MycoKeys 95: 27-45 (2023) er-reviewed open-access journal doi: 10.3897/mycokeys.95.96400 < MycoKkeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Morphological and phylogenetic analyses reveal two new species and a new record of Apiospora (Amphisphaeriales, Apiosporaceae) in China Rongyu Liu'*, Duhua Li’, Zhaoxue Zhang’, Shubin Liu’, Xinye Liu', Yixin Wang', Heng Zhao’, Xiaoyong Liu’, Xiuguo Zhang!”, Jiwen Xia’, Yujiao Wang' I College of Life Sciences, Shandong Normal University, Jinan, 250358, China 2 Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, College of Plant Protection, Shandong Agricultural University, Taian, 271018, China 3 Institute of Microbiology, School of Ecology and Nature Conservation, Beijing Forestry University, Beijing, 100083, China Corresponding authors: Jiwen Xia (xiajiwen1@126.com); Yujiao Wang (18354285903@163.com) Academic editor: Xinlei Fan | Received 17 October 2022 | Accepted 3 January 2023 | Published 27 January 2023 Citation: Liu R, Li D, Zhang Z, Liu S, Liu X, Wang Y, Zhao H, Liu X, Zhang X, Xia J, Wang Y (2023) Morphological and phylogenetic analyses reveal two new species and a new record of Apiospora (Amphisphaeriales, Apiosporaceae) in China. MycoKeys 95: 27—45. https://doi.org/10.3897/mycokeys.95.96400 Abstract The genus Apiospora includes endophytes, pathogens and saprobes, with a wide host range and geographic distribution. In this paper, six Apiospora strains isolated from diseased and healthy tissues of bamboo leaves from Hainan and Shandong provinces in China were classified using a multi-locus phylogeny based on a combined dataset of ITS, LSU, tef7 and tu62, in conjunction with morphological characters, host associa- tion and ecological distribution. Two new species, Apiospora dongyingensis and A. hainanensis, and a new record of A. pseudosinensis in China, are described based on their distinct phylogenetic relationships and morphological analyses. Illustrations and descriptions of the three taxa are provided, along with compari- sons with closely related taxa in the genus. Keywords Apiospora dongyingensis, Apiospora hainanensis, Ascomycota, bamboo, taxonomy Copyright Rongyu Liu 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. 28 Rongyu Liu et al. / MycoKeys 95: 27—45 (2023) Introduction Apiospora Sacc., the type genus of Apiosporaceae K.D. Hyde, J. Frohl., Joanne E. Taylor & M.E. Barr, was introduced by Saccardo with A. montagnei Sacc. as the type species (Saccardo 1875). The sexual morphs of Apiospora are characterized by multi-locular perithecial stromata with hyaline ascospores surrounded by a thick gelatinous sheath (Dai et al. 2016, 2017; Pintos and Alvarado 2021). The asexual morphs of Apiospora are characterized by their basauxic conidiogenesis, and globose to subglobose conidia, which are usually lenticular or obovoid in the side view, and pale brown to brown in color (Kunze 1817; Hyde et al. 1998; Dai et al. 2016). Most species of Apiospora are quite similar to each other in morphology, thus it is difficult to distinguish them with- out molecular phylogenetic data. Until the studies of Pintos and Alvarado (2021) and Jiang et al. (2022a), the closely related genera Apiospora, Arthrinium Kunze and Neoarthrinium Ning Jiang were considered a single taxon because of their similar morphological character- istics, especially the basauxic conidiogenesis. However, the conidia of Apiospora and Neoarthrinium are more or less rounded in the face view and lenticular in the side view, whereas the conidia of Arthrinium are variously shaped (angular, curved, fusiform, globose, polygonal, navicular). In addition, the conidiophores of several Arthrinium and Neoarthrinium species have thick blackish septa, which are rarely observed in Apiospora (Pintos and Alvarado 2021; Tian et al. 2021; Jiang et al. 2022a). Apiospora species have a worldwide distribution and can be found on various hosts, while Arthrinium species are rarely found in tropical and subtropical habitats and commonly occur on Cyperaceae Juss. and Juncaceae Juss. (Ramos et al. 2010; Dai et al. 2017; Wang et al. 2018; Hyde et al. 2020; Pintos and Alvarado 2021; Tian et al. 2021). Four Neoarthrinium species have been discovered on four hosts from three distantly related host plant families in China, Colombia and Great Britain (Jiang et al. 2022a). Most Apiospora species are associated with plants as endophytes, pathogens or saprobes (Agut and Calvo 2004; Dai et al. 2016, 2017; Tian et al. 2021). Some species are economically important plant pathogens, for example, A. arundinis causes bamboo brown culm streak, chestnut leaf spot and barley kernel blight (Martinez-Cano et al. 1992; Chen et al. 2014; Jiang et al. 2021), while A. sacchari causes damping-off of durum wheat (Mavragani et al. 2007). Some species have also been isolated from lichens, air, soil, seaweeds and animal tissues, and a few species are human pathogens which can cause cutaneous infections (Tian et al. 2021). The aim of this study was to explore the diversity of Apiospora species in symp- tomatic and asymptomatic bamboo leaves collected in Hainan and Shandong prov- inces (China). We describe two newly discovered species, Apiospora dongyingensis and A. hainanensis, and a new record of A. pseudosinensis in China based on phylogenetic data and morphology. Three species of Apiospora in China 29 Materials and methods Isolation and morphological studies The samples were collected at the Diaoluoshan National Nature Reserve, Hainan Prov- ince, and the Dongying Botanical Garden, Shandong Province (China). ‘The strains of Apiospora were isolated from single spores and fungal tissue obtained from diseased and healthy bamboo leaves following the methods described by Chomnunti et al. (2014). Sampled spores were suspended in sterile distilled water, spread onto potato dextrose agar (PDA) plates, and incubated for one day at 25 °C. After germination, the spores were transferred to a new PDA plate to obtain a pure culture. Additionally, about 25 mm‘ tissue fragments were taken from the margin of leaf lesions and their surface sterilized by consecutive immersions in a 75% ethanol solution for 60 s, 5% sodium hypochlorite solution for 30 s, and then rinsed in sterile distilled water for 60 s (Mu et al. 2021). The surface sterilized plant tissue was dried with sterilized paper and moved on the PDA plates. All the PDA plates were incubated at 25 °C for 3-4 days in dark- ness, and then hyphae were picked out of the periphery of the colonies and grown on new PDA plates (Jiang et al. 2022b). After 7 days of incubation, the morphological characters of the colonies were record- ed on PDA with a digital camera (Canon G7X). Morphological descriptions were based on cultures sporulating on water agar (WA). The size of the conidiogenous cells and conidia were shown as minimum-maximum. Color notations were done using the color charts of Rayner (1970). The micro-morphological characters of the colonies were stud- ied using a stereomicroscope (Olympus SZX10) and a microscope (Olympus BX53), both fitted with high-definition color digital cameras. Grown cultures of Apiospora were stored in 10% sterilized glycerin and sterile water at 4 °C for further studies in the future. All specimens were deposited in the Herbarium of the Department of Plant Pathol- ogy, Shandong Agricultural University (HSAUP). Living cultures were deposited in the Shandong Agricultural University Culture Collection (SAUCC). Taxonomic informa- tion on the new taxa was submitted to MycoBank (http://www.mycobank.org). DNA extraction and amplification Genomic DNA was extracted from fungal mycelia grown on PDA, using a modified cetyltrimethylammonium bromide (CTAB) protocol as described in Guo et al. (2000). DNA sequences of four different loci were obtained, including the nrDNA internal transcribed spacer regions 1 and 2 with the intervening 5.8S subunit (ITS), a partial sequence of the large subunit nrDNA subunit (LSU), a partial sequence of the transla- tion elongation factor 1-alpha gene (zef1), and a partial sequence of the beta-tubulin gene (tub2). They were all amplified with the primer pairs and polymerase chain reac- tion (PCR) program listed in Table 1. 30 Rongyu Liu et al. / MycoKeys 95: 27-45 (2023) PCR was performed using an Eppendorf Master Thermocycler (Hamburg, Ger- many). Amplification reactions contained 12.5 wL 2x Taq Plus Master Mix I (Va- zyme, Nanjing, China), 1 wL of each forward and reverse primers (10 pM) (Tsingke, Qingdao, China), 1 wL of template genomic DNA, and distilled deionized water to a total volume of 25 uL. The PCR products were visualized on 1% agarose electropho- resis gels. Bi-directional sequencing was conducted by the Tsingke Company Limited (Qingdao, China). Consensus sequences were obtained using MEGA 7.0 (Kumar et al. 2016). All sequences generated in this study were deposited in GenBank (Table 2). Table |. Gene regions and respective primer pairs used in the study. Locus PCR primers PCR: thermal cycles: (Annealing temperature in bold) Reference ITS ITSS/ITS4 (94 °C: 30 s, 55 °C: 30 s, 72 °C: 45 s) x 29 cycles White et al. 1990 LSU LROR/LR5 ~— (94 °C: 30 s, 48 °C: 50 s, 72 °C: 1 min 30 s) x 35 cycles _- Vilgalys and Hester 1990; Cubeta et al. 1991 tefl EF1-728F/EF2 = (95 °C: 30 s, 51 °C: 30 s, 72 °C: 1 min) x 35 cycles © O’Donnell et al. 1998; Carbone and Kohn 1999 tub2 Bt-2a/Bt-2b (95 °C: 30 s, 56 °C: 30 s, 72 °C: 1 min) x 35 cycles Glass and Donaldson 1995 Table 2. Isolates and GenBank accession numbers used in the phylogenetic analyses. Species Isolate/Strain Host/Substrate Origin GenBank accession numbers ITS LSU tef1 tub2 Apiospora acutiapica KUMCC 20-0210 — Bambusa bambos China MT946343 MT946339 MT947360 MT947366 (Type) A, agari KUC21333 (Type) Agarum cribrosum Korea MH498520 MH498440 MH544663 MH498478 A, aquatica S-642 (Type) Submerged wood China MK828608 MK835806 NA NA A, arctoscopi KUC21331 (Type) — Egg of Arctoscopus Korea MH498529 MH498449 MN868918 MH498487 japonicus A. arundinis CBS 124788 Living leaves of Fagus Switzerland KF144885 KF144929 KF145017 KF144975 sylvatica A. aurea CBS 244.83 (Type) Air Spain AB220251 KF144935 KF145023 KF144981 A. balearica CBS 145129 (Type) Undetermined Spain MKO014869 MK014836 MK017946 MKO017975 Poaceae A. biserialis CGMCC 3.20135 Bamboo China MW481708 MW478885 MW522938 MW522955 (Type) A. camelliae-sinensis LC5007 (Type) Camellia sinensis China KY494704 KY494780 KY705103 KY705173 A. chiangraiense MFLUCC21-0053 Dead culms of Thailand MZ542520 MZ542524 NA MZ546409 (Type) bamboo A, chromolaenae MFLUCC 17-1505 Chromolaena odorata Thailand MT214342 MT214436 NA NA (Type) A. cordylines GUCC 10027 Leaves of Cordyline China MT040106 NA MT040127 MT040148 (Type) fruticosa A. cyclobalanopsidis CGMCC 3.20136 — Cyclobalanopsidis China MW481713 MW478892 MW522945 MW522962 (Type) glauca A. descalsii CBS 145130 (Type) Ampelodesmos Spain MK014870 MK014837 MK017947 MK017976 mauritanicus A. dichotomanthi LC4950 (Type) Dichotomanthus China KY494697 KY494773 KY705096 KY705167 tristaniaecarpa A, dongyingensis SAUCC 0302 Leaf of bamboo China OP563375 OP572424 OP573264 OP573270 (Type) SAUCC 0303 Leaf of bamboo China OP563374 OP572423 OP573263 OP573269 A, esporlensis CBS 145136 (Type) Phyllostachys aurea Spain MK014878 MK014845 MKO017954 MKO017983 A. euphorbiae IMI 285638b Bambusa sp. Bangladesh AB220241 AB220335 NA AB220288 A, fermenti KUC21289 (Type) Seaweed Korea MFG615226 MF615213 MH544667 MFG615231 Species A. gaoyouensis A, garethjonesii A, gelatinosa A. guiyangensis A. guizhouensis A. hainanensis A, hispanica A, hydei A. hyphopodii A, hysterina A. A. A. iberica intestini italica A, jatrophae A, jiangxiensis A, A, A, A, A, PSPS PSS kogelbergensis koreana locuta-pollinis longistroma malaysiana marianiae marit marina mediterranea minutispora montagne mori muttiloculata mytilomorpha neobambusae neochinense neogarethjonesii neosubglobosa Three species of Apiospora in China Isolate/Strain CFCC 52301 (Type) JHB004 (Type) HKAS 111962 (Type) HKAS 102403 (Type) LC5322 (Type) SAUCC 1681 (Type) SAUCC 1682 IMI 326877 (Type) CBS 114990 (Type) MFLUCC 15-0003 (Type) ICPM 6889 (Type) AP10118 (Type) CBS 135835 (Type) CBS 145138 (Type) CBS 134262 (Type) L.C4577 (Type) CBS 113333 (Type) KUC21332 (Type) LC11683 (Type) MFLUCC 11-0481 (Type) CBS 102053 (Type) AP18219 (Type) CBS 497.90 (Type) KUC21328 (Type) IMI 326875 (Type) 17E-042 (Type) AP301120 (Epitype) AP19421 CPC 18900 MELU 18-2514 (Type) MELUCC 21-0023 (Type) DAOM 214595 (Type) LC7106 (Type) CFCC 53036 (Type) HKAS 102408 (Type) JHB007 (Type) Host/Substrate Phragmites australis Culms of dead bamboo Culms of dead bamboo Dead culms of Poaceae Air in karst cave Leaf of bamboo Leaf of bamboo Maritime sand Culms of Bambusa tuldoides Dead culms of bamboo Bamboo Arundo donax Gut of grasshopper Arundo donax Jatropha podagrica Maesa sp. Dead culms of Restionaceae Egg of Arctoscopus japonicus Brassica campestris Culms of decaying bamboo Macaranga hullettii stem colonised by ants Dead stems of Phleum pratense Atmosphere, pharmaceutical excipients, home dust and beach sands Seaweed Air Soil Arundo micrantha Arundo micrantha Culms of Phragmites australis Dead leaves of Morus australis Dead culms of Bambusae Dead blades of Andropogon sp. Leaf of bamboo Fargesia ginlingensis Dead culms of Bambusae Bamboo Origin China China China China China China China Spain China Thailand New Zealand Portugal India Italy India China South Africa Korea China Thailand Malaysia Spain Spain Korea Spain South Korea Spain Spain Italy China Thailand India China China China China 31 GenBank accession numbers ITS MH197124 KY356086 MW481706 MW240647 KY494709 OP563373 OP563372 AB220242 KF144890 KRO069110 MKO014874 MK014879 KRO11352 MK014880 JQ246355 KY494693 KF144892 MH498524 MF939595 KU940141 KF144896 ON692406 MH873913 MH498538 AB220243 LC517882 ON692408 ON692418 KF144909 MW114313 OL873137 KY494685 KY494718 MK819291 MK070897 KY356090 LSU NA KY356091 tefl MH236793 NA MW478888 MW/522941 MW240577 MW759535 KY494785 OP572422 OP572421 AB220336 KF144936 NA MK014841 MKO014846 KR149063 MK014847 NA KY494769 KF144938 MH498444 NA KU863129 KF144942 ON692422 KF144947 MH498458 AB220337 NA ON692424 ON692425 KF144956 MW114393 OL873138 NA KY494794 NA MK070898 KY356095 KY705108 OP573262 OP573261 NA KF145024 NA MKO017951 MK017955 KRO11351 MKO017956 NA KY705092 KF145026 MH544664 MF939616 NA KF145030 ONG77180 KF145035 MH544669 NA LC518889 ONG77182 ONG677183 KF145043 NA NA NA KY806204 MK818545 NA NA tub2 MH236789 NA MW522958 MW775604 KY705178 OP573268 OP573267 AB220289 KF144982 NA MKO017980 MK017984 KRO11350 MKO017985 NA KY705163 KF144984 MH498482 MF939622 NA KF144988 ONG77186 KF144993 MH498496 AB220290 LC518888 ONG77188 ONG77189 KF145001 NA OL874718 NA KY705186 MK818547 NA NA D2 Species A. obovata A. ovata A, paraphaeosperma A. phyllostachydis A, piptatheri A, pseudomarii A, pseudoparenchymatica A, pseudorasikravindrae A. pseudosinensis A, pseudosinensis A, pseudospegazzinii A. pterosperma A, pusillisperma A. qinlingensis A. rasikravindrae A. sacchari A. saccharicola A, sargassi A. sasae A. septata A. serenensis A. setariae A. sichuanensis A, sorghi A, sphaerosperma A, stipae A, subglobosa A. subrosea A. thailandica A. vietnamensis A. xenocordella A. yunnana Arthrinium caricicola Notes: Strains in this study are marked in bold. NA = not available. Rongyu Liu et al. / MycoKeys 95: 27-45 (2023) Isolate/Strain LC4940 (Type) CBS 115042 (Type) MFLUCC13-0644 (Type) MFLUCC 18-1101 (Type) CBS 145149 (Type) GUCC 10228 (Type) C7234 (Type) KUMCC 20-0208 (Type) CPC 21546 (Type) SAUCC 0221 SAUCC 0222 CBS 102052 (Type) CPC 20193 (Fype) KUC21321 (Type) CFCC 52303 (Type) LC5449 CBS 212.30 CBS191.73 KUC21228 (Type) CBS 146808 (Type) CGMCC 3.20134 (Type) IMI 326869 (Type) CFCC 54041 (Type) HKAS 107008 (Type) URM 93000 (Type) CBS114314 CBS 146804 (Type) MFLUCC 11-0397 (Type) LC7292 (Type) LC5630 IMI 99670 (Type) CBS 478.86 (Type) MEFLUCC 15-0002 (Type) CBS 145127 Host/Substrate Lithocarpus sp. Arundinaria hindsii Dead clumps of Bambusa sp. Phyllostachys heteroclada Piptatherum miliaceum Leaves of Aristolochia debilis Leaf of bamboo Bambusa dolichoclada Leaf of bamboo Leaf of bamboo Leaf of bamboo Macaranga hullettii stem colonized by ants Lepidosperma gladiatum Seaweed. Fargesia ginlingensis Soil in karst cave Phragmites australis Air Sargassum fulvellum Dead culms of Sasa veitchii Bamboo Food, pharmaceutical excipients, atmosphere and home dust Decaying culms of Setaria viridis Dead culms of Poaceae Sorghum bicolor Leaf of Hordeum vulgare Dead culm of Stipa gigantea Dead bamboo culms Leaf of bamboo Rotten wood Citrus sinensis Soil from roadway Decaying bamboo culms Carex ericetorum Origin China China Thailand China Spain China China China Netherlands China China Malaysia Australia Korea China China UK Netherlands Korea Netherlands China Spain China China Brazil Iran Spain Thailand China China Vietnam Zimbabwe China China GenBank accession numbers ITS KY494696 KF144903 KX822128 MK351842 MK014893 MT040124 KY494743 MT946344 KF144910 OP563377 OP563376 KF144911 KF144913 MH498533 MH197120 KY4947 13 KF144916 KF144920 KT207746 MW883402 MW481711 AB220250 MT492004 MW240648 MK371706 KF144904 MW883403 KRO69112 KY494752 KY494714 KX986096 KF144925 KU940147 MKO014871 LSU KY494772 KF144950 KX822124 MH368077 MKO014860 NA KY494819 NA KF144957 OP572426 OP572425 KF144958 KF144960 MH498453 NA KY494789 KF144962 KF144966 KT207696 MW883797 MW/478890 AB220344 NA MW240578 NA KF144951 MW883798 KRO069113 KY494828 KF144970 KX986111 KF144970 KU863135 MKO014838 tefl KY705095 KF145037 NA MK340918 MK017969 MT040145 KY705139 MT947361 KF145044 OP573266 OP573265 KF145045 KF145046 MN868930 MH236795 KY705112 KF 145047 KF145051 MH544677 MW890104 MW522943 NA NA MW759536 NA KF145038 MW890082 NA KY705148 KY705113 NA KF145055 NA MK017948 tub2 KY705166 KF144995 NA MK291949 NA MT040166 KY705211 MT947367 MN868936 OP573272 OP573271 KF145002 KF145004 MH498491 MH236791 KY705182 KF145005 KF145009 KT207644 MW890120 MW522960 AB220297 NA MW775605 MK348526 KF144996 MW890121 NA KY705220 KY806200 KY019466 KF145013 NA MKO017977 Three species of Apiospora in China 33 Phylogenetic analyses Newly generated ITS, LSU, tef? and tub2 sequences from the six strains studied were aligned with all reference sequences of Apiospora and related species available in GenBank using the MAFFT v.7.11 online software (http://mafft.cbre.jp/alignment/ server/, Katoh et al. 2019) with the default settings, manually correcting the result- ing alignment where necessary. Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic analyses were conducted individually on each locus (ITS, LSU, tef/ and tub2) and on a combined dataset including all of them. The best-fitting evolu- tionary model of each partition was determined using MrModeltest v. 2.3 (Nylander 2004). ML and BI were run on the CIPRES Science Gateway portal (https://www. phylo.org/) using RaxML-HPC2 on XSEDE (8.2.12) (Miller et al. 2012; Stamata- kis 2014) and MrBayes on XSEDE (3.2.7a), respectively (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003; Ronquist et al. 2012). For ML analyses the default parameters were used, while BI was carried out using a Markov chain Monte Carlo (MCMC) algorithm. BI analyses included four MCMC chains and were run for 5,000,000 generations until the average standard deviation of split frequencies was below 0.01 with trees saved every 1000 generations. The burn-in fraction was set to 0.25 and posterior probabilities (PP) were determined from the remaining trees. The resulting 50% majority-rule tree was plotted using FigTree v. 1.4.4 (http://tree.bio. ed.ac.uk/software/figtree) and edited with Adobe Illustrator CS6.0. Results Phylogenetic analyses Among the six strains of Apiospora isolated from the samples studied, two new species were discovered, and another one found for the first time in China after the combined analysis of ITS, LSU, zef7 and tub2 DNA sequences from 89 isolates of Apiospora plus Arthrinium caricicola Kunze & J.C. Schmidt (CBS 145127) as the outgroup taxon. A total of 2241 characters including gaps were compared in the phylogenetic anal- ysis, viz. ITS: 1-706, LSU: 707-1513, tef1: 1514-1932, tub2: 1933-2241. Of these characters, 1436 were constant, 271 were variable and parsimony-uninformative, and 534 were parsimony-informative. For the BI and ML analyses, the substitution model GTR+I+G was selected for all partitions. The BI analysis reached the established convergence after 3935000 generations, resulting in 39351 sampled trees, of which 295 14 trees were used to calculate the posterior probabilities. The ML tree topology agreed with that obtained from the BI analysis, and therefore, only one tree (the ML) is presented (Fig. 1). The four strains (SAUCC 0302, SAUCC 0303, SAUCC 1681 and SAUCC 1682) studied in the present work represent two independent clades, interpreted as newly discovered independent species. ‘These are described below and accommodated under the new names Apiospora dongyingensis and 34 Rongyu Liu et al. / MycoKeys 95: 27-45 (2023) 94/0.99[- Apiospora sphaerosperma CBS 114314 62/0.92 Apiospora serenensis IMI 326869 92/1]! Apiospora aquatica S-642 acl Apiospora saccharicola CBS 191.73 Apiospora dichotomanthi LC4950 SAUCC 0221 1900/1! SAUCC 0222 Apiospora pseudosinensis bag CPC 21546 Apiospora iberica AP10118 400/0.95| SAUCC 0302 100/1}' SAUCC 0303 62/- Apiospora camelliae-sinensis LC5007 53/0.96 Apiospora dongyingensis sp. nov. Apiospora cyclobalanopsidis CGMCC 3.20136 93/- Apiospora setariae CFCC 54041 85/1 Apiospora jiangxiensis LC4577 1 79/1 Apiospora obovata LC4940 63/0.96 Apiospora arctoscopi KUC21331 Apiospora guiyangensis HKAS 102403 Apiospora locuta-pollinis LC11683 91/- Apiospora pseudomarii GUCC 10228 S7-| Apiospora hispanica IMI 326877 96/0.99 90/4 Apiospora mediterranea IM| 326875 Raia marii CBS 497.90 oa AGio ane gaoyouensis CFCC 52301 Apiospora sacchari CBS 212.30 uel o Aniaeeeee guizhouensis LC5322 Apiospora biserialis CGMCC 3.20135 -/0.91 OTA pilosa gelatinosa HKAS 111962 Apiospora septata CGMCC 3.20134 1400/1) SAUCC 1681 SAUCC 1682 wah Apiospora piptatheri CBS 145149 Apiospora longistroma MFLUCC 11-0481 Apiospora hainanensis sp. nov. Apiospora pseudospegazzinii CBS 102052 Apiospora fermenti KUC21289 96/- Apiospora acutiapica KUMCC 20-0210 g99/- Apiospora pseudorasikravindrae KUMCC 20-0208 Apiospora marina KUC21328 oe Apiospora paraphaeosperma MFLUCC 13-0644 ee Apiospora rasikravindrae LC5449 61/- Apiospora neochinense CFCC 53036 4100/1 Apiospora sargassi KUC21228 AplOs ra cordylines GUCC 10027 99/1 = Apieeeaed hydei CBS 114990 Be Apiospora aurea CBS 244.83 Apiospora minutispora 17E-042 a Apiospora montagnei AP301120 100/1| Apiospora montagnei CPC 18900 100/1 Apiospora montagne! AP19421 Apiospora descalsii CBS 145130 92/1 Apiospora balearica CBS 145129 Figure |. Phylogram of Apiospora based on combined ITS, LSU, tef? and tub2 genes. ML bootstrap support values (MLBS > 50%) and Bayesian posterior probability (BYPP = 0.90) are shown as first and second position above nodes, respectively. Strains from this study are shown in red, ex-type or ex-epitype cultures are indicated in bold face. Some branches were shortened according to the indicated mulipliers. Three species of Apiospora in China 35 100/1 cy 10X i Tox’ Plospora neosubglobosa JHB007 61/0.98 Apiospora subglobosa MFLUCC 11-0397 Apiospora mori MFLU 18-2514 Apiospora multiloculata MFLUCC 21-0023 Apiospora jatrophae CBS 134262 1400/1 Apiospora sichuanensis HKAS 107008 66/0.97 2x 2x 98/1 Apiospora pseudoparenchymatica LC7234 Apiospora hyphopodii MFLUCC 15-0003 Apiospora mytilomorpha DAOM 214595 Apiospora garethjonesii JHB004 Apiospora neogarethjonesii HKAS 102408 Apiospora subrosea LC7292 Apiospora neobambusae LC7106 63/.[ Apiospora sasae CBS 146808 100/1|“ Apiospora hysterina ICPM 6889 Apiospora yunnana MFLUCC 15-0002 Apiospora ovata CBS 115042 Apiospora marianiae AP18219 75/- 100/1 99/1 79/0.94 81/1 Apiospora pterosperma CPC 20193 65/17 Aplospora euphorbiae IMI 285638b 76/- |' Apiospora malaysiana CBS 102053 92/0.96] Apiospora vietnamensis IMI 99670 83/4 Apiospora chromolaenae MFLUCC 17-1505 94/4 Apiospora phyllostachydis MFLUCC 18-1101 an neat Apiospora thailandica LC5630 Apiospora italica CBS 145138 Apiospora agari KUC21333 87/0.96 gee 2 6X 4 Apiospora arundinis CBS 124788 z 10X 100/1 921-7 Apiospora kogelbergensis CBS 113333 97/1 Apiospora xenocordella CBS 478.86 Apiospora esporlensis CBS 145136 Apiospora koreana KUC21332 4100/1 sh Apiospora qinlingensis CFCC 52303 Apiospora stipae CBS 146804 400/41 Apiospora intestini CBS 135835 100/1 Apiospora pusillisperma KUC21321 Apiospora chiangraiense MFLUCC 21-0053 6X Apiospora sorghi URM 93000 Arthrinium caricicola CBS 145127 0.08 Figure |. Continued. A. hainanensis. Another two strains (SAUCC 0221 and SAUCC 0222) clustered with A. pseudosinensis (CPC 21546) with full support (MLBS: 100% and BYPP: 1), and are therefore considered no different from this species. Taxonomy Apiospora dongyingensis R.Y. Liu, J.W. Xia & X.G. Zhang, sp. nov. MycoBank No: 846065 Fig. 2 Etymology. Named after Dongying City (China) where the type was collected. 36 Rongyu Liu et al. / MycoKeys 95: 27-45 (2023) Figure 2. Apiospora dongyingensis (SAUCC 0302, ex-holotype culture) a leaf of host plant b, ¢ surface # (b) and reverse (C) sides of colony after incubation for 7 days on PDA d conidiomata formed in culture e, f conidiogenous cells and conidia g, h conidia. Scale bars: 10 um (eh). Type. China, Shandong Province: Dongying Botanical Garden, on diseased leaves of bamboo, 13 July 2022, R.Y. Liu, holotype HSAUP 0302, ex-type living culture SAUCC 0302. Description. Asexual morph: On WA, hyphae 1.3—3.6 pm diam., hyaline, branched, septate. Conidiophores cylindrical, septate, verrucose, flexuous, sometimes reduced to conidiogenous cells. Conidiogenous cells globose to subglobose, erect, blastic, aggregated in clusters on hyphae, hyaline to pale brown, smooth, branched, 8.2-13.9 x 4.2-8.2 um, mean + SD: 9.6 + 1.6 x 6.7 £ 1.1 pm (n = 40). Conidia Three species of Apiospora in China 37 globose, subglobose to lenticular, with a longitudinal germ slit, occasionally elon- gated to ellipsoidal, brown to dark brown, smooth to finely roughened, 8.0—16.5 x 5.5—-9.0 um, mean + SD: 9.4 = 1.9 x 7.3 = 1.0 um, L/W = 1.3-1.9 (n = 40). Sexual morph: Undetermined. Culture characteristics. Colonies on PDA flat with entire margin, aerial myce- lium white to gray, floccose cottony; surface and reverse gray in the center and grayish margin. PDA attaining 78.5—-86.5 mm in diameter after 7 days at 25 °C, growth rate 11.0-12.5 mm/day. Additional specimen examined. Cuina, Shandong Province: Dongying Botani- cal Garden, on diseased leaves of bamboo, 13 July 2022, R.Y. Liu, paratype HSAUP 0303, ex-paratype living culture SAUCC 0303. Notes. Apiospora dongyingensis is closely related but phylogenetically dis- tinct from A. camelliae-sinensis (M. Wang, F. Liu & L. Cai) Pintos & P. Alvarado and A. cyclobalanopsidis (Y. Feng & Jian K. Liu) X.G. Tian & Tibpromma (Fig. 1). A. dongyingensis differs from A. camelliae-sinensis by 18 nucleotides (13/518 in ITS, 2/804 in LSU, 2/374 in tefl and 1/265 in tub2) and A. cyclobalanopsidis by 58 nucleo- tides (17/518 in ITS, 4/799 in LSU, 26/377 in tef7 and 11/266 in tub2). Morphologi- cally, it differs from A. camelliae-sinensis and A. cyclobalanopsidis in its conidia (globose, subglobose to lenticular, 8.0—-16.5 x 5.5—9.0 um in A. dongyingensis vs. globose to sub- globose, 9.0-13.5 x 7.0-12.0 um in A. camelliae-sinensis and surface view globose to ellipsoid, 8-12 um long and side view lenticular, 10-14 um long in A. cyclobalanopsidis; Wang et al. 2018; Feng et al. 2021; Pintos and Alvarado 2021; Tian et al. 2021). Apiospora hainanensis R.Y. Liu, J.W. Xia & X.G. Zhang, sp. nov. MycoBank No: 846066 Fig. 3 Etymology. Named after Hainan Province (China) where the type was collected. Type. China, Hainan Province: Diaoluoshan National Nature Reserve, on dis- eased leaves of bamboo, 23 June 2021, R.Y. Liu, holotype HSAUP 1681, ex-type liv- ing culture SAUCC 1681. Description. Asexual morph: On WA, hyphae 1.2—3.4 um diam., hyaline, branched, septate. Conidiophores cylindrical, septate, verrucose, flexuous, sometimes reduced to conidiogenous cells. Conidiogenous cells globose to subglobose, erect, blastic, aggregated in clusters on hyphae, hyaline to pale brown, smooth, branched, 6.4—8.8 x 5.2—7.1 um, mean + SD: 7.9 = 1.1 x 6.1 £ 0.9 um (n = 40). Conidia glo- bose, subglobose to lenticular, with a longitudinal germ slit, occasionally elongated to ellipsoidal, brown to dark brown, smooth to finely roughened, 5.5-8.5 x 5.0— 7.5 um, mean + SD: 6.8 + 0.9 x 6.7 = 0.7 um, L/W = 1.0-1.1 (n = 40). Sexual morph: Undetermined. Culture characteristics. Colonies on PDA flat with entire margin, aerial myce- lium white to grey, floccose cottony; reverse white to pale honey colored. PDA attain- ing 77.5—-85.5 mm in diameter after 7 days at 25 °C, growth rate 10.5—12.5 mm/day. 38 Rongyu Liu et al. / MycoKeys 95: 27-45 (2023) Se aa Figure 3. Apiospora hainanensis (SAUCC 1681, ex-holotype culture) a leaf of host plant b, € surface g Pe Rh (b) and reverse (C) sides of colony after incubation for 7 days on PDA d conidiomata formed in culture e, f conidiogenous cells and conidia g, h conidia. Scale bars: 10 um (e=h). Additional specimen examined. Cuina, Hainan Province: Diaoluoshan Nation- al Nature Reserve, on diseased leaves of bamboo, 23 June 2021, R.Y. Liu, paratype HSAUP 1682, ex-paratype living culture SAUCC 1682. Notes. The two strains (SAUCC 1681 and SAUCC 1682) of A. hainanensis clustered together with significant support in an isolated branch basal to A. sacchari and related species of the phaeospermum clade (Pintos and Alvarado 2022; Fig. 1). Other species in a more or less similar phylogenetic position include A. septata (Y. Feng & Jian K. Liu) X.G. Tian & Tibpromma, A. piptatheri (Pintos & P. Alvarado) Three species of Apiospora in China 39 Pintos & P. Alvarado, A. longistroma (D.Q. Dai & K.D. Hyde) Pintos & P. Alva- rado, A. pseudospegazzinii (Crous) Pintos & Alvarado and A. fermenti (S.L. Kwon, S. Jang & J.J. Kim) S.L. Kwon & J.J. Kim. Morphologically, it differs from A. septata, A. piptatheri, A. longistroma, A. pseudospegazzinii and A. fermenti in its conidia (glo- bose, subglobose to lenticular, 5.5-8.5 x 5.0-7.5 um in A. /ainanensis vs. surface view globose to ellipsoid, 8-13 um long and side view lenticular, 8-14 um long in A, septata, globose to ellipsoidal, 6-8 x 3—5 um in A. piptatheri, asexual morph unde- termined in A. /ongistroma, surface view globose, 7—9 um diam. and side view lenticu- lar, 5-6 um diam. in A. pseudospegazzinii, surface view globose to elongate ellipsoid, 7.5-9 x 7-9 um and side view lenticular, 6-7 um diam. in A. fermenti; Crous and Groenewald 2013; Dai et al. 2016; Pintos et al. 2019; Feng et al. 2021; Kwon et al. 2021, 2022; Pintos and Alvarado 2021; Tian et al. 2021). Apiospora pseudosinensis (Crous) Pintos & P. Alvarado, Fungal Systematics and Evolution 7: 207. (2021) Fig. 4 = Arthrinium pseudosinense Crous, in Crous & Groenewald, IMA Fungus 4(1): 148 (2013). Description. Asexual morph: On WA, hyphae 1.1—2.9 um diam., hyaline, branched, septate. Conidiophores cylindrical, septate, verrucose, flexuous, sometimes reduced to conidiogenous cells. Conidiogenous cells globose to subglobose, erect, blastic, aggre- gated in clusters on hyphae, hyaline to pale brown, smooth, branched, 9.4—11.0 x 6.1— 8.8 um, mean + SD: 10.4 + 0.7 x 7.7 = 1.1 um (n = 40). Conidia globose, subglobose to lenticular, with a longitudinal germ slit, occasionally elongated to ellipsoidal, brown to dark brown, smooth to finely roughened, 7.5—-11.5 x 7.0-9.5 um, mean + SD: 10.1 + 1.3 x 8.3 £ 0.6 um, L/W = 1.1-1.3 (n = 40). Sexual morph: Undetermined. Culture characteristics. Colonies on PDA flat with irregular margin, aerial myce- lium white to pale yellow, floccose cottony; reverse pale yellow to yellow. PDA attain- ing 69.5-78.5 mm in diameter after 7 days at 25 °C, growth rate 9.5—11.5 mm/day. Specimens examined. Cuina, Shandong Province: Dongying Botanical Garden, on diseased leaves of bamboo, 15 July 2022, R.Y. Liu, HSAUP 0221, living culture SAUCC 0221; China, Hainan Province: Diaoluoshan National Nature Reserve, on diseased leaves of bamboo, 29 June 2021, R.Y. Liu, HSAUP 0022, living culture SAUCC 0022. Notes. Apiospora pseudosinensis was originally described from bamboo leaves col- lected in the Utrecht Botanical Garden of the Netherlands (Crous and Groenewald 2013; Pintos and Alvarado 2021). In the present study, DNA sequences obtained from two strains (SAUCC 0221 and SAUCC 0222) collected also from bamboo leaves, were not significantly different from those of A. pseudosinensis (Fig. 1). Morphologically, our strains were similar to the original description (conidia 8-10 x 7-10 pm diam. in sur- face view, 7-8 um diam. in side view). We therefore consider the newly found strains as A. pseudosinensis (Crous and Groenewald 2013; Pintos and Alvarado 2021). 40 Rongyu Liu et al. / MycoKeys 95: 27-45 (2023) o. "F PN fi ens} Figure 4. Apiospora pseudosinensis (SAUCC 0221) a leaf of host plant b, ¢ surface (b) and reverse (C) sides of colony after incubation for 7 days on PDA d conidiomata formed in culture e, f conidiogenous cells and conidia g, h conidia. Scale bars: 10 um (eh). Discussion The family Apiosporaceae was proposed to accommodate genera with apiosporous hyaline ascospores and a basauxic, Arthrinium-like conidiogenesis (Hyde et al. 1998). Crous and Groenewald (2013) synonymized Apiospora with Arthrinium on the ba- sis of the one fungus-one name policy (Hawksworth et al. 2011). Crous and Groe- newald (2013) also resolved the genetic identity of multiple species of Arthrinium Three species of Apiospora in China 4] (= Apiospora), analysing ex-type collections, and confirmed that most species occur in Poaceae (R.Br.) Barnh. hosts, although some were known from many other plant host families. However, with the aid of additional genetic data from the type species of Arthrinium, Ar. caricicola, Apiospora and Arthrinium were separated again as two distinct genera (Pintos and Alvarado 2021). Arthrinium species have variously shaped conidia and inhabit Cyperaceae and Juncaceae in temperate, cold or alpine habitats. Most Apiospora species have rounded/lenticular conidia and inhabit mainly Poaceae (and many other host plant families) in a wide range of habitats, including tropical and subtropical regions (Pintos and Alvarado 2021; Samarakoon et al. 2022). An epi- type for the type species of Apiospora, A. montagnei, was recently proposed by Pintos and Alvarado (2022). There are many Apiospora species found on bamboos across the world (Table 2). Bamboos (Poaceae) are distributed in tropical and subtropical to mild temperate re- gions, with the heaviest concentration and largest number of species in China. Due to their abundance and economic importance, it is of great significance to study and identify the fungi growing on bamboo (Feng et al. 2021). In the present study, two new species (Apiospora dongyingensis and A. hainanensis) are introduced, and another one (A. pseudosinensis) is reported for the first time in China. All of them were collected from bamboo leaves and described based on their phylogenetic data and morphologi- cal characters. ‘The descriptions and molecular data for species of Apiospora represent an important resource for understanding the diversity of bamboo fungi. Acknowledgements This work was supported by the National Natural Science Foundation of China (no. 31900014, U2002203 and 31750001). 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The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.95.96400.suppl1