MycoKeys 33: 39-67 (202 | ) er-reviewed open-access journal doi: 10.3897/mycokeys.83.69906 < MycoKkeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research A taxonomic study of Nemania from China, with six new species Yin Hui Pi'?, Si Han Long', You Peng Wu', Li Li Liu', Yan Lin', Qing De Long', Ji Chuan Kang’, Ying Qian Kang*, Chu Rui Chang', Xiang Chun Shen'”, Nalin N. Wijayawardene'**, Xu Zhang', Qi Rui Li!” I State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, Gui- yang 550004, China 2 The High Efficacy Application of Natural Medicinal Resources Engineering Center of Guizhou Province (The Key Laboratory of Optimal Utilization of Natural Medicine Resources), School of Pharmaceutical Sciences, Guizhou Medical University, University Town, Guian New District, Guizhou, China 3 Engineering and Research Center for Southwest Bio-Pharmaceutical Resources of National Education Ministry of China, Guizhou University, Guiyang, Guizhou 550025, China 4 Key Laboratory of Environmen- tal Pollution Monitoring and Disease Control, Ministry of Education of Guizhou and Guizhou Talent Base for Microbiology and Human Health, School of Basic Medical Sciences, Guizhou Medical University, Guiyang, China 5 Center for Yunnan Plateau Biological Resources Protection and Utilization, College of Biological Resource and Food Engineering, Qujing Normal University, Qujing, Yunnan 655011, China 6 Section of Ge- netics, Institute for Research and Development in Health and Social Care, No: 393/3, Lily Avenue, Off Robert Gunawardane Mawatha, Battaramulla 10120, Sri Lanka Corresponding author: Qi Rui Li (lqrnd2008@163.com) Academic editor: Ekaphan Kraichak | Received 9 June 2021 | Accepted 29 July 2021 | Published 24 August 2021 Citation: Pi YH, Long SH, Wu YP, Liu LL, Lin Y, Long QD, Kang JC, Kang YQ, Chang CR, Shen XC, Wijayawardene NN, Zhang X, Li QR (2021) A taxonomic study of Nemania from China, with six new species. MycoKeys 83: 39-67. https://doi.org/10.3897/mycokeys.83.69906 Abstract During an investigation of Xylariaceae from 2019 to 2020, isolates representing eight Nemania (Xylari- acese) species were collected from Yunnan, Guizhou and Hainan Provinces in China. Morphological and multi-gene phylogenetic analyses, based on combined ITS, «-actin, rp62 and §-tubulin sequences, con- firmed that six of them are new to science, viz. Nemania camelliae, N. changningensis, N. cyclobalanopsina, N. feicuiensis, N. lishuicola and N. rubi; one is a new record (JN. caries) for China and one is a known spe- cies (NV. diffusa). Morphological descriptions and illustrations of all species are detailed. In addition, the characteristics of Nemania are summarised and prevailing contradictions in generic concepts are discussed. Keywords phylogeny, six new species, taxonomy, Xylariaceae Copyright YinHui Pi 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. 40 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) Introduction Nemania Gray was established by Gray (1821) for a heterogeneous assemblage of taxa and was afhliated with Xylariaceae Tul. & C. Tul. Since the early taxonomic description of this genus was ambiguous, taxonomists have often regarded some species of Nema- nia as synonyms of Hypoxylon Bull. For example, Nemania angusta (Petch) Y.M. Ju & J. D. Rogers was regarded as a synonym of Hypoxylon angustum Petch. (Miller 1961; Whalley et al. 1983; Ju and Rogers 2002). Subsequently, the generic concept of Nema- nia was modified by Pouzar (1985a, b) and Petrini and Rogers (1986). Granmo et al. (1999) and Ju and Rogers (2002) provided a comprehensive background to Nemania and accepted 37 species. Sanchez-Ballesteros et al. (2000) used the internal transcribed spacers (ITS) sequence to perform a phylogenetic study of Nemania, which supported the segregation of Nemania from Hypoxylon. However, their conclusion was based only on ITS sequences and Xylaria Hill & Schrank was not included in this study. Hence, the generic placement of Nemania in the Xylariaceae was unclear. Hsieh et al. (2005) used 8-tubulin and «-actin to evaluate the phylogenetic relationship of several xylari- aceous genera. It was found to be particularly useful in xylariaceous fungi as limited success in using ribosomal DNA genes to delineating genera and resolving generic relationships (Tang et al. 2007). Tang et al. (2007) re-established the phylogenetic re- lationships of Nemania with related genera, based on the combined dataset of ITS and rpb2 which supported the separation of Nemania from Hypoxylon. However, Tang et al. (2007) stated that Nemania is closely related to Xylaria and phylogenetically distinct from Annulohypoxylon Y.M. Ju et al., Daldinia Ces. & De Not. and Hypoxylon. Ulti- mately, the boundaries of the genus became relatively clear and Nemania has been ac- cepted as a distinct genus in Xylariaceae (Ju and Rogers 2002). The major morphologi- cal characteristics of Nemania include dark brown to black stromata, carbonaceous or at least brittle and not yielding pigments in 10% potassium hydroxide (KOH) (Ju and Rogers 2002), white soft tissue existing between or below the perithecia, ascospores usually pale brown and most of them have no obvious germ-slit and spore dehiscence in 10% KOH (Tang et al. 2007). Nemania accepted 37 species by 2002, which occurs mainly distributed on the rotting wood of angiosperms (Ju and Rogers 2002; Tang et al. 2007). There are a few species introduced from China in recent years. Two new species (lV. flavitextura Y.M. Ju, H.M. Hsieh & J.D. Rogers and NV. primolutea Y.M. Ju, H.M. Hsieh & J.D. Rog- ers), collected from Taiwan, were reported by Ju et al. (2005). One new species and two new record species were discovered and described by Du et al. (2016) and Ariyawansa et al. (2015) in China. Recently, two new species (NV. yunnanensis Tibpromma & Lu and NV. aquilariae Vibpromma & Lu), collected from Yunnan Province, China, were discovered by Tibpromma et al. (2021). Ninety-three epithets of Nemania are listed on Index Fungorum (2021) (accession date: 06. 2021). Only 17 species of Nemania with gene sequences were retrieved from the NCBI database (https://www.ncbi.nlm.nih. gov) and morphological methods are the main distinguishing method for Nemania. Morphologically, it is mainly distinguished according to the germ slit, the size of the ascospores and the characteristics of the stromata. A taxonomic study of Nemania from China 4] In this study, eight species of Nemania, collected from Guizhou, Hainan and Yun- nan Provinces in China, are introduced. Six new species are identified, based on mor- pho-molecular analyses, while NV. caries is reported as a new record for China; NV. diffusa has been previously reported from China (Du 2015). Detailed morphological descrip- tions, illustrations and phylogenetic information of all species are provided in this paper. Materials and methods Collection, isolation and morphology Samples of rotting wood with fungi were collected from October 2019 to December 2020 in various nature reserves of Guizhou, Hainan and Yunnan Provinces, China. These samples were placed in sealed bags and the coordinates of sampling sites (such as latitude, longitude and altitude) were recorded. Specimens were taken to the labo- ratory for examination. Microscopic observations were made with fungi mounted in distilled water. A drop of Melzer’s Reagent was added to determine whether or not the ascus apical ring blued (the amyloid iodine reaction) and the reaction and morphology of the ring could be observed. Fragments of stroma and perithecial wall were placed in 10% KOH ona microscope slide and the extractable pigment observed. Pure cultures were obtained with the single spore isolation method (Long et al. 2019) and the cul- tures were grown on oatmeal agar (OA) and potato dextrose agar (PDA). Morphological examination of fungi on the rotting wood followed the methods of Xie et al. (2020). The characteristics of the stromata were observed with an Olympus SZ61 stereomicroscope and photographed using a fitted Canon 700D digital camera. The photomicrographs of asci and ascospores were taken with a Nikon digital camera (700D) fitted to a light microscope (Nikon Ni). Adobe Photoshop CS6 was used to arrange all the microphotographs. Measurements were performed using the Tarosoft image framework (v. 0.9.0.7). At least 30 ascospores, asci and ascus apical apparatus were measured for each specimen. To prepare herbarium materials, the colonies grown on PDA were transferred to three 1.5 ml microcentrifuge tubes filled with sterile water and stored at 4 °C or with 10% glycerol at —20 °C. Herbarium materials were deposited in the Herbarium of Guizhou Medical University (GMB) and Herbarium of Kunming Institute of Bota- ny, Chinese Academy of Sciences (KUN). Living cultures were deposited at Guizhou Medical University Culture Collection (GMBC). DNA extraction, PCR amplification and sequencing The BIOMIGA Fungal Genomic DNA Extraction Kit (GD2416, Biomiga, USA) was used to extract genomic DNA from fresh fungal mycelium, according to the manufac- turer's instructions. The extracted DNA was stored at —20 °C. Target regions of internal transcribed spacers (ITS) and RNA polymerase I second largest subunit (7p62) regions were amplified symmetrically using primers of ITS4/ 42 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) ITS5 (White et al. 1990; Gardes and Bruns 1993) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. ACT512F and ACT783R (Hsieh et al. 2005) and T11 and T22 (Tanaka et al. 2009; Hsieh et al. 2010) primers were used for the amplification of the a-actin gene (ACT) and $-tubulin (TUB2), respectively. The components of the poly- merase chain reaction (PCR) mixture and thermal cycling programme were performed as described by Pi et al. (2020). The amplified PCR fragments were sent to Sangon Biotech (Shanghai) Co., China, for sequencing. All newly-generated sequences of ITS, a-actin, 7pb2 and £-tubulin regions were uploaded to the GenBank database and the accession numbers are shown in Table 1. Sequence alignment and phylogenetic analyses Except for newly-generated sequences, all sequences used for phylogenetic analysis were downloaded from GenBank, based on published literature and the highest hit rate of ITS in the GenBank database. Sequence data for the construction of the phyloge- netic tree are listed in Table 1. Sequence alignments were generated using the MAFFT v.7.110 online programme (http://mafft.cbrc.jp/alignment/server/, Katoh and Stand- ley 2013) under default settings. Multiple sequence alignments of ITS, «-actin, 7pb2 and $-tubulin were analysed individually and in combination, manually adjusted to achieve the maximum alignment and to minimise gaps using the BioEdit v.5 (Hall 1999). The file formats were converted in ALTER (Alignment Transformation Envi- Ronment) (http://www.sing-group.org/ALTER/). The Maximum Likelihood analysis was carried out with GTR+G+I model of site substitution by using RAxML 7.4.2 black box (https://www.phylo.org/, Stamatakis et al. 2008) and Bayesian Inference Table |. Taxa of Nemania and related genera used for phylogenetic analyses and their GenBank accession numbers. Species Strain number GenBank Accession number References ITS rpb2 §-tubulin o-actin Amphirosellinia fushanensis AST 91111209 (HT) GU339496 GQ848339 GQ495950 GQ452360 Hsieh et al. (2010) Am. nigrospora HAST 91092308 (HT) GU322457 GQ848340 GQ495951 GQ452361 Hsieh et al. (2010) Astrocystis bambusae HAST 89021904 GU322449 GQ844836 GQ495942 GQ449239 Hsieh et al. (2010) As. bambusicola MFLUCC 17-0127 (HT) MF467942 MF467946 N/A N/A Hyde et al. (2017) As. concavispora MFLUCC 14-0174 KP297404 KP340532 KP406615 N/A Daranagama et al. (2015) As. mirabilis HAST 94070803 GU322448 GQ844835 GQ495941 GQ449238 Hsieh et al. (2010) Brunneiperidium gracilentum MFLUCC 14-0011 (HT) KP297400 KP340528 KP406611 N/A Daranagama et al. (2015) B. involucratum MFLUCC 14-0009 KP297399 KP340527 KP406610 N/A Daranagama et al. (2015) Collodiscula bambusae GZUH0102 KP054279 KP276675 KP276674 N/A Li et al. (2015b) C. fangjingshanensis GZUH0109 (HT) KR002590 KRO02592 KRO02589 N/A Li et al. (2015a) C. leigongshanensis GZUHO0107 (HT) KP054281 KR002588 KR002587 N/A Li et al. (2015a) C. tubulosa GACP QRO111 (HT) MN017302 MN018403 MN018405 MNO018402 Xie et al. (2020) Daldinia bambusicola CBS 122872 (HT) KY610385 KY624241 AY951688 KU684037 Hsieh et al. (2005), Wendt et al. (2018) Dematophora buxi JDR99 GU300070 GQ844780 GQ470228 GQ398228 Hsieh et al. (2010) De. necatrix CBS 349.36 AY909001 KY624275 KY624310 N/A Pelaez et al. (2008), Wendt et al. (2018) Species Discoxylaria myrmecophila Entoleuca mammata Hypoxylon pulicicidum Kretzschmariella culmorum Nemania abortiva NN. aenea N. aenea var. aureolutea N. aquilariae N. beaumontii N. bipapillata N. camelliae N. caries N. changningensis N. chestersii N. cyclobalanopsina N. diffusa N. feicuiensis N. fusoidispora N. illita N. rubi N. lishuicola IN. macrocarpa NN. maritima N. plumbea N. primolutea NN. serpens N. sphaeriostoma NN. yunnanensis Podosordaria mexicana Pod. muli Poronia pileiformis Por. punctata Rosellinia aquila R. merrillii R. sanctae-cruciana Stilbohypoxylon elaeicola S. quisquiliarum Xylaria allantoidea X. apoda X. compunctum X. cubensis X. digitata X. juruensis Notes: Type specimens are marked with HT (holotype), ET (epitype). N/A: sequences not available. A taxonomic study of Nemania from China Strain number JDR 169 JDR 100 CBS 122622 (HT) JDR 88 BISH 467 (HT) CBS 680.86 ATCC 60819 KUMCC 20-0268 (HT) HAST 405 HAST 90080610 GMB0067 GMB0068 (HT) GMB0069 GMB0070 GMB0056 (HT) GMB0057 JE 04024 GMB0061 GMB0062 (HT) HAST 91020401 GMB0071 GMB0072 GMB0058 GMB0059 (HT) GZUH0098 YMJ 236 GMB0063 GMB0064 (HT) GMB0065 (HT) GMB0066 WSP 265 HAST 89120401 (ET) JE TH-04-01 YMJ 91102001 (HT) HAST 235 JDR 261 KUMCC 20-0267 (HT) WSP 176 WSP 167 (HT) WSP 88113001 (ET) CBS 656.78 (HT) MUCL 51703 HAST 89112601 HAST 90072903 HAST 94082615 HAST 89091608 HAST 94042903 HAST 90080804 CBS 359.61 JDR 860 HAST 919 HAST 92042501 ITS GU322433 GU300072 JX183075 KX430043 GU292816 AJ390427 AJ390428 MW729422 GU292819 GU292818 MW851888 MW851889 MW851873 MW851874 MW851875 MW85 1876 AJ390430 MW851882 MW851883 GU292817 MW851877 MW851878 MW851879 MW851880 MW851881 EF026122 MW851884 MW851885 MW85 1886 MW85 1887 GU292823 GU292822 DQ641634 EF026121 GU292820 GU292821 MW729423 GU324762 GU324761 GU324760 KT281904 KY610392 GU300071 GU292824 GU322440 EF026120 GU324743 GU322437 KT281903 GU991523 GU322456 GU322439 GenBank Accession number rpb2 GQ844819 GQ844782 KY624280 KX430045 GQ844768 N/A N/A MW717891 GQ844772 GQ844771 MW836056 MW836055 MW836069 MW836071 MW836061 MW836062 DQ631949 MW836058 MW836051 GQ844769 MW836067 MW836068 MW836064 MW836063 MW836070 GQ844770 MW836060 MW836059 MW836065 MW836066 GQ844776 GQ844775 DQ631952 GQ844767 GQ844773 GQ844774 MW717892 GQ853039 GQ853038 GQ853037 KY624278 KY624285 GQ844781 GQ844777 GQ844827 GQ853021 GQ848356 GQ844823 KY624230 GQ848365 GQ848338 GQ844825 §-tubulin GQ487710 GQ470230 JX183072 KX430046 GQ470219 N/A N/A MW881142 GQ470222 GQ470221 MW836030 MW836029 MW836035 MW836036 MW836027 MW836028 DQ840089 MW836026 MW836025 GQ470220 MW836031 MW836032 MW836024 MW836023 MW836037 EF025608 MW836022 MW836021 MW836033 MW836034 GQ470226 GQ470225 DQ840084 EF025607 GQ470223 GQ470224 MW881141 GQ844840 GQ844839 GQ502720 KX271281 KX271253 GQ470229 GQ470227 GQ495933 EF025606 GQ502692 GQ495930 KX271255 GQ502700 GQ495949 GQ495932 o-actin GQ438747 GQ398230 JX183071 KX430044 GQ374123 N/A N/A MW717889 GQ389694 GQ389693 MW836047 MW836046 MW836051 MW836050 MW836042 MW836043 N/A MW836039 MW836038 GQ389692 MW836053 MW836052 MW836045 MW836044 MW836054 EF025593 MW836041 MW836040 MW836048 MW836049 GQ389698 GQ389697 N/A EF025592 GQ389695 GQ389696 MW717890 GQ455451 GQ455450 GQ455449 N/A N/A GQ398229 GQ389699 GQ438754 EF025591 GQ452377 GQ438751 N/A GQ455444 GQ449245 GQ438753 43 References Hsieh et al. (2010) Hsieh et al. (2010) Bills et al. (2012), Wendt et al. (2018) Johnston et al. (2016) Hsieh et al. (2010) Tang et al. (2007) Tang et al. (2007) Tibpromma et al. (2021) Wendt et al. (2018) Hsieh et al. (2010) This study This study This study This study This study This study Tang et al. (2007, 2009) This study This study Hsieh et al. (2010) This study This study This study This study Ariyawansa et al. (2015) Hsieh et al. (2010) This study This study This study This study Hsieh et al. (2010) Hsieh et al. (2010), Li et al. (2015a, b) Tang et al. (2007, 2009) Hsieh et al. (2010) Hsieh et al. (2010), Li et al. (2015a, b) Hsieh et al. (2010) Tibpromma et al. (2021) Hsieh et al. (2010) Hsieh et al. (2010) Hsieh et al. (2010) Senanayake et al. (2015) Wendt et al. (2018) Hsieh et al. (2010) Hsieh et al. (2010) Hsieh et al. (2010) Ju et al. (2007), Hsieh et al. (2010) Hsieh et al. (2010) Hsieh et al. (2010) Senanayake et al. (2015) Hsieh et al. (2010) Hsieh et al. (2010) Hsieh et al. (2010) 44 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) (BI) analysis was performed with MrBayes v.3.1.2 (Huelsenbeck and Ronquist 2001). The branch support was evaluated with a bootstrapping method of 1000 replicates (Hillis and Bull 1993). Posterior probabilities (PP) were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.2.2 (Ronquist et al. 2012). The nu- cleotide substitution model was estimated by MrModeltest v.2.3 (Posada and Crandall 1998). Six simultaneous Markov chains were run for 2000000 generations and the trees were sampled each 100" generation. The first 25% of trees were discarded dur- ing the burn-in phase of each analysis. The phylogenetic trees were viewed in Figtree v.1.4.0 and arranged by Photoshop CS6. ‘The alignments and respective phylogenetic trees were uploaded in TreeBASE (submission number: 28371). Results Phylogenetic analyses The multiple-genes sequence alignments of ITS, «-actin, 7pb2 and 8-tubulin included 67 taxa, 2,041 positions including gaps (ITS: 1-486, «-actin: 487-677, rpb2: 678- 1,715, 8-tubulin: 1,716-2,041). Daldinia bambusicola Y.M. Ju et al. (CBS 122872) and Hypoxylon pulicicidum J. Fourn. et al. (CBS 122622) were selected as the outgroup taxa. A best-scoring ML tree is represented in Fig. 1. RAxML bootstrap support value > 75% and Bayesian posterior probabilities (BYPP) value = 0.90 are shown above the branches and indicated as thickened lines. In the phylogenetic tree (Fig. 1), Nemania Gray is a sister taxon to the genera Rosellinia De Not., Dematophora R. Hartig and Entoleuca Syd. Nemania was divided into six sub-clades. In clade N1, N. bipapillata (Berk. & M.A. Curtis) Pouzar, N. ca- melliae sp. nov. and WN. lishuicola sp. nov. grouped with high statistical values (100/1). In clade N2, N. fusoidispora Q.R. Li et al. and N. illita (Schwein.) Pouzar. grouped with high statistical values (100/1). Clade N3 contained the frequent species NV. diffusa (Sowerby) S.F. Gray along with NV. cyclobalanopsina sp. nov. grouping with high statis- tical values (100/1). In clade N4, WN. feicuiensis sp. nov. with N. abortiva J.D. Rogers et al., NV. aquilariae Vibpromma & Lu and WN. primolutea Y.M. Ju et al. grouped with high statistical values (100/1). Within clade N5, N. macrocarpa Y.M. Ju & J.D. Rog- ers clustered in a well-supported sub-clade with N. maritima Y.M. Ju & J.D. Rogers with high statistical values (100/1). Clade N6 comprised NV. changningensis sp. nov., N. yunnanensis Tibpromma & Lu, N. caries (Schwein.) Y.M. Ju & J.D. Rogers, N. rubi sp. nov., VV. plumbea A.M.C. Tang et al., N. chestersii J.D. Rogers & Whalley) Pouzar, N. serpens (Pers.) Gray with N. aenea (Nitschke) Pouzar, N. aenea var. aureolutea (L.E. Petrini & J.D. Rogers) Y.M. Ju & J.D. Rogers, NV. sphaeriostomum (Schwein.) Lar.N. Vassiljeva & S.L. Stephenson and NV. beaumontii (Berk. & M.A. Curtis) Y.M. Ju & J.D. Rogers grouping with high support values (100% ML, 1 BYPP). A taxonomic study of Nemania from China 10 100/1, Nemania camelliae GMB0067 80/0.97| |'Nemania camelliae GMB0068 0/1 Nemania bipapillata HAST 90080610 Nemania lishuicola GMB0066 100/1' Nemania lishuicola GMB0065 -- Nemania fusoidispora GZUH0098 100/1| 100/1— Nemania illita YMJ 236 100, 99/0.99 100/1 = 1 100/1 100/1 0 0/1 eee vip Nemania diffusa GMB0072 Nemania diffusa GMB0071 Nemania diffusa HAST 91020401 Nemania cyclobalanopsina GMB0061 100/1'Nemania cyclobalanopsina GMB0062 00/1 -- Nemania primolutea YMJ 91102001 _ Nemania aquilariae KUMCC 20-0268 100/1, Nemania feicuiicola GMB0058 Nemania feicuiicola GMB0059 Nemania abortiva BISH 467 Nemania macrocarpa WSP 265 97/1 Teal 78/0.98 100/1 98/1 100/1 100/1 100/1 93/0.99 89/0.99 97/1 87/0.94 -/0.99 98/1 Nemania maritima HAST 89120401 Nemania caries GMB0070 Nemania caries GMB0069 Nemania yunnanensis KUMCC 20-0267 Nemania changningensis GMB0056 100/1' Nemania changningensis GMB0057 100/1) Nemania rubi GMB0064 Nemania rubi GMB0063 Nemania plumbea JF-TH-04-01 Nemania chestersii JF 04024 — Nemania serpens HAST 235 Nemania aenea var. aureolutea ATCC 60819 Nemania aenea CBS 680.86 Nemania sphaeriostoma JDR 261 Nemania beaumontii HAST 405 100/1-— Entoleuca mammata JDR 100 Rosellinia merrillii HAST 89112601 Rosellinia aquila MUCL 51703 Rosellinia sanctae-cruciana HAST 90072903 Dematophora buxi JDR 99 Dematophora necatrix CBS 349.36 Collodiscula fangjingshanensis GZUH0109 Collodiscula tubulosa GACP QRO111 Collodiscula leigongshanensis GZUH0107 Collodiscula bambusae GZUH0102 Astrocystis mirabilis HAST 94070803 Astrocystis bambusae HAST 89021904 100/1, Astrocystis bambusi¢ola MFLUCC 17-0127 Astrocystis concavispora MFLUCC 14-0174 Kretzschmariella culmorum JDR 88 Amphirosellinia fushanensis HAST 91111209 Amphirosellinia nigrospora HAST 91092308 100/1 82/1 Xylaria digitata HAST 919 Xylaria allantoidea HAST 94042903 Stilbohypoxylon quisquiliarum HAST 89091608 ——— Xylaria cubensis JDR 860 100/1-— Xvlaria juruensis HAST 92042501 as Stilbohypoxylon elaeicola HAST 94082615 as Aylaria apoda HAST 90080804 100/1 100/1 100/1 94/1 85 Discoxylaria myrmecophila JDR 169 -———— Brunneiperidium gracilentum MFLUCC 14-0011 —— Brunneiperidium involucratum MFLUCC 14-0009 Podosordaria muli WSP 167 Podosordaria mexicana WSP 176 Xylaria compunctum CBS 359.61 100/1 Poronia punctata CBS 656.78 Poronia pileiformis WSP 88113001 Daldinia bambusicola CBS 122872 — Hypoxylon pulicicidum CBS 122622 Nl N2 N3 N4 N5 N6 45 Outgroup Figure |. RAxML tree based on analysis of a combined dataset of ITS, «-actin, rpb2 and $-tubulin sequences from taxa of Nemania and related genera. Bayesian posterior probability (PP) = 0.90 is marked at the node and the maximum likelihood bootstrap support (BS) values greater than = 75%; a dash (“-”) indicates a value < 0.90 (PP) or < 75% (BS). The strain number is indicated after the species name. The here-studied strains are in bold and new species are indicated in red. 46 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) Taxonomy Nemania camelliae Y.H. Pi & Q.R. Li, sp. nov. MycoBank No: 840086 Fig. 2 Etymology. Refers to the host genus name, camellia. Material examined. Cutna, Guizhou Province, Tongren City, Fanjingshan Na- ture Reserve (27°47'11.41"N, 108°43'43.90"E, altitude: 515 m), on dead wood of Ca- mellia sp., 15 October 2020, Y.H. Pi, 2020FJS26 (GMB0068, holotype; GMBC0068, ex-type living culture; KUN-HKAS 112689, isotype). Description. Saprobic on the surface of decaying wood of Camellia sp. Sexual morph: Stromata pulvinate to effused-pulvinate, rarely perithecioid, orbicular to ir- regularly elongated, often coalescent; single distribution or confluent into irregularly elongated compound stromata, 1.5-4 mm long x 1—2 mm wide x 0.5—1 mm high, surface dull black, hard-textured, with inconspicuous to moderately exposed perithe- cial contours and usually sloping margins, internally black between ascomata, carbo- naceous; subperithecial tissue black, conspicuous; does not release a coloured pigment in 10% KOH. Perithecia 0.65—0.95 mm diam. x 0.65—0.7 mm high, subglobose to depressed-spherical. Ostioles finely papillate, black, conspicuously sunken in a shallow discoid depression; ostiolar area blackish, shiny, frequently flattened. Asci 180-290 x 6-11 um (av. = 230 x 7.5 um, n = 30), 8-spored, unitunicate, long-cylindrical, long- stipitate, the spore-bearing parts 80—95 pm long, apically rounded with a J+, apical apparatus, 2-3 x 2.5—4 um (av. = 2.5 x 3 um, n = 30), jar shape. Ascospores 10-14 x 45-7 um (av. = 12 x 5.5 um, n = 30), uniseriate, unicellular, ellipsoid to slightly fu- soid, inequilateral, with slightly narrow rounded ends, smooth, brown to dark brown, with a fairly conspicuous, straight, almost spore-length germ slit on the least convex side; lacking a sheath and appendage; perispore indehiscent in 10% KOH. Asexual morph: Undetermined. Culture characteristics. The colony grows on PDA medium with a diameter of 6 cm after one week at 25 °C; white, cottony, circular, flocculent or velvety, with light yellow to slightly yellow at the centre. Not sporulating on OA nor on PDA. Other examined material. CHINA, Guizhou Province, Tongren City, Fanjin- gshan Nature Reserve (27°42'10.26"N, 108°31'35.34"E, altitude: 426 m), on dead wood of Camellia sp., 16 October 2020, Y.H. Pi, 2020FJS54-1 (GMB0067), living culture, GMBC0067. Notes. Phylogenetic analyses showed that Nemania camelliae form a distinct clade with NV. bipapillata (82% ML, 0.97 BYPP, Fig. 1). Morphologically, N. camelliae is similar to NV. immersidiscus Van der Gucht et al. in having a small discoid depression around the ostiolar papilla. However, the stromata of NV. camelliae are entirely carbona- ceous, whereas those of NV. immersidiscus contain white soft tissue between and beneath the perithecia Ju and Rogers 2002). Moreover, NV. immersidiscus has slightly thinner ascospores [(10—)11—14(—16) x (4~-)4.5—5.5 um)]. A taxonomic study of Nemania from China 47 Figure 2. Nemania camelliae (GMBO0068, holotype) A type material B, C stromata on the surface of host D transverse section of stroma E longitudinal section of stroma FH asci with ascospores I pigments in 10% KOH J ascospore with indehiscent perispore in 10% KOH K ascus apical apparatus (stained in Melzer’s Reagent) L, M ascospores N, O colonies on PDA (N-upper, O-lower). Scale bars: 0.5 mm (C-E); 10 um (F=H, J=-M). 48 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) Nemania caries (Schwein.) Y.M. Ju & J.D. Rogers, Nova Hedwigia 74(1—2): 90 (2002) MycoBank No: 477305 Fig. 3 Synonyms. Sphaeria caries Schwein., Trans. Am. phil. Soc., New Series 4(2): 194 (1832). Hypoxylon caries (Schwein.) Sacc., Syll. fung. (Abellini) 1: 393 (1882). Hypoxylon balansae Speg., Anal. Soc. cient. argent. 26(1): 30 (1888). Description. Saprobic on the surface of decaying wood. Sexual morph: Stromata ir- regularly effused-pulvinate, 5.5-18 mm long x 3-9 mm wide x 0.4-0.6 mm thick, with conspicuous perithecial mounds, surface blackish-grey, carbonaceous, interior white, loosely fibrous to cottony; mature stromata lacking KOH extractable pigments. Perithecia 0.25—0.5 mm wide x 0.4-0.6 mm high, obovoid. Ostioles slightly higher than stromatal surface and with openings conic-papillate, black, inconspicuous, with- out encircling disc. Asci 130-200 x 7-13 um (av. = 150 x 9.5 um, n = 30), 8-spored, cylindrical, unitunicate, long-stipitate, the spore-bearing parts 65-95 um long, api- cally rounded with a J+, short-cylindrical apical apparatus, 1.5—-2.5 x 1-2.5 um (av. = 2 x 1.5 um, n = 30). Ascospores 9—13.5 x 3—7 wm (av. = 11.5 x 5 pm, n = 30), brown to light brown, smooth, with an inconspicuous, straight, germ slit 1/3 spore-length, nearly equilateral, with broadly rounded ends; perispore indehiscent in 10% KOH. Asexual morph: Undetermined. Culture characteristics. Colonies grow on PDA at 25 °C for two weeks, with a diameter of 4 cm. Colony on the surface is white or light orange, shallow, flat, zonnate, with irregular edges and orange on the reverse side. The colony reverse is orange. Not sporulating on OA nor on PDA. Material examined. Cuina, Yunnan Province, Changning County, Lancang Riv- er Nature Reserve (25°01'13.56"N, 99°35'25.12"E, altitude: 2626 m), on dead wood, 6 October 2019, Y.H. Pi, 2019LC369 (GMB0070, KUN-HKAS 112680), living cul- ture, GMBC0070; CHINA, Yunnan Province, Changning County, Lancang River Nature Reserve (25°01'13.33"N, 99°35'26.55"E, altitude: 2641 m), on dead wood, 6 October 2019, Y.H. Pi, 2019LC401 (GMB0069, KUN-HKAS 112682), living cul- ture, GMBC0069. Known distribution. Hawaii (Rogers and Ju 2012), Martinique (Fournier et al. 2018), Paraguay, USA (Ju and Rogers 2002), Yunnan Province, China (this paper). Notes. The phylogenetic analyses show Nemania caries groups with N. changnin- gensis with high statistical support (100% ML, 1 BYPP, Fig. 1) and the comparison calculation within the alignment found that there is a 4% difference in ITS sequences between NV. changningensis and N. caries. Morphologically, N. caries resembles N. colu- brina J. Fourn. & Lechat which has medium brown ascospores and a similar size of ascospores. However, NV. colubrina differs from N. caries by ellipsoid-inequilateral ascospores with narrowly-rounded ends (Ju and Rogers 2002; Fournier et al. 2018). Nemania caries is distinguished from N. plumbea by its dimension of ascospores, the A taxonomic study of Nemania from China Figure 3. Nemania caries (GMBO0070) A type material B, C stromata on the surface of host D transverse sections of stromata E longitudinal section of stroma F—H asci with ascospores I pigments in 10% KOH J ascospore with indehiscent perispore in 10% KOH K ascus apical apparatus (stained in Melzer’s Reagent) L, M ascospores N, O Colonies on PDA (N-upper, O-lower). Scale bars: 0.5 mm (C=E); 10 um (F—H, J—M). 50 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) latter has larger ascospores (13—16 x 5.4—6.6 um) with narrowly-rounded ends (Tang et al. 2007). The specimens we collected from the Lancang River Nature Reserve in Yunnan fit the definition of NV. caries well and represent the first record from China. Nemania changningensis Y.H. Pi & Q.R. Li, sp. nov. MycoBank No: 840087 Fig. 4 Etymology. Refers to the collection location, Changning County. Material examined. Cuina, Yunnan Province, Changning County, Lancang Riv- er Nature Reserve (25°01'35.02"N, 99°33'15.42"E, altitude: 2670 m), on dead wood, 3 October 2019, Y.H. Pi, 2019LC203 (GMB0056, holotype; GMBC0056, ex-type living culture; KUN-HKAS 112668, isotype). Description. Saprobic on the surface of decaying wood. Sexual morph: Stroma- ta effused-pulvinate, confluent into irregularly elongated compound stromata, up to 18-35 mm long x 2-4 mm wide x 0.3—-0.5 mm high, irregularly lobed, plane or with inconspicuous perithecial mounds and sloping margins; surface covered with white tis- sue, persistent layer, with blackish-grey carbonaceous sub-surface showing through in places; the tissue beneath the perithecial layer inconspicuous, greyish-white in places, the underlying wood blackened; mature stromata lacking KOH extractable pigments. Perithecia 0.45—0.6 mm diam. x 0.4—0.55 mm high, subglobose to depressed-spher- ical. Ostioles slightly higher than stromatal surface and with openings papillate, of- ten surrounded by white tissue, inconspicuous, black, without encircling disc. Asci 100-140 x 7-10 um (av. = 111 x 8.5 um, n = 30), 8-spored, unitunicate, cylindrical, short-stipitate, the spore-bearing parts 70-90 um long, the apical apparatus of im- mature asci blue in Melzer’s Reagent, but not blue in mature asci. Ascospores 10-13 x 4—6.5 um (av. = 11.5 x 5.5 um, n = 30), uniseriate unicellular, smooth, light brown, slightly inequilateral, with broadly rounded ends, inconspicuous or lack a germ slit; perispore indehiscent in 10% KOH. Asexual morph: Undetermined. Culture characteristics. The colony grows slowly on the PDA with a diameter of 4.5 cm after 2 weeks at 25 °C. The colony on the surface is white, thick and flat in the middle, edges are shallow, irregular bands and rosettes. Colony reverse is orange and intermediate colour darker. Not sporulating on OA nor on PDA. Other examined material. Cumna, Yunnan Province, Changning County, Lancang River Nature Reserve (25°01'30.36"N, 99°35'30.53"E, altitude: 2586 m), on dead wood, 4 October 2019, Y.H. Pi, 2019LC342 (GMB0057), living culture, GMBC0057. Notes. In the phylogenetic analyses, N. changningensis is on a separate branch and grouped with WN. caries with high support values (100% ML, 1 BYPP, Fig. 1). In term of ascospores dimension, NV. changningensis resembles N. caries, but differs in the peri- thecia of NV. caries (obovoid, 0.3—0.6 mm diam. x 0.5—0.7 mm high), in the surface not covered with white tissue and in its apical apparatus of mature asci bluing in Melzer’s Reagent (Miller 1961; Ju and Rogers 2002). A taxonomic study of Nemania from China Figure 4. Nemania changningensis (GMB0056, holotype) A type material B, C stromata on the surface of host D transverse sections of stromata E longitudinal section of stroma F=H asci with ascospores I pig- ments in 10% KOH J, K asci apical apparatus (stained in Melzer’s Reagent) L, M ascospores N, O colo- nies on PDA (N-upper, O-lower). Scale bars: 0.5 mm (C=E); 10 um (F-H, J—-M). 52 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) Nemania cyclobalanopsina Y.H. Pi & Q.R. Li, sp. nov. MycoBank No: 840088 Fig. 5 Etymology. Refers to its host, Cyclobalanopsis glauca. Material examined. Cuina, Yunnan Province, Changning County, Lancang Riv- er Nature Reserve (25°01'9.46"N, 99°35'29.47"E, altitude: 2623 m), on dead wood of C. glauca, 6 October 2019, Y.H. Pi, 2019LC357 (GMB0062, holotype; GMBC0062, ex-type living culture; KUN-HKAS 112679, isotype). Description. Saprobic on the surface of decaying branches of C. glauca (Thunb.) Oerst. Sexual morph: Stromata effused-pulvinate, orbicular to ellipsoid or irregularly lobed, 6-26 mm long x 3.5—10 mm wide x 0.5—1 mm thick, occasionally confluent into larger compound stromata, with steep to sloping margins; surface light blackish, slightly blood colour; outer crust carbonaceous; interior black, entire tissue carbo- naceous around the perithecia; mature stromata lacking KOH-extractable pigments. Perithecia 0.2-0.3 mm diam. x 0.38-0.46 mm high, subglobose obovoid or tubu- lar. Ostioles higher than stromatal surface and with coarsely rounded-papillate, black, without encircling disc. Asci 90-160 x 7-11 um (av. = 125 x 9 um, n = 30), 8-spored, unitunicate, cylindrical, long-stipitate, the spore-bearing parts 65-85 um long, api- cally rounded with a J+, short-cylindrical to slightly tubular apical apparatus stained in Melzer’s Reagent, 1.5—2.5 x 2-3 um (av. = 2 x 2.3 um, n = 30). Ascospores 9-14 x 4.5-7.5 um (av. = 11 x 6 um, n = 30), uniseriate, unicellular, ellipsoid-inequilater- al with broadly rounded ends, smooth, brown to dark brown, with a conspicuous, straight germ slit slightly less than spore-length to almost spore-length on the convex side; lacking a sheath and appendage; perispore indehiscent in 10% KOH. Asexual morph: Undetermined. Culture characteristics. Colonies on PDA medium in size with a diameter of 6 cm after two weeks at 25 °C; the surface is white, intermediate thick, cottony, dense, with undulate or ring edge, flat, low, whitish-yellow, reverse of the colony yellow at the centre. Not sporulating on OA nor on PDA. Other examined material. Cu1na, Yunnan Province, Changning County, Lan- cang River Nature Reserve (25°52'17.40"N, 99°35'20.53"E, altitude: 1489 m), on dead wood of C. glauca, 4 October 2019, Y.H. Pi, 2019LC357-1 (GMB0061), living culture, GMBC0061. Notes. In our phylogenetic analyses, NV. cyclobalanopsina grouped with N. diffusa (100% ML, 1 BYPP, Fig. 1). Morphologically, NV. cyclobalanopsina differs from N. dif- fusa by its blackish stromatal surfaces and coarsely rounded-papillate ostioles. Moreo- ver, NV. diffusa has larger perithecia (0.3—-0.6 x 0.4-0.8 mm) (Granmo et al. 1999; Ju and Rogers 2002). In the multi-gene phylogenetic analysis, N. cyclobalanopsina ap- peared in a separate branch which is distinct from NV. diffusa (Fig. 1). Moreover, there is a 3% difference in ITS sequences between JN. diffusa and N. cyclobalanopsina. (Vu et al. 2019; Jeewon and Hyde 2016). A taxonomic study of Nemania from China 53 Figure 5. Nemania cyclobalanopsina (GMB0062, holotype) A type material B, C stromata on the sur- face of host D transverse sections of stromata E longitudinal sections of stromata F-H asci with ascospores I pigments in 10% KOH J ascospore with indehiscent perispore in 10% KOH K ascus apical apparatus (stained in Melzer’s Reagent) L, M ascospores N, O colonies on PDA (N-upper, O-lower). Scale bars: 0.5 mm (C-E); 10 um (F-H, J-M). 54 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) Nemania diffusa (Sowerby) S.F. Gray, Nat. Arr. Brit. Pl.: 517 (1821) MycoBank No: 477312 Fig. 6 Synonyms. Sphaeria diffusa Sowerby, Col. fig. Engl. Fung. Mushr. (London) 3(no. 25): tab. 373, fig. 10 (1802) Sphaeria unita Fr., Elench. fung. (Greifswald) 2: 67 (1828) Sphaeria exarata Schwein., Trans. Am. phil. Soc., New Series 4(2): 192 (1832) Hypoxylon exaratum (Schwein.) Sacc., Syll. fung. (Abellini) 1: 392 (1882) Ustulina linearis Rehm, Hedwigia 31(6): 310 (1892) Hypoxylon lilacinofuscum Bres., Fl. Trident. Nov. 2: 43 (1892) Hypoxylon cohaerens vat. brasiliense Starback, Bih. K. svenska VetenskAkad. Handl., Afd. 3 27(no. 9): 8 (1901) Hypoxylon vestitum Petch, Ann. R. bot. Gdns Peradeniya 8: 156 (1924) Nemania unita (Fr.) Krieglst. & Enderle, Mitteilungsblatt der Arbeitsgemeinschaft Pilzkunde Niederrhein 1: 64 (1989) Description. Saprobic on the surface of rotten wood. Sexual morph: Stromata ef- fused-pulvinate, clear outline, ellipsoid or irregularly lobed, occasionally confluent into a larger compound stromata, 2—20 mm long x 2—9 mm wide x 0.5—1 mm thick, with conspicuous perithecial mounds, carbonaceous between the perithecia, surface dark brown or brown; the inter-perithecial tissue blackish, carbonaceous; does not release a coloured pigment in 10% KOH. Perithecia 0.3-0.55 diam. x 0.4-0.7 mm high, subglobose to obovoid. Ostioles finely conic-papillate, black, shiny. Asci 130-250 x 6-10 um (av. = 170 x 8 um, n = 30), 8-spored, unitunicate, cylindrical, long-stipi- tate, the spore-bearing parts 70-90 um, apically rounded with a J+ apical apparatus, 1.5—2.5 x 2—3.5 pum (av. = 2 x 2.6 um, n = 30), tubular with a faint upper rim, bluing in Melzer’s Reagent. Ascospores 9.5—13 x 4.5—7 pm (av. = 11 x 5.5 um, n = 30), uni- cellular, ellipsoid-inequilateral, with narrowly-rounded ends, smooth, brown to dark brown, with a conspicuous, straight germ slit spore-length to slightly less than spore- length on the ventral side; lacking a sheath and appendage; perispore indehiscent in 10% KOH. Asexual morph: Undetermined. Culture characteristics. Colonies grow on PDA at 25 °C for a week reaching a diameter of 5 cm. Colonies are cotton white in colour, flocculent or velvety, dense, circular, radial. On the reverse, white edge, light yellow in the middle. Not sporulating on OA nor on PDA. Material examined. Cutna, Guizhou Province, Tongren City, Fanjingshan Na- ture Reserve (27°53'46.59"N, 108°431'16.29"E, altitude: 1058 m), on dead wood, 14 October 2020, Y.H. Pi, 2020FJS1 (GMB0072, KUN-HKAS 112686), living culture, GMBC0072; CHINA, Yunnan Province, Changning County: Lancang River Nature Reserve (21°54'17.44"N, 107°54'10.05"E, altitude: 1382 m), on dead wood, 1 Oc- tober 2019, Y.H. Pi, 2019LC008 (GMB0071, KUN-HKAS 112658), living culture, GMBC0071. A taxonomic study of Nemania from China Figure 6. Nemania diffusa (GMB0072) A specimen B, C stromata on the surface of host D transverse sections of stromata E longitudinal sections of stromata FH asci with ascospores | pigments in 10% KOH J ascospore with indehiscent perispore in 10% KOH K ascus apical apparatus (stained in Melzer’s Reagent) L, M ascospores N, O colonies on PDA (N-upper, O-lower). Scale bars: 0.5 mm (C=E); 10 um (F—H, J—M). 56 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) Notes. The new collection morphologically resembles NV. diffusa (Gray 1821), hav- ing effused-pulvinate carbonaceous stromata with inconspicuous perithecial mounds, brown to dark brown ellipsoid-inequilateral ascospores (9.5—13.5 x 5-6 um), with narrowly-rounded ends and a long germ slit on the ventral side (Granmo et al. 1999; Ju and Rogers 2002). Fournier et al. (2018) predicted that N. diffusa might be a spe- cies complex as it is difficult to identify, based solely on morphology, thus, it should be evaluated after extensive sampling and using DNA-based taxonomy. In phylogenetic analyses of combined ITS, 7p62, 8-tubulin and «-actin genes (Fig. 1), new collections clearly showed its close kinship with NV. diffusa. Only a 2% difference of ITS sequences existed between our strains and N. diffusa (HAST 91020401, authoritative strain). Therefore, we regard the new collection as N. diffusa. Nemania carbonacea Pouzat. can be confused with NV. diffusa by having the same dark ascospores and nearly spore- length germ slits. However, N. carbonacea has white, soft stromatal tissue between the perithecia (Ju and Rogers 2002). Nemania feicuiensis Y.H. Pi & Q.R. Li, sp. nov. MycoBank No: 840089 Fig. 7 Etymology. Refers to the collection location, Emerald Park, Chinese name of jade, feicui. Material examined. Cuina, Hainan Province, Wuzhishan City, Emerald Park (18°48'9.64"N, 109°31'6.59"E, altitude: 352 m), on dead wood, 14 November 2020, Y.H. Pi, 2020FCGY12-2 (GMB0059, holotype; GMBC0059, ex-type living culture; KUN-HKAS 112698, isotype). Description. Saprobic on the surface of decaying wood. Sexual morph: Stromata effused-pulvinate, superficial, orbicular to ellipsoid or irregularly lobed, 5—27 mm long x 2.5-10 mm wide x 0.3-0.5 mm thick, surface blackish-grey, with inconspicuous perithecial outer mounds, crust weakly carbonaceous; interior black, stromatal tissue between the perithecia carbonaceous; mature stromata lacking KOH extractable pig- ments. Perithecia 0.3—0.55 mm diam. x 0.25—0.37 mm high, subglobose to depressed- spherical. Ostioles higher than stromatal surface and with openings slightly papillate, black, conspicuous, without encircling disc. Asci 130-180 x 7—11.5 um (av. = 145 x 9 um, n = 30), 8-spored, unitunicate, cylindrical, long-stipitate, the spore-bearing parts 65-85 um long, apically rounded with a J+ apical apparatus, 1—2.5 x 2—3 um (av. = 1.8 x 2.4 um, n = 30), long-cylindrical. Ascospores 9.5—13 x 4—7.5 wm (av. = 11 x 6 um, n = 30), uniseriate, unicellular, ellipsoid or slightly inequilateral, with broadly rounded ends, smooth, brown to dark brown, with a conspicuous, straight, almost spore-length germ slit on the flattened side; lacking a sheath and appendage; perispore indehiscent in 10% KOH. Asexual morph: Undetermined. Culture characteristics. Colonies grow slowly on PDA at 25 °C for 2 weeks, with a diameter of 5 cm. Colonies are cotton white in colour, flocculent or velvety, slightly convex, circular, shallow edges, radial, white to light yellow on the reverse, light brown in the middle. Not sporulating on OA nor on PDA. A taxonomic study of Nemania from China Figure 7. Nemania feicuiensis (GMBO0059, holotype) A type material B, C stromata on the surface of host D transverse sections of stromata E longitudinal sections of stromata F—H asci with ascospores | pigments in 10% KOH J ascospore with indehiscent perispore in 10% KOH K ascus apical apparatus (stained in Melzer’s Reagent) L, M ascospores N, O colonies on PDA (N-upper, O-lower). Scale bars: 0.5 mm (C-E); 10 um (F-H, J-M). 58 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) Other examined material. Cuina, Hainan Province, Wuzhishan City, Emerald Park (18°47'8.26"N, 109°31'5.34"E, altitude: 426 m), on dead wood, 16 November 2020, Y.H. Pi, 2020FCGY20 (GMB0058), living culture, GMBC0058. Notes. ‘The phylogenetic tree (Fig. 1) shows that NV. feicuiensis and N. primolutea are closely related (100% ML, 1 BYPP). In morphology, NV. feicuiensis differs from N. primolutea in that the latter has luteous stromatal surface and slightly smaller as- cospores (10-13 x 4.5—-5.5 um) with narrowly-rounded ends (Ju et al. 2005). Fur- thermore, in the multi-gene phylogenetic analysis, NV. feicuiensis appeared in a separate branch which is distinct from NV. primolutea (Fig. 1). Nemania feicuiensis is similar to N. diffusa in stromatal anatomy and ascospores size, but differs by ascospores shape (broadly rounded ends vs. narrowly rounded ends) and the larger perithecia of N. dif- fusa (0.3-0.6 x 0.4—-0.8 mm) (Ju and Rogers 2002). Nemania lishuicolaY .H. Pi & Q.R. Li, sp. nov. MycoBank No: 840090 Fig. 8 Etymology. Refer to the host, quercus. Material examined. Cuina, Yunnan Province, Changning County: Lancang River Nature Reserve (25°01'7.93"N, 99°35'30.74"E, altitude: 2629 m), on dead bark of Quercus sp., 4 October 2019, Y.H. Pi, 2019LC263 (GMB0065, holotype; GMBC0065, ex-type living culture; KUN-HKAS 112673, isotype). Description. Saprobic on the surface of decaying wood of Quercus sp. Sexual morph: Stromata pulvinate, attached to substrate along entire area of the base, con- taining one to several perithecia, frequently confluent, 1.5—4 mm long x 1-2 mm wide x 0.5—1 mm thick, with conspicuous perithecial mounds, carbonaceous be- tween the perithecia, surface dull black and slightly shiny at maturity, the inter- perithecial tissue blackish, carbonaceous; not releasing a coloured pigment in 10% KOH. Perithecia 0.7-0.95 mm diam. x 0.65—0.85 mm high, subglobose to de- pressed-spherical. Ostioles coarsely papillate in discoid areas, ostiolar area blackish, shiny, frequently flattened, usually around a circle of white tissue. Asci 150-300 x 7-12 pm (av. = 200 x 9 um, n = 30), 8-spored, unitunicate, cylindrical, long-stip- itate, spore-bearing parts 95-130 um long, apically rounded with a J+ apical ap- paratus, 2—3 x 2—3.5 um (av. = 2.5 x 3 um, n = 30), tubular with a faint upper rim. Ascospores 12.5—17 x 5—8.5 um (av. = 15 x 6.5 um, n = 30), uniseriate, unicellular, ellipsoid-inequilateral, with broadly rounded ends, smooth, brown to dark brown, with a conspicuous, straight germ slit spore-length to slightly less than spore-length on the flattened side; lacking a sheath and appendage; perispore indehiscent in 10% KOH. Asexual morph: Undetermined. Culture characteristics. Colonies grow on PDA, a diameter of 6 cm after one week at 25 °C, white, velvety to hairy, zonnate, rosette, high convex in centre, dense, white to cream from above, white irregular edge with light yellow to slightly yellow at centre from the below. Not sporulating on OA nor on PDA. A taxonomic study of Nemania from China a) Figure 8. Nemania lishuicola (GMB0065, holotype) A type material B stromata on the surface of host C pigments in 10% KOH D transverse sections of stromata E longitudinal sections of stromata F ascospore with indehiscent perispore in 10% KOH Gel asci with ascospores J ascus apical apparatus (stained in Melzer’s Reagent) K, L colonies on PDA (K-upper, L-lower) M=P ascospores. Scale bars: 0.5 mm (B, D, E); 10 um (F-J, M—-P). 60 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) Other examined material. Cina, Yunnan Province, Changning County: Lan- cang River Nature Reserve (25°01'30.75"N, 99°35'21.53"E, altitude: 2608 m), on dead bark of Quercus sp., 4 October 2019, Y.H. Pi, 2019LC253 (GMB0066), living culture, GMBC0066. Notes. Phylogenetic analyses of combined ITS, 7pb2, 8-tubulin and «-actin genes (Fig. 1) show that NV. Lishuicola has a close relationship with N. bipapillata with high support values (100 MLBP, 1% BYPP). Morphologically, N. lishuicola differs from N. bipapillata by its larger ascospores (12.5—17 x 5—8.5 um vs. 10.5-13.5 x 4.5—6 um) (Miller 1961; Ju and Rogers 2002). Nemania rubryY.H. Pi & Q.R. Li, sp. nov. MycoBank No: 840091 Fig. 9 Etymology. Refers to the name of host genus, rubus. Material examined. Cutna, Guizhou Province, Pingba County (26°25'13.38"N, 106°24'25.23"E, altitude: 1255 m), on dead branches of Rubus lambertianus Ser., 5 September 2020, Y.H. Pi, 2020PB70 (GMB0064, holotype; GMBC0064, ex-type liv- ing culture; KUN-HKAS 112695, isotype). Description. Saprobic on dead branches of R. lambertianus. Sexual morph: Stromata effused-pulvinate, irregular shape, multi-peritheciate, scattered, separate to confluent into larger compound stromata, 2.5—-15 mm long x 2—9 mm wide x 0.4—0.6 mm thick; sur- face blackish, weakly carbonaceous, with unexposed perithecial contours, uneven and ir- regular, internally whitish between ascomata, tissue, soft-textured; not releasing a coloured pigment in 10% KOH. Perithecia 0.25—0.35 mm diam. x 0.2—0.3 mm high, subglobose. Ostioles papillate, black, obtusely conical to hemispherical, without encircling disc. Asci 85-160 x 7-11 um (av. = 130 x 9 um, n = 30), 8-spored, unitunicate, cylindrical, long- stipitate, spore-bearing parts 60-85 ym long, apically rounded with a J+, long-cylindrical apical apparatus, 1.5—2.5 x 2-3 um (av. = 1.5 x 2.5 um, n = 30). Ascospores 9-12 x 4-6 um (av. = 10 x 4.8 um, n = 30), uniseriate to irregularly-biseriate unicellular, smooth, olivaceous when fresh, turning brown to medium brown after a period of time, ellipsoid- inequilateral with often broadly-rounded ends, lacking a germ slit sheath and appendage; perispore indehiscent in 10% KOH. Asexual morph: Undetermined. Culture characteristics. Colonies grow slowly on PDA medium with a diameter of 5 cm after 10 days at 25 °C. Colonies surface were white to pale orange, circular, cot- tony, low, dense, cottony mycelium, reverse with light orange mycelium. Not sporulat- ing on OA nor on PDA. Other examined material. Curina, Guizhou Province, Pingba County (26°25'10.24"N, 106°24'25.21"E, altitude: 1052 m), on dead wood, 5 September 2020, Y.H. Pi, 2020PB22 (GMB0063), living culture, GMBC0063. Notes. In our phylogenetic analysis, Nemania rubi formed a distinct branch, which is sister to VV. changningensis and N. caries (Fig. 1). In morphology, N. rudi is similar to A taxonomic study of Nemania from China 61 Figure 9. Nemania rubi (GMB0064, holotype) A type material B, C stromata on surface of host D transverse sections of stromata E longitudinal section of stromata F—H asci with ascospores I pigments in 10% KOH J ascospore with indehiscent perispore in 10% KOH K ascus apical apparatus (stained in Melzer’s Reagent) L, M ascospores N, O colonies on PDA (N-upper, O-lower). Scale bars: 0.5 mm (C=E); 10 um (F—H, J—-M). 62 YinHui Pi et al. / MycoKeys 83: 39-67 (2021) N. caries, but is distinct in having a long-cylindrical apical apparatus and the inequi- lateral ascospores lacking a germ slit (Miller 1961; Ju and Rogers 2002). In addition, the perithecia of NV. caries are obovoid (0.3—0.6 x 0.5—-0.7 mm) and its height is greater than the width (Tang et al. 2007). The ascomata surface of NV. rubi ascomata is uneven with inconspicuous perithecial mounds, which is similar to those of N. plumbea, but the latter has larger ascospores (13-16 x 5.4—6.6 um) with germ slits on the concave side (Tang et al. 2007). Discussion In this study, newly-collected Nemania species from Hainan, Yunnan and Guizhou Provinces were subjected to morpho-molecular analyses. Six new species were intro- duced while reporting one new record from China. Nemania showed a closer affinity to Roselinia than to Kretzschmaria Fr. and Xylaria (U’Ren et al. 2016), which is also supported in the phylogenetic analysis, based on ITS, 7p62, -tubulin and «-actin sequences. Although no asexual morphs were observed in this study, Nemania has geniculisporium-like asexual morphs which are a common character in members of Xylariaceae (Fournier et al. 2018). Nemania forms a single branch in the phylogenetic analysis, which supports that it is a monophyletic genus. However, Nemania genus is separated into six clades (NI-N6, Fig. 1), each of which have relatively-uniform morphological characteristics. N1 clade is represented by NV. bipapillata and taxa in this clade have carbonaceous interior to the stromata, ostioles encircled with a disc and dark brown ascospores with a long germ slit. The species within clade N2 are distinguished from other Nemania species with fusoid- inequilateral and pale brown ascospores and by having white soft tissues between the perithecia. The species in clades N3, N4 and N5 have little difference in morphology and may be confused. Most taxa in clades N4 and N5 have usually brown, dark brown or blackish-brown ascospores with a germ slit longer than 2/3 spore length (Granmo et al. 1999; Ju and Rogers 2002; Fournier et al. 2018). The taxa in NG clade have light brown or medium brown ascospores with a germ slit shorter than 2/3 spore length or seemingly lacking (Ju and Rogers 2002). Interestingly, the ascospores of most taxa in N6 clade are olivaceous brown when fresh, turning medium brown after desiccation. Separation of members of Nemania, based on morphology, is relatively difficult and confusing (Fournier et al. 2018). In some early literature, the new species lacked the description of some key morphological characteristics (Du et al. 2016). Moreover, sequences are available for only a few species in GenBank, thus species identification, based on DNA sequences, is also problematic. Hence, it is essential to re-collect old species that lack ex-type cultures and DNA sequences and to epitypify them. The similarity of morphological features between species is high, which makes it difficult for existing morphological taxonomic features to identify species. For exam- ple, species in clade N3, which includes N. diffusa and N. cyclobalanopsina, are difficult to identify, based solely on morphological characteristics, although their ITS sequence A taxonomic study of Nemania from China 63 differences can reach more than 3% (Jeewon and Hyde 2016; Vu et al. 2019). In this clade, we tentatively use multiple-genes sequence as the main classification basis for species. Molecular data should be the main identification basis for Nemania species, especially for clade N3. It is worth noting that we should compare sequences with that from type or authoritative strains. Acknowledgements This research was supported by the National Natural Science Foundation of China (32000009 and 31960005); the Fund of the Science and Technology Foundation of Guizhou Province ([2020]1Y059); the Fund of Special Project of Academic New Seedling Cultivation and Innovation Exploration in Guizhou Medical University [2018]5779-64; Guizhou Province Ordinary Colleges and Universities Youth Science and Technology Talent Growth Project [2021]154; National Natural Science Founda- tion of China [No. U1812403-4-4], the Fund of High-Level Innovation Talents [No. 2015-4029], the Base of International Scientific and Technological Cooperation of Guizhou Province [No. [2017]5802]; Yinggian Kang is grateful to the 111 Project (D20009) and Talent Base Project of Guizhou Province, China (FCJD2018-22). 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