves of Musa sp. (banana) were collected in northern Thailand during an investigation of saprobic fungi. Preliminary morphological observations revealed that three specimens belong to Dictyoarthrin- ium. Phylogenetic analyses of combined SSU, LSU, ITS and tefl-« sequence data revealed that Dicty- oarthrinium forms a clade in Didymosphaeriaceae (Massarineae, Pleosporales, Dothideomycetes) sister to Spegazzinia. Based on contrasting morphological features with the extant taxa of Dictyoarthrinium, coupled with the multigene analyses, Dictyoarthrinium musae sp. nov. is introduced herein. Our study Copyright Binu C. Samarakoon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 102 Binu C. Samarakoon et al. | MycoKeys 71: 101-118 (2020) provides the first detailed molecular investigation for Dictyoarthrinium and supports its placement in Didymosphaeriaceae (Massarineae, Pleosporales, Dothideomycetes). Previously, Dictyoarthrinium was classified in Apiosporaceae (Xylariales, Sordariomycetes). Keywords Banana, Dictyoarthrinium sacchari, DNA sequences, Musaceae, one new species, saprobes, taxonomy Introduction Hughes (1953) documented seven hyphomycete genera (Arthrinium, Catenospegazzin- ia, Cordella, Dictyoarthrinium, Endocalyx, Pteroconium and Spegazzinia) that had unique basauxic conidiogenous cell development. Hyde et al. (1998) accommodated Dictyoarthrinium, Endocalyx, Scyphospora (= Arthrinium) and Spegazzinia in Apio- sporaceae (Xylariales, Sordariomycetes), based on morphological characteristics. Based on molecular phylogenetic data (LSU and ITS), Cordella and Pteroconium were syn- onymised under Arthrinium by Crous and Groenewald (2013) and Arthrinium was confirmed as the asexual morph of Apiospora. With the availability of molecular data (SSU, LSU, ITS and tefl-«), Tanaka et al. (2015) transferred Spegazzinia to Didymos- phaeriaceae. Wijayawardene et al. (2018) and Hyde et al. (2020) accommodated Ar- thrinium, Dictyoarthrinium and Endocalyx, all with basauxic conidiogenous cell devel- opment, in Apiosporaceae. Dictyoarthrinium was introduced by Hughes (1952) with D. quadratum as the type species. Dictyoarthrinium africanum was simultaneously introduced. Damon (1953) re- examined the type material, descriptions and illustrations of Téetracoccosporium sacchari (Johnston and Stevenson 1917) and mentioned that 7’ sacchari was congeneric with Dic- tyoarthrinium quadratum. ‘Therefore, Damon (1953) combined 7’ sacchari as Dictyoar- thrinium sacchari. Damon (1953) also named D. guadratum as the heterotypic synonym of D. sacchari. Rao and Rao (1964) introduced D. Lilliputeum and D. microsporum, while Kobayasi et al. (1971) introduced D. rabaulense as novel taxa to the genus. Somrithipol (2007) introduced D. synnematicum and currently seven epithets of Dictyoarthrinium are listed in Index Fungorum (2020). All Dictyoarthrinium species were introduced, based only on morphological data. Vu et al. (2019) sequenced D. sacchari (CBS 529.73) and submitted LSU data to GenBank as the only valid molecular record for the genus. Dictyoarthrinium is characterised by basauxic conidiogenous cell development (Hughes 1952; Damon 1953; Matsushima 1971). Basauxic development is demon- strated by conidiogenous cells in which elongation occurs at a basal growing point after formation of a single, terminal blastic conidium at its apex (Cole 1976). Con- idiophores of Dictyoarthrinium are minutely verruculose, subhyaline and transversely septate (Ellis 1971). Usually, the septa are dark brown and appear as thick stripes on the conidiophore. Conidiophore mother cells are often hyaline or pale brown and cup-shaped (Hughes 1952) or subspherical (Ellis 1971). The length of conidiophores Dictyoarthrinium belongs in Didymosphaeriaceae (Pleosporales, Dothideomycetes) 103 varies within the genus, but in some species, the dimensions are more or less similar. Conidia of Dictyoarthrinium arise from the conidiophore at terminal or lateral parts. Conidiogenesis is monoblastic or polyblastic and integrated (Ellis 1971). Conidia are simple, solitary, dematiaceous and often four-celled. Some taxa (e.g. D. africanum) have 16-celled conidia (Hughes 1952). The surface of conidia is verruculose and most species have warts on the surface. However, the conidia of D. rabaulense are densely echinulate with long spines (Kobayasi 1971). The conidia vary in shape from square to spherical, subspherical or oblong. Most conidia appear flattened on one side. As a spe- cific feature, only D. synnematicum possesses synnemata with filaments (Somrithipol 2007). Stroma, setae and hyphopodia have not been observed in Dictyoarthrinium. Many Dictyoarthrinium species are saprobes that colonise dead plant materials, although D. rabaulense was recorded even from soil and air (Kobayasi et al. 1971; Ellis 1976). Most Dictyoarthrinium species occur on monocotyledonous plants. The genus is widely distributed across the tropics, mainly in terrestrial environments (Ellis 1971; 1976). The sexual morph of Dictyoarthrinium is unknown. Hosts, substrates and geo- graphical distributions of extant Dictyoarthrinium species are listed in Table 1. A study was undertaken to determine the saprobic fungi associated with Musa sp. (banana) in Thailand, during the dry season. Three hyphomycetous taxa that morpho- Table |. Hosts, substrates and geographical distribution of Dictyoarthrinium species. Species Hosts/substrates Geographical References distribution Dictyoarthrinium Miscanthus, Panicum, Paspalum Argentina, Ghana, | Hughes (1952); Ellis (1971); McKenzie africanum S. virgatum, Saccharum, leaf litter of Typha | Solomon Islands, and Jackson (1986); Urtiaga (1986); Hughes latifolia Venezuela Tarda et al. (2019) D. lilliputeum Leaf litter of Bambusa India Rao and Rao (1964); Sushma et al. P. Rag. Rao and D. Rao D. microsporum Dead leaves of Borassus flabellifer India Rao and Rao (1964) P. Rag. Rao and D. Rao (2020) D. rabaulense Brassica campestris, Dendrocalamus Bismarck Kobayasi et al. (1971); Ellis (1976); Matsush. strictus, Gossypium, Xylia xylocarpa, ait | Archipelago, Britain, Bhat (2010) and soil Congo, India, New Caledonia, Nigeria, Tanzania. D. sacchari (J.A. Dead stems and leaves of Ananas, Brazil, Cuba, Hughes (1952); Subramanian (1952); Stev.) Damon = Bambusa, Borassus, Cassia, Cosmos Federated Ghana, Nair and Tyagi (1961); Srivastava D. quadratum S. bipinnatus, Cymbopogon, Delonix India, Malaysia, et al. (1964); Dennis (1970); Ellis Hughes elata, Dracaena, Erythrina, Lithachne Pakistan, Puerto (1971); Matsushima (1971); Stevenson pauciflora, Musa acuminata, Rico, Solomon (1975); Srivastava and Gupta (1981); M. paradisiaca, Neolitsea scrobiculata, Islands, Spain, Arnold (1986); McKenzie and Jackson Pandanus, Persea mechrantha, Phragmites, | States of Micronesia, | (1986); Paul and Singh (1986); Gene Prunus amygdalus, Saccharum sp., Thailand, Venezuela, | et al. (1990); McKenzie and Jackson S. officinarum, S. spontanium, Zinnia, Zambia (1990); Ahmad et al. (1997); Pande and leaf litter of Typha latifolia, decaying Rao (1998); Lumyong et al. (2003); plant materials of dicots Saravanan and Vittal (2007); Leao- Ferreira et al. (2010); Tarda et al. (2019) D. synnematicum Decaying leaves of Musa sp. India, Thailand Somrithipol (2007) Somrith. 104 Binu C. Samarakoon et al. | MycoKeys 71: 101-118 (2020) logically resembled Dictyoarthrinium were examined. According to our phylogenetic analyses of combined SSU, LSU, ITS and tefl-« sequence data, Dictyoarthrinium clus- tered in Didymosphaeriaceae (Pleosporales, Dothideomycetes) with strong statistical support, sister to Spegazzinia. Hence, we propose to transfer Dictyoarthrinium from Apiosporaceae (Xylariales, Sordariomycetes) to Didymosphaeriaceae (Pleosporales, Dothideomycetes) and introduce Dictyoarthrinium musae sp. nov. as a saprobe recorded from Musa sp. We also provide detailed morphological illustrations, descriptions and DNA sequence data for D. sacchari, recorded on Musa sp. from Thailand, which further validates the novel taxonomic placement of Dictyoarthrinium in Didymosphaeriaceae. Materials and methods Sample collection, morphological studies and isolation Dead leaves of Musa sp. were collected from Thailand during the dry season (Decem- ber to August) of 2018 and 2019. Specimens were transferred to the laboratory in cardboard boxes. Samples were examined with a Motic SMZ 168 Series microscope. Powder-like masses of fungal conidia were mounted in water for microscopic studies and photomicrography. The specimens were examined using a Nikon ECLIPSE 80i compound microscope and photographed with a Canon 550D digital camera fitted to the microscope. Measurements were made with the Tarosoft (R) Image Frame Work programme and images used for figures were processed with Adobe Photoshop CS3 Extended v. 10.0 software (Adobe Systems, USA). Single spore isolation was carried out following the method described in Chomnun- ti et al. (2014). Germinated spores were individually transferred to potato dextrose agar (PDA) plates and incubated at 25 °C in daylight. Colony characteristics were observed and measured after 3 weeks at 25 °C. Herbarium specimens were deposited in the Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand. Living cultures were deposited in the Culture Collection of Mae Fah Luang University (MFLUCC). Faces of fungi numbers (Jayasiri et al. 2015) and MycoBank numbers (http://www.MycoBank. org) were obtained for the respective taxa. DNA extraction, PCR amplification and sequencing Fungal isolates grown on potato dextrose agar (PDA) for 4 weeks at 25 °C were used to extract total genomic DNA. DNA was extracted from 50 to 100 mg of axenic myce- lium of the 4-weeks-old growing cultures. The mycelium was ground to a fine powder in liquid nitrogen and fungal DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit-BSC14S1 (BioFlux, PR. China) according to the manufacturer’s instructions. Four gene regions, the internal transcribed spacer (ITS), partial 18S small sub unit (SSU), partial 28S large sub unit (LSU) and partial translation elongation fac- Dictyoarthrinium belongs in Didymosphaeriaceae (Pleosporales, Dothideomycetes) 105 tor l-alpha gene (efl-«) were amplified using ITS5/ITS4 (White et al. 1990), NS1/ NS4 (White et al. 1990), LROR/LRS5 (Vilgalys and Hester 1990) and EF1-983F /EF1- 2218R (Rehner 2001) primers, respectively. Polymerase chain reactions (PCR) were conducted according to the following pro- tocol. The total volume of the PCR reaction was 25 ul and consisted of 12.5 pl of 2 x Power Taq PCR MasterMix (a premix and ready to use solution, including 0.1 Units/ ulTaq DNA Polymerase, 500 um dNTP Mixture each (dATP, dCTP dGTP, dTTP), 20 mM Tris-HCl pH 8.3, 100 mMKCI, 3 mM MgCl, stabiliser and enhancer), 1 ul of each primer (10 pM), 2 pl genomic DNA extract and 8.5 pl double distilled water (ddH,O). ‘The reaction was conducted by running for 40 cycles. The annealing temperature was 56 °C for ITS and LSU, 57.2 °C for tefl-« and 55 °C for SSU and initially 95 °C for 3 min, denaturation at 95 °C for 30 seconds, annealing for 1 min, elongation at 72 °C for 30 seconds and final extension at 72 °C for 10 min for all gene regions. PCR amplification was confirmed on 1% agarose electrophoresis gels stained with ethidium bromide. The amplified PCR fragments were sent to a commercial se- quencing provider (TsingKe Biological Technology Co., Beijing, China). The nucleo- tide sequence data acquired were deposited in GenBank. Sequence alignment Sequences obtained in this study were subjected to BLAST search in GenBank (htt- ps://blast.ncbi.nlm.nih.gov/Blast.cgi). BLAST search results and initial morphological studies supported that our isolates belong to Didymosphaeriaceae. Other sequences used in the analyses were obtained from GenBank based on recently published papers (Tanaka et al. 2015; Jayasiri et al. 2019) (Table 2) and BLAST search results. The sin- gle gene alignments were done by MAFFT v. 7.036 (http://mafft.cbrc.jp/alignment/ server/large.html; Katoh et al. 2019) using the default settings and later refined, where necessary, using BioEdit v. 7.0.5.2 (Hall 1999). Table 2. Selected taxa with their corresponding GenBank accession numbers in the family Didymospha- eriaceae that are used in the phylogenetic analyses. Type strains are indicated as superscript T and newly- generated strains are indicated in bold. Taxa Culture collection ITS LSU SSU tefl-o Alloconiothyrium aptrootii CBS 980.95" JX496121 JX496234 NA NA A. aptrootii CBS 981.95" JX496122 JX496235 NA NA Austropleospora archidendri CBS 168.777 JX496049 JX496162 NA NA A. keteleeriae MFLUCC 18-1551" NR_163349 MK348021 MK347910 =MK360045 Bambusistroma didymosporum MFLU 15-0057 KP761733 KP761730 KP761737 KP761727 B. didymosporum MELU 15-0058 KP761734 KP761731 KP761738 KP761728 Bimuria novae zelandiae GBshoreng: MH861181 AY016356 AY016338 DQ47 1087 Chromolaenicola lampangensis MFLUCC 17-1462" MN325016 MN325004 MN325010 MN335649 C. thailandensis MFLUCC 17-1510" _MN325018 MN325006 MN325012 MN335651 Cylindroaseptospora leucaenae MFLUCC 17-2424" NR_163333 NG_066310 MK347856 MK360047 106 Taxa Deniquelata barringtoniae D. vittalii Dictyoarthrinium musae D. musae D. sacchari D. sacchari Didymocrea sadasivanii Didymosphaeria rubi-ulmifolii D. rubi-ulmifolii Kalmusia italica K. variisporum Kalmusibambusa triseptata Karstenula rhodostoma K. rhodostoma Laburnicola hawksworthii L. muriformis Letendraea cordylinicola L. cordylinicola Montagnula bellevaliae M. cirsii M. scabiosae Neokalmusia brevispora N. scabrispora Neptunomyces aureus NN. aureus Paracamarosporium fagi P fagi Paraconiothyrium cyclothyrioides Paramassariosphaeria anthostomoides P. anthostomoides Paraphaeosphaeria rosae P rosicola Phaeodothis winteri Pseudocamarosporium propinquum P pteleae Pseudopithomyces entadae P. rosae Spegazzinia bromeliacearum S. deightonii S. intermedia S. lobulata S. musae S. neosundara S. radermacherae S. tessarthra Tremateia arundicola TL. guiyangensis LT. murispora Verrucoconiothyrium nitidae Xenocamarosporium acaciae X. acaciae Culture collection MFLUCC 11-0422! NFCCI4249' MFLUCC 20-0105" MFLUCC 20-0106" MFLUCC 20-0107 CBS 529.73 CBS 438.65" MFLUCC 14-0023! MFLUCC 14-0024 MFLUCC 14-0560! CBS 121.517" MFLUCC 13-0232! CBS 690.94 CBS 691.94 MFLUCC 13-0602! MFLUCC 14-0921' MFLUCC 11-0150 MFLUCC 11-0148" MFLUCC 14-0924! MFLUCC 13-0680 MFLUCC 14-0954" KT 1466' KT 1023 CMG12! CMG13 CPC 24890 CPC 24892! GBS972.95" CBS 615.86 MELU 16-0172! MFLUCC 17-2549! MFLUCC 15-0042! CBS 182.58 MFLUCC 13-0544 MFLUCC 17-0724! MFLUCC 17-0917' MFLUCC 15-0035" URM 8084! MFLUCC 20-0002 CBS 249.89! CBS 361.587 MFLUCC 20-0001" MFLUCC 15-0456! MFLUCC 17-2285! SH 287 MELU 16-1275" GZAASO1" GZCC 18-2787" CBS:119209 CBS:139895 MFLUCC 17-2432 ITS NR_111779 MF406218 MT482323 MT482324 MT482325 NA MH858658 NA NA KP325440 NR_145165 KY682697 NA LC014559 KU743194 KU743200 KM213996 NR_154118 KT443906 KX274242 KT443907 LC014573 LC014575 MK912121 MK912122 KR611886 KRG11887 JX496119 MH862005 KU743206 MG828937 NR_157528 NA KJ747049 NR_157536 NA MG828953 MK804501 MN956768 MH862171 MH857812 MN930512 KX965728 MK347740 JQ673429 KX274241 KX274240 NR_165916 EUS552112 NR_137982 MK347766 LSU NG_042696 MF182395 MT482320 MT482321 MT482322 MH872479 DQ384103 KJ436586 KJ436585 KP325441 JX496143 KY682695 GU301821 AB807531 KU743195 KU743201 KM213999 NG_059530 KT443902 KX274249 KT443903 AB524600 AB524593 NA NA KR611904 KR611905 JX496232 GU205223 KU743207 MG829046 MG829047 GU301857 KJ813280 MG829061 NG_066305 MG829064 MK809513 MN956772 MH873861 MH869344 MN930514 KX954397 MK347957 AB807584 KX274248 KX274247 MK972751 NA NG_058163 MK347983 Binu C. Samarakoon et al. | MycoKeys 71: 101-118 (2020) SSU JX254656 MF622059 MT482326 MT482327 MT482328 NA NA NG_063557 KJ436587 KP325442 NA KY682696 GU296154 AB797241 KU743196 KU743202 KM214002 KM214001 KT443904 KX274255 KT443905 AB524459 ABS524452 NA NA NA NA AY642524 GU205246 KU743208 MG829152 MG829153 GU296183 KJ819949 MG829166 MK347835 MG829168 NA MN956770 NA NA MN930513 KX986341 MK347848 AB797294 KX274254 KX274253 MK972750 NA NA MK347873 tefl-o NA MF182398 MT495602 MT495603 NA NA NA NA NA NA NA NA GU349067 AB808506 NA NA NA NA KX949743 KX284707 NA AB539112 AB539106 MK948000 MK948001 NA NA NA NA NA MG829223 NA NA NA MG829233 MK360083 NA NA NA NA NA NA NA MK360088 AB808560 KX284706 KX284705 MK986482 NA NA MK360093 *Abbreviations of culture collections: CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, CPC: Work- ing collection of Pedro Crous housed at CBS, GZAAS: Guizhou Academy of Agricultural Sciences Herbarium, China, KT: K. Tanaka, MFLU: Mae Fah Luang University, Chiang Rai, Thailand, MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand, SH: Academia Sinica People’s Republic of China. Shanghai, URM: Universidade Federal de Pernambuco. Dictyoarthrinium belongs in Didymosphaeriaceae (Pleosporales, Dothideomycetes) 107 Phylogenetic analyses Maximum Likelihood (ML) trees were generated using the RAxML-HPC2 on XSEDE (8.2.8) (Stamatakis et al. 2008; Stamatakis 2014) in the CIPRES Science Gateway platform (Miller et al. 2010) using GTR+1+G model of evolution. Bootstrap supports were obtained by running 1000 pseudo-replicates. Maximum Likelihood bootstrap values (ML) = 60% are given above each node of the phylogenetic tree in blue (Fig. 1). Bayesian analysis was conducted with MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001) to evaluate posterior probabilities (PP) (Rannala and Yang 1996; Zhaxybayeva and Gogarten 2002) by Markov Chain Monte Carlo sampling (BMCMC). Two parallel runs were conducted, using the default settings, but with the following adjustments: four si- multaneous Markov chains were run for 2,000,000 generations, trees were sampled every 100" generation and 20,001 trees were obtained. ‘The first 4,000 trees, representing the burn-in phase of the analyses, were discarded. The remaining 16,001 trees were used for calculating PP in the majority rule consensus tree. Branches with Bayesian posterior prob- abilities (BYPP) = 0.95 are indicated above each node of the phylogenetic tree (Fig. 1). Phylogenetic trees were visualised with the Fig Tree v1.4.0 programme (Rambaut 2011). Results Phylogenetic analyses The combined SSU, LSU, ITS and ¢efl-« matrix comprised 61 sequences that represents the genera in Didymosphaeriaceae. The best scoring RAxML tree is shown (Fig. 1) with a final ML optimisation likelihood value of -19278.64. The matrix had 1091 distinct alignment patterns, with 39.08% of undetermined characters or gaps. Estimated base fre- quencies were: A = 0.234095, C = 0.252628, G = 0.278053, T = 0.235224; substitution rates AC = 1.252730, AG = 2.198875, AT = 1.318760, CG = 0.953798, CT = 5.276095, GT = 1.000000; proportion of invariable sites I = 0.491333; gamma distribution shape parameter « = 0.446418. All trees (ML and BYPP) were similar in topology and did not differ at the generic relationships, which are in agreement with multi-gene phylogeny of Tanaka et al. (2015) and Jayasiri et al. (2019). All Dictyoarthrinium strains analysed herein clustered as a highly-supported monophyletic clade (ML = 100%, BYPP = 1.00) in Didymosphaeriaceae (Fig. 1) sister to Spegazzinia (ML = 75%, BYPP = 0.98). We have included LSU sequence data of D. sacchari (CBS 529.73) of Vu et al. (2019) in our phy- logenetic analyses. According to GenBank, CBS 529.73 was classified in Apiosporaceae (Sordariomycetes). In our analyses, D. sacchari (CBS 529.73) clustered with MFLUCC 20-0105, MFLUCC 20-0106 and MFLUCC 20-0107 strains in Didymosphaeriaceae with a strong statistical support (ML = 100%, BYPP = 1.00). Our strain MFLUCC 20- 0107 grouped with D. sacchari (CBS 529.73). The novel isolates of D. musae (MFLUCC 20-0105 and MFLUCC 20-0106) were sister to D. sacchari (CBS 529.73 and MFLUCC 20-0107) with strong statistical support (ML = 100%, BYPP = 1.00). 108 Binu C. Samarakoon et al. / MycoKeys 71: 101-118 (2020) 100/1.00)""" Tremateia guiyangensis GZAASO1 Tremateia Tremateia murispora GZCC 18-2787 71/0.99| [9941-60 Deniquelata barringtoniae MFLUCC 11-0422 | | Deniquelata vittalii NFcc\4249 Bimuria novae-zelandiae CBs 107.79 Pseudopithomyces rosae MFLUCC 15-0035 | pea ee entadae MFLUCC 17-0917 jLt_/9.95 ! feOKaAIMU / OFeEVISDOrea Neeranrern ee KT 1023 ae aU winteri CBS 182.58 51 0:95 100/1. 00 WaRESREGTE cirsii MFLUCC 13-0680 00/1.00} 'Montagnula scabiosae MFLUCC 14-0954 Montagnula bellevaliae MFLUCC 14-0924 100/1.00 ;Letendraea cordylinicola MFLUCC 11-0148 Letendraea cordylinicola MFLUCC 11-0150 Pseudocamarosporium propinquum MELUCC 13-0544 71/¢ Paracamarosporium fagi CPC 24892 100/0.99| _1|'Paracamarosporium fagi CPC 24890 48|Didymosphaeria rubi-ulmifolii MFLUCC 14-0024 0.99 Paraphaeosphaeria rosae MFLUCC 17-2549 3 Paraphaeosphaeria rosicola MFLUCC 15-0042 ‘ Karstenula rhodostoma CBS 690.94 q/{1).00| Austropleospora keteleeriae MFLUCC 18-1551 Austropleospora archidendri cBs 168.3 77 araco @ Vriu VCIO Vidlellel R 95 -00 Chromolaenicola thailandensis MFLUCC 17-1510 ul Chromolaenicola lampangensis MFLUCC 17-1462 || ‘rucoco OTNYTIU OO CB ZU Neptunomyces aureus CMG13 00/1.00' Xenocamarosporium acaciae CPC 24755 Xenocamarosporium acaciae MFLUCC 17-2432 a. Uy VaOMDUSA eCptata FLU 3-U 85/0.98 | ik 00/1.00 p0/1.00) Alloconiothyrium aptrootii CBS 981.95 0.99 Alloconiothyrium aptrootii CBS 980.95 Kalmustia variisporum CBS 121.517 Laburnicola muriformis MFLUCC 14-0921 Laburnicola hawksworthii MFLUCC 13-0602 ol E | ~ Spegazzinia bromeliacearum URM 8084 85/100 - Spegazzinia deightonii MFLUCC 20-0002 81/0.98 Spegazzinia musae MFLUCC 20-0001 “" Spegazzinia neosundara MFLUCC 15-0456 100/1.00| Dictyoarthrinium sacchari MFLUCC 20-0107 100/1.00 Dictyoarthrinium sacchari CBS 529.73 100/1.00_| Dictyoarthrinium musae MFLUCC 20-0105 Dictyoarthrinium musae MFLUCC 20-0106 100/1.00 | Bambusistroma didymosporum MFLU 15-0058 aan Bambusistroma didymosporum MFLU 15-0057 Figure 1. Maximum Likelihood tree revealed by RAxML from an analysis of SSU, LSU and ITS and tefl-« sequence data of the genera of Didymosphaeriaceae, showing the phylogenetic position of Dictyoarthrinium musae (MFLUCC 20-0105, MFLUCC 20-0106) and D. sacchari (MFLUCC 20-0107). ML bootstrap supports (= 60%) and Bayesian posterior probabilities (= 0.95 BYPP) are given above the branches, respectively. The tree is rooted with Bambusistroma didymosporum (MFLU 15-0057 and MFLU 15-0058). Strains generated in this study are indicated in brown bold type. Ex-type strains are indicated Paramassariosphaeria anthostomoides MFLUCC 16-0172 Cylindroaseptospord leucaenae MFLUCC 17-2424 Deniquelata Bimuria Pseudopithomyces okaimusia Phaeodothis UIGY O OD Montagnula Letendraea Paracamarosporium Viqymospnaeriaa Paraphaeosphaeria Austropleospora araconiothvrium Chromolaenicola UICOCO oe} VTiu Cylindroaseptospora Jentunomvyces Xenocamarosporium 0 USIDQMDU Alloconiothyrium Kal nu Tf Laburnicola Dictyoarthrinium Outgroup in black bold. The scale bar represents the expected number of nucleotide substitutions per site. Dictyoarthrinium belongs in Didymosphaeriaceae (Pleosporales, Dothideomycetes) 109 Taxonomy Dictyoarthrinium musae Samarakoon, Chomnunti & K.D. Hyde, sp. nov. MycoBank No: 835764 Facesoffungi Number: FoF08467 Figure 2 Etymology. Name reflects the host genus, Musa (Musaceae). Holotype. MFLU 20-0437 Description. Saprobic on dead leaves of Musa sp. Sexual morph: Undetermined. Asexual morph: Co/onies compact or effuse, black, often pulvinate. Mycelium super- ficial, a close network of branched and anastomosing hyphae. Stromata none. Setae and hyphopodia absent. Conidiophores 30-140 x 1-2 um (x 81.5 x 1.6 um, n = 25), basauxic, arising usually singly from subspherical, subhyaline to light brown conidio- phore mother cells, 4.5—4.8 x 4.3-4.5 um (x = 4.6 x 4.4 um, n = 10), macronematous, mononematous, straight or flexuous, narrow, cylindrical, rough, subhyaline to pale brown, with thick brown or dark brown transverse septa that appear as stripes with dis- tances of 6.3—5.8 «um at apex and 2.3—3 um at base of the conidiophore. Conidiogenous cells 4.14.5 x 4.3-4.7 um (x = 4.4 x 4.5 um, n = 10), blastic, integrated, terminal and intercalary, cylindrical, smooth, denticles absent, hyaline. Conidia 7-11.5 x 6.5—9 um (x = 8.7 x 7.9 um, n = 40), solitary, dry, acropleurogenous, simple, square, rounded at the corners, 4-celled, spherical or subspherical, often flattened in one plane, pale to dark brown at maturity, verrucose, with light brown to dark brown warts, immature conidia often 1-celled and subhyaline. Terminal conidium with four cells, sometimes absent or fallen before lateral conidia, mature conidia split along one line of the septa, most conidia arranged obliquely downwards on the conidiophore, conidial formation observed as a bunch starting after conidiophore 1—3 septate. Culture characteristics. Conidia germinating on PDA within 18 hrs. Colonies on PDA reaching a diameter of 50 mm after 14 days at 25 °C, slightly raised, hairy, filamen- tous, moderately dense, middle light grey, periphery white; reverse white to greyish-white. Material examined. THAILAND. Chiang Rai. On dead leaves of Musa sp. (Musaceae), 7 December 2018, M. C. Samarakoon, BNS265 (MFLU 20-0437, holotype), ex-type living culture (MFLUCC 20-0105); ibid. 20 February 2019, B. C. Samarakoon BNS2239 (MFLU 20-0438, paratype), ex-paratype living culture (MFLUCC 20-0106). Notes. Based on BLAST search results of SSU, LSU, ITS and tefl-a sequence data, Dictyoarthrinium musae (MFLUCC 20-0105 and MFLUCC 20-0106) showed high similarity as follows: SSU = 99.15% to Paraconiothyrium hawatiense (CBS 120025), LSU = 95.57% to Cylindroaseptospora siamensis (MFLUCC 17-2527), ITS = 98.24% to Kalmusia italica (isolate 5), tefl-x = 97.75% to Spegazzinia neosundara (MFLUCC 13-0211) with 100%, 100%, 87% and 99% query covers, respectively. In the mul- tigene phylogeny, the Dictyoarthrinium clade was sister to Spegazzinia (ML = 75%, BYPP = 0.98). Within the Dictyoarthrinium clade, D. musae (MFLUCC 20-0105 and MFLUCC 20-0106) separated from the sister taxon, D. sacchari with strong statisti- 110 Binu C. Samarakoon et al. | MycoKeys 71: 101-118 (2020) Figure 2. Dictyoarthrinium musae (MFLU 20-0437, holotype) a conidia on the host b conidiophore and conidia with conidiophore mother cell C—f conidia with conidiophores on stalk g developmental stage of an immature lateral conidium h four-celled terminal conidium i conidiophore j conidiophores and conidia with terminal conidium k, I conidiophores without terminal conidium m attachment of a mature lateral conidium n=q warted four-celled mature conidia r § mature conidia that split at septa t colony on PDA after 21 days. Scale bars: 500 um (a); 50 um (b, c); 20 um (d=g, i); 10 um (h); 5 um (js). cal support (ML = 100%, BYPP = 1.00). ITS sequence comparison revealed 7.84% base pair differences between D. musae and D. sacchari (MFLUCC 20-0107), which is in agreement with the new species concept outlined by Jeewon and Hyde (2016). Dictyoarthrinium musae differs from D. sacchari by its unique conidial development in the apex. The terminal conidia of D. musae are always 4-celled and similar in colour Dictyoarthrinium belongs in Didymosphaeriaceae (Pleosporales, Dothideomycetes) 111 to mature lateral conidia. In addition, the terminal conidia of D. musae are sometimes absent or fallen before the lateral conidia. In contrast, the terminal conidia of D. sac- chari can be 2-celled or 4-celled, pale brown with respect to lateral mature conidia and always persist on the conidiophore. In addition, the mature conidia of D. musae split along one line of the septa and this specific feature is absent in D. sacchari. Dictyoar- thrinium musae has a subhyaline, spherical conidiophore mother cell while D. sacchari has a distinct cup-shaped, brown conidiophore mother cell. Therefore, based on con- trasting morphological differences to D. sacchari and strong statistical support from our molecular phylogeny, D. musae is herein introduced as a new species. Dictyoarthrinium sacchari (J.A. Stev.) Damon, Bull. Torrey bot. Club 80: 164 (1953) Facesoffungi Number: FoF08468 Figure 3 Description. Saprobic on dead leaves of Musa sp. Sexual morph: Undetermined. Asex- ual morph: Colonies compact or effuse, black, often pulvinate. Mycelium superficial, a close network of branched and anastomosing hyphae. Stromata none. Setae and hyphopo- dia absent. Conidiophores 50-110 x 1-2 um (x = 72.0 x 1.6 um, n = 15), basauxic, arising from cup-shaped, brown, distinct conidiophore mother cells, 3.4-4.4 x 2.9-4.7 um (x = 4 x 3.7 um, n = 10), macronematous, mononematous, usually straight or flexuous, narrow, cylindrical, rough-walled, subhyaline to pale brown, with dark brown transverse septa as stripes with distances of 6.3—5.8 um at apex and 2.3—3 um at base of the con- idiophore. Conidiogenous cells 4—4.5 x 4.3-4.7 um (x = 4.4 x 4.5 um, n = 10), blastic, integrated, terminal and intercalary, cylindrical, smooth, hyaline. Conidia at maturity 8.5-11.5 x 8.5-10 um (x = 9.9 x 9.3 um, n = 40), solitary, dry, acropleurogenous, simple, square, rounded at the corners, 4-celled, but difficult to distinguish the cells due to their blackish-brown nature, spherical or subspherical, often flattened in one plane, blackish-brown at maturity, with brown warts on surface of the cells, terminal conidium always 4-celled or 2-celled, light brown when compared with lateral conidia, most co- nidia arranged perpendicular to the conidiophore, some directed obliquely upwards. Culture characteristics. Conidia germinating on PDA within 18 hrs. Colonies on PDA reaching a diameter of 55 mm after 14 days at 25 °C, raised, moderately dense, entire margined, brownish-grey at maturity; reverse white to greyish-white. Material examined. THatLanp, Chiang Mai. On mid-rib of a dead leaf of Musa sp. (Musaceae), S. Phongeun, 18 July 2018, BNS2287, (MFLU 20-0439), living cul- ture MFLUCC 20-0107. Notes. Based on BLAST search results of SSU, LSU, ITS and tefl-a sequence data, our strain (MFLUCC 20-0107) showed high similarity to the taxa in GenBank as fol- lows (SSU = 99.26% to Paraconiothyrium brasiliense (isolate GF1), LSU = 96.14% to Alloconiothyrium aptrooti (CBS 981.95), ITS = 93.00% to Kalmusia italica (MFLUCC 13-0066). In the multigene phylogeny, MFLUCC 20-0107 groups with Dictyoarthrin- ium sacchari, sister to D. musae with strong statistical support (ML = 100%, BYPP = 112 Binu C. Samarakoon et al. / MycoKeys 71: 101-118 (2020) Figure 3. Dictyoarthrinium sacchari (MFLU 20-0439) a conidia on the host b developmental stage of terminal conidium attached to the conidiophore c=f Conidiophores and conidia (e, with distinct mother cell) g, h mature conidiophores with four-celled terminal conidium i conidiophore with two celled terminal conidium j developmental stages of conidia on conidiophore k colony on PDA after 21 days l=q conidia. Scale bars: a = 1000 pm (a); 20 um (b,j); 50 um (c=i); 5 um (leq). 1.00). Our strain shares similar morphological features with D. sacchari (Subrama- nium 1952; Ellis 1971) and did not differ significantly. There are slight differences in conidial dimensions and the length of conidiophores of our collection and other D. sacchari collections by previous studies. Conidial dimensions and the length of conidi- ophores may differ due to diverse environmental effects and host associations. LSU sequence data of D. sacchari (CBS 529.73) are identical with our strain (MFLUCC 20-0107). Unfortunately, ITS, SSU and tefl-« sequence data of CBS 529.73 are not Dictyoarthrinium belongs in Didymosphaeriaceae (Pleosporales, Dothideomycetes) 113 Figure 4. Morphology of conidia and conidiophores of previously described Dictyoarthrinium species a, d D. microsporum b, i D. synnematicum ¢, e D. lilliputeum f,j D. africanum g, h, k D. rabaulense. Scale bars: 20 um (a, c, d, e); 10 pm (b, i). Magnification x 650 (f, g, h, j, k). Redrawn from Rao and Rao (1964), Ellis (1971), Kobayasi et al. (1971) and Somrithipol (2007). available in GenBank to compare with our strain. LSU data of Dictyoarthrinium musae have 2.24% of base pair difference with D. sacchari (CBS 529.73 and MFLUCC 20- 0107). Dictyoarthrinium sacchari was reported on Musa sp. from Thailand in Lumyong et al. (2003) without morpho-molecular justifications. In this study, we document D. sacchari with detailed morphological illustrations, description, herbarium material and a living culture coupled with DNA sequence data (SSU, LSU, ITS) for a better taxonomic resolution. 114 Binu C. Samarakoon et al. | MycoKeys 71: 101-118 (2020) Discussion Both Dictyoarthrinium and Spegazzinia are characterised by basauxic conidiophores (Hughes 1952; Ellis 1971; Tanaka et al. 2015). Spegazzinia often has stellate («) and disc-shaped (8) conidia (Ellis 1971; Tanaka et al. 2015). The conidia of Dictyoarthrini- um (except D. africanum) share some similar characteristics with disc-shaped, 8 conidia of Spegazzinia. Both conidia are brown, 4-celled and constricted at the septa. Conidia of Dictyoarthrinium have characteristic hyaline or brown warts. Rarely, some taxa of Spegazzinia, for example, S. deightonii, also bear blunt ended spines. Most disc-shaped conidia of Spegazzinia are not warted. In addition, stellate conidia of Spegazzinia are always 4—5-celled and spinulose (Ellis 1971; Tanaka et al. 2015). There are contrast- ing morphological features of the basauxic conidiophores of both genera. The conidi- ophores of Dictyoarthrinium are hyaline to subhyaline with septa that appear as dark brown or light brown stripes throughout the conidiophore. The conidiophores (in stellate conidia) of Spegazzinia are more elongated, narrow, aseptate and dematiaceous. Dictyoarthrinium quadratum (type of Dictyoarthrinium) is the heterotypic syno- nym of D. sacchari. Dictyoarthrinium quadratum has a terminal mature conidium with one to two cells. As described in Hughes (1952), these 2-celled conidia remain on the conidiophore, even when other conidia fall off. This feature is absent in D. musae. The terminal conidium of D. musae always ends up with four cells. The conidia of D. quadratum are obliquely upwardly directed, whereas the conidia of D. musae are obliquely downwardly directed (Fig. 2). The conidiophores of D. guadratum are erect and straight while D. musae has more curved conidiophores. Dictyoarthrinium africanum differs significantly from D. musae by having 16-celled conidia. The conidia of D. rabaulense are completely black and densely echinulate with spines sometimes up to 4 um long (Ellis 1976). However, D. musae has brown warts on the surface of conidia, while D. /illiputeum has hyaline warts. Dictyoarthrinium mi- crosporum has longer conidiophores (250 pm) than D. musae. Morphological features of Dictyoarthrinium species are illustrated in Fig. 4. A key to the species of Dictyoar- thrinium is provided below. Key to the species of Dictyoarthrinium 1 SVMS ATA IPRES CMG. ce esi ue ab anes tee ueerdaestagdtiae oa teevetenee D. synnematicum — Sy Met ataeaseilbte c Aedtns oh ther sttie out edute dla am bidentate tenet cheeses 2 2. Crsndia 2 ort called teem a cbs heat uch a enoslo tab ues vnc olds eav Rich te denial 3 -- Conidia TG celled Ss, .op-tasnc. a tices aps oneceanoest ote vsnnd atte ne eee Ee D. africanum 3 Gonidiatwich- Drow iw akiger ayrs.eeus sa tepere eae nue ose t eter mente seer ui nates aan. é — Wonidia-with: lryal (neswat ts: sccs_wceceecastecauadeb ceteree Miata sons Setteaheds D. lilliputeum 2 Conidiophores Upto *1GO- pi OG cig. ae ed ceatie one. Uecbucseey antevcgaeesedoerasnces paces 5 Conidicphoresup 10°250 uit long. isssvcetises ses oziieestcnden D. microsporum Dictyoarthrinium belongs in Didymosphaeriaceae (Pleosporales, Dothideomycetes) 115 5 Terminal conidium always 4-celled, mature conidia split along one line of PMCS 08 or Maree an Mt eel a Oe eet CAD at Mee ate READ cain ila D. musae = Terminal conidium 2- or 4-celled, mature conidia do not split along septa... BERR rs Seed os Aahih atc sAc cde Me enter aad fos Ms deta Rat Rak ni stah hey iaad D. sacchari To date, the taxonomy and phylogeny of most genera that have basauxic conidio- genesis (Hughes 1952) have been resolved with their correct taxonomic placements. Dictyoarthrinium and Endocalyx represented the sole unresolved genera. We transferred Dictyoarthrinium to Didymosphaeriaceae based on morphological and molecular evi- dence. This study uses multigene sequence data of SSU, LSU, ITS and ¢efl-« for the first time to confirm the taxonomic placement of Dictyoarthrinium in Didymosphaeriaceae. Acknowledgements Samantha C. Karunarathna would like to thank the CAS President's International Fel- lowship Initiative (PIFI) young staff under the grant number: 2020FYC0002 for fund- ing his postdoctoral research and the National Science Foundation of China (NSFC, project code 31851110759) for partially funding this work. Rungtiwa Phookamsak thanks CAS President's International Fellowship Initiative (PIFI) for young staff (grant no. Y9215811Q1), the National Science Foundation of China (NSFC) project code 31850410489 (grant no. Y811982211) and Chiang Mai University for their partial sup- port of this research work. Dhanushka Wanasinghe thanks CAS President's International Fellowship Initiative (PIFI) for funding his postdoctoral research (number 2019PC0008), the National Science Foundation of China and Chinese Academy of Sciences for finan- cial support under the grant 41761144055. K.D Hyde thanks Thailand research grants entitled “The future of specialist fungi in a changing climate: baseline data for generalist and specialist fungi associated with ants, Rhododendron species and Dracaena species (Grant No: DBG6080013) and “Impact of climate change on fungal diversity and bio- geography in the Greater Mekong Sub region (Grant No: RDG6130001). Binu C. Sa- marakoon offers her sincere gratitude to S. Phongeun, G. Samarakoon, Seetha Malani, Thiue Samarakoon and A.J Gajanayake for the valuable support they have given. References Ahmad S, Iqbal SH, Khalid AN (1997) Fungi of Pakistan. Sultan Ahmad Mycological Society of Pakistan, 248 pp. Arnold GRW (1986) Lista de Hongos Fitopatogenos de Cuba. 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