683 MycoKeys MycoKeys 121: 93-109 (2025) DOI: 10.3897/mycokeys.121.158724 Research Article Morphological and phylogenetic analyses reveal two new species of Tubeufia (Tubeufiales, Tubeufiaceae) from freshwater habitats in China Xiao-Yan Ma’®, Fu Tian'®, Jiang-Fen Feng'®, Min-Min Wang'®, Huan-Huan Shi'®, Jian Ma’#® 1 School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang 550003, China 2 Guizhou Key Laboratory of Agricultural Microbiology, Guizhou Academy of Agricultural Sciences, Guiyang 550009, China 3 Guizhou Industry Polytechnic College, Guiyang 550008, China Corresponding authors: Fu Tian (tianfu@git.edu.cn); Jian Ma (yanmajian@163.com) OPEN Qaccess This article is part of: Exploring the Hidden Fungal Diversity: Biodiversity, Taxonomy, and Phylogeny of Saprobic Fungi Edited by Samantha C. Karunarathna, Danushka Sandaruwan Tennakoon, Ajay Kumar Gautam Academic editor: Samantha C. Karunarathna Received: 12 May 2025 Accepted: 2 July 2025 Published: 21 August 2025 Citation: Ma X-Y, Tian F, Feng J-F, Wang M-M, Shi H-H, Ma J (2025) Morphological and phylogenetic analyses reveal two new species of Tubeufia (Tubeufiales, Tubeufiaceae) from freshwater habitats in China. Mycokeys 121: 93-109. https://doi. org/10.3897/mycokeys.121.158724 Copyright: © Xiao-Yan Ma et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Tubeufia is a genus of helicosporous hyphomycetes distinguished by a high degree of morphological variation in its asexual morphs. During an investigation of helicosporous fungi in tropical regions, four fungal strains were isolated from submerged decaying wood in Hainan Province, southern China. Multigene phylogenetic analyses based on a combined ITS-LSU-tef1-a-rpb2 sequence dataset were conducted to determine the phylogenetic placement of the four fungal strains, which showed two distinct new spe- cies. Morphological characteristics were also used to support their taxonomic delimita- tion in addition to the molecular phylogeny. Two novel species, Tubeufia yanuodaensis and T. yinggelingensis, are described and illustrated. Key words: 2 new species, helicosporous hyphomycetes, phylogeny, saprobic fungi, taxonomy Introduction Tubeufia was described by Penzig and Saccardo (1897) with T. javanica as the type species based on morphological characteristics. Currently, Tubeufia com- prises 86 species, of which 27 are found in freshwater habitats, 44 in terres- trial habitats, and 15 in both freshwater and terrestrial habitats (Penzig and Saccardo 1897; Barr 1979; Rossman 1987; Lu et al. 2017, 2018a, 2018b, 2022, 2023; Lu and Kang 2020; Tian et al. 2022; Ma et al. 2024; Lu et al. 2025). These species are primarily saprobic on decaying wood in tropical and temperate re- gions—predominantly in China and Thailand (Chaiwan et al. 2017; Dai et al. 2017; Doilom et al. 2017; Lu et al. 2017, 2018a, 2018b, 2022, 2023; Luo et al. 2017; Kuo and Goh 2018a, 2018b; Tibpromma et al. 2018; Lu and Kang 2020; Li et al. 2022; Tian et al. 2022; Ma et al. 2023, 2024; Lu et al. 2025). Tubeufia species are widely distributed in Austria, Bermuda, Brazil, Canada, China, Colombia, Cuba, India, New Zealand, Panama, Peru, South Africa, Sri Lan- ka, Tanzania, Thailand, Trinidad, Uganda, Venezuela, and the USA (Morgan 1892; Talbot 1956; Moore 1957; Rao and Rao 1964; Deighton and Pirozynski 1966; Munk 93 Xiao-Yan Ma et al.: Introducing two new Tubeufia species 1966; Ellis 1971; Panwar et al. 1973; Barr 1979, 1980; Barr and Rogerson 1983; Goos 1985, 1990; Matsushima 1987, 1993; Rossman 1987; Holubova-Jechova 1988; Ma et al. 2023, 2024; Lu et al. 2025). The asexual morph of Tubeufia is con- sidered the most morphologically diverse among helicosporous hyphomycetes (Ma et al. 2023, 2024). For instance, the conidial morphology of Tubeufia species exhibits a wide range of forms, including dorsiventrally curved, coiled, ovate, el- lipsoid to ovoid, spherical to obclavate, and subreniform conidia, occasionally bearing one or more small, globose secondary conidia (Tsui and Berbee 2006; Tsui 2007; Zhao et al. 2007; Lu et al. 2018b; Ma et al. 2023, 2024). In this study, two saprobic Tubeufia species were collected from submerged decaying wood in Hainan Province, China. Based on morphological character- istics and multigene phylogenetic analyses, two novel species, Tubeufia yanuo- daensis and T. yinggelingensis, are described and illustrated. Comparisons with closely related taxa are also provided to support their taxonomic placements. Materials and methods Sample collection, specimen examination, and isolation Samples of submerged decaying wood pieces were collected from Hainan Province in southern China between October 2021 and July 2022, and the col- lection details were recorded (Rathnayaka et al. 2024). The wood samples were taken to the mycology laboratory at Guizhou Institute of Technology for exam- ination. Fresh specimens were incubated in zip-lock bags and sterile, moist plastic boxes at room temperature for two weeks. The microscopic features were examined and photographed using a stereomicroscope (SMZ-168, Nikon, Japan) and an ECLIPSE Ni compound microscope (Nikon, Tokyo, Japan) with a Canon 90D digital camera. Single spore isolations were performed on PDA plates following the meth- ods described by Senanayake et al. (2020), and the germinated helicosporous conidia were aseptically transferred to fresh PDA plates. Mycelium was grown on PDA and incubated at 25 °C for 30-39 days—the morphological character- istics, such as color, shape, and size, were recorded. Dried fungal specimens were deposited in the Herbarium of Kunming Institute of Botany, Chinese Acad- emy of Sciences (Herb. HKAS), Kunming, China, and the Herbarium of Guizhou Academy of Agriculture Sciences (Herb. GZAAS), Guiyang, China. Pure cultures were deposited in the Guizhou Culture Collection, China (GZCC), Guiyang, Chi- na. The MycoBank numbers of newly obtained species were registered in the MycoBank database (https://www.mycobank.org/). DNA extraction, PCR amplification, and sequencing Fresh fungal mycelia were scraped from colonies grown on PDA plates and transferred to a 1.5 mL microcentrifuge tube using a sterilized lancet for ge- nomic DNA extraction. Genomic DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, China). ITS5/ITS4, LROR/LR5, fRPB2-5F/ fRPB2-7cR, and EF1-983F/EF1-2218R were employed to amplify the internal transcribed spacer (ITS; White et al. 1990), large ribosomal subunit (LSU; Vilga- lys and Hester 1990), RNA polymerase II second largest subunit (rpb2; Liu et al. MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 94 Xiao-Yan Ma et al.: Introducing two new Tubeufia species 1999), and translation elongation factor 1-a (tef7-a; Rehner and Buckley 2005) sequence fragments, respectively. DNA preparation was conducted in a 25 uL mixture, which included 1 uL DNA, 1 uL of the forward and reverse primers each, and 22 uL of 1.1x T3 Supper PCR Mix (Qingke Biotech, Chongging, China). The conditions for the polymerase chain reaction (PCR) correspond to those reported by Ma et al. (2023). The PCR products were purified and sequenced with the same primers at Beijing Tsingke Biotechnology Co., Ltd. Phylogenetic analyses The forward and reverse sequence data of our new taxa were checked and assembled using BioEdit v.7.0.5.3 (Hall 1999) and SeqMan v.7.0.0 (DNASTAR, Madison, WI, USA; Swindell and Plasterer 1997), respectively. The sequences incorporated in this study were downloaded from GenBank (Table 1; https:// www.ncbi.nlm.nih.gov/). Multiple sequences were aligned using MAFFT v.7.473 (https://mafft.cbrc.jp/alignment/server/; Katoh and Standley 2013; Ka- toh et al. 2019). The dataset was trimmed using trimAl v.1.2rev59 software (Ca- pella-Gutiérrez et al. 2009). A combined sequence dataset was created using SequenceMatrix-Windows-1.7.8 software (Vaidya et al. 2011). The Maximum Likelihood (ML) tree was constructed using the IQ-TREE web server (http:// iqtree.cibiv.univie.ac.at/; Nguyen et al. 2015). Bayesian Inference (BI) analysis was conducted following the methodology described by Ma et al. (2022). Phylogenetic trees were visualized using FigTree v.1.4.4 and edited with Adobe Illustrator CC 2019 (v.23.1.0; Adobe Systems, USA). Photo plates were assembled using Adobe Photoshop CC 2019 (Adobe Systems, USA). Measure- ments were made using Tarosoft (R) Image Frame Work software. Phylogenetic analysis results The phylogenetic positions of the newly isolated taxa were inferred based on par- tial nucleotide sequences of the LSU, ITS, tef7-a, and rpb2 gene regions. A total of 88 strains, including our newly isolated strains and two outgroups, were analyzed. The concatenated sequence matrix consisted of 3,339 characters (LSU = 844 bp, ITS = 538 bp, tef7-a = 912 bp, and rpb2 = 1,045 bp). The best-scoring ML tree gen- erated is shown in Fig. 1, with a final log-likelihood value of —23,679.213. The multigene phylogenetic tree (Fig. 1) shows that the four new collections represent two distinct species within Tubeufia. The four newly obtained iso- lates, GZCC 23-0488 and GZCC 24-0165, form a sister lineage to GZCC 23-0525 and GZCC 24-01656 with 100% ML/1.00 BYPP support. Taxonomy Tubeufia yanuodaensis X.Y. Ma, J. Ma & Y.Z. Lu, sp. nov. MycoBank No: 904047 Fig. 2 Etymology. “yanuodaensis” refers to the type locality, “Yanuoda Rainforest Cul- tural Tourist District”. Holotype. HKAS 128924. MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 95 Xiao-Yan Ma et al.: Introducing two new Tubeufia species Table 1. Taxa used in this study, along with their corresponding GenBank accession numbers for the DNA sequences. GenBank Accessions Taxon rpb2 Acanthohelicospora aurea MF589911 Tubeufa bambusicol MH551097 Tubeufia baomeilingensis PP596515 Tubeufia chiangmaiensis MH551100 Tubeufia chiangraiensis PV367525 Tubeufa chlamytdospora MH551101 Tubeufa coffeae pv367528 Tubeufia cylindrothecia MH551143 Tubeufia cylindrothecia MH551102 Tubeufa dentculata PP596520 Tubeufa dictyospor MH551104 Tubeufa dictyospor MH551103 Tubeufa entadae ~MFLU1@2100"———)«MKS47727_ | MK3794S Tubeufia fangchengensis MH551109 Tubeufaflformis Luce te-1128" =~ ~—=««KY092407 | KY117028 | MF535264 Tubeufia formosiformis ~-BCRCFU30850 —=S==«iLC371249 | Lca24i01 | = - ~—S—«La94219 Tubeufia formosiformis ~ BCRCFU30757" —=s=«iLC'193730 | LC201751 | - ~—S—S=«L494220 Tubeufia formosiformis BORG FU30851" —~=«LC371250.-LCA24102. | =; ~S*S*C«SC9ADTB ____ MFLUCC 16-0252" MH275089 _MH412786 Tubeufia freycinetiae MFLUCC 16-0252" MH275089 | MH260323 | MH412786 ; Tubeufa geniulat perc rusces = stcaase7 | Tubeufa geniculat —newuete = stoaasers | S| Tubeufia guangxiensis MG012011 Tubeufia guangxiensis MH551111 Tubeufia guttulata GZCC 23-0404" OR046678 | ORO46684 Tubeufia hainanensis GZCC 22-2015" OR046679 | ORO46685 Tubeufia hechiensis MFLUCC 17-0052" MH558785 | MH558910 | MH550978 | MH551112 Tubeufia hyalospora MFLUCC 15-1250" MH558786 MH550979 - Tubeufia inaequalis MFLUCC 17-0053" MH550982 | MH551115 Tubeufia inaequalis MFLUCC 17-1989 MH550983_ | MH551116 Tubeufia javanica MFLUCC 12-0545" KJ880034 KJ880037 - Tubeufia krabiensis MFLUCC 16-0228" MH558792 | MH558917 | MH550985 | MH551118 Tubeufia latispora MFLUCC 16-0027° KY092417 KY092412 KY117033 MH551119 Tubeufia laxispora MFLUCC 16-0219 KY092414 KY092409 KY117030 MF535286 MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 96 Xiao-Yan Ma et al.: Introducing two new Tubeufia species Taxon Tubeufia laxispora Tubeufia lilliputea Tubeufia liyui Tubeufia longihelicospora Tubeufia longihelicospora Tubeufia longiseta Tubeufia machaerinae Tubeufia mackenziei Tubeufia muriformis Tubeufia nigroseptum Tubeufia pandanicola Tubeufia parvispora Tubeufia parvispora Tubeufia roseohelicospora Tubeufia roseohelicospora Tubeufia rubra Tubeufia rubra Tubeufia sahyadriensis Tubeufia sahyadriensis Tubeufia sessilis Tubeufia subrenispora Tubeufia sympodihylospora Tubeufia sympodihylospora Tubeufia sympodihylospora Tubeufia sympodilaxispora Tubeufia sympodilaxispora Tubeufia taiwanensis Tubeufia tectonae Tubeufia tectonae Tubeufia tratensis Tubeufia tropica Tubeufia xylophila Tubeufia xylophila Tubeufia yanuodaensis Tubeufia yanuodaensis Tubeufia yinggelingensis Tubeufia yinggelingensis Tubeufiaceae sp. GenBank Accessions Strain ITS LSU tefl-a rpb2 MFLUCC 16-0232" KY092413 KY092408 KY117029 MF535287 NBRC 32664 AY916483 AY856899 - - GZCC 22-2030° OP888466 OP888465 OP856589 OP856588 MFLUCC:21-0151 OL606156 OL606149 OL964520 0L964526 MFLUCC 16-0753" MZ538531 MZ538565 | MZ567106 3 MFLUCC 15-0188" KU940133 - - - MFLUCC 17-0055 MH558795 | MH558920 | MH550988 | MH551122 MFLUCC 16-0222" KY092415 KY092410 KY117031 MF535288 GZCC 22-2039° ORO030843 ORO030836 ORO046680 OR046686 CGMCC 3.204307 MZ092716 MZ853187 | OM022002 | OM022001 MFLUCC 16-0321' MH275091 MH260325 - = MFLUCC 17-2009 MH558798 | MH558923 | MH550991 MH551125 MFLUCC 16-0324" MH275090 | MH260324 | MH412787 | MH412761 MFLUCC 17-1797 MH558800 | MH558925 | MH550993 | MH551127 MFLUCC 15-1247 KX454177 KX454178 - MH551144 GZCC 16-0083 MH558802 | MH558927 | MH550995 | MH551129 GZCC 16-0081' MH558801 MH558926 | MH550994 | MH551128 NFCCI 4252 RAJ/99.1° MH033849 | MH033850 | MHO033851 : NFCCI RAJ 99.2 MN393081 MN393082 | MN393083 : MFLUCC 16-0021° MH558803 - MH550996 | MH551130 CGMCC 3.25560" PP781938 PP781939 PP785815 PP785813 GZCC 16-0051 MH558805 | MH558929 | MH550998 | MH551132 MFLUCC 17-0044" MH558806 | MH558930 | MH550999 | MH551133 GZCC 16-0049 MH558804 | MH558928 | MH550997 | MH551131 GZCC 16-0058 MH558807 | MH558931 MH551000 | MH551134 MFLUCC 17-0048" MH558808 | MH558932 | MH551001 MH551135 BCRC FU30844' LC316605 - - - MFLUCC 17-1985 MH558810 | MH558934 | MH551003 | MH551137 MFLUCC 12-0392" KU144923 KU764706 KU872763 - MFLUCC 17-1993" MH558811 MH558935 | MH551004 | MH551138 GZCC 23-0219" PP626675 PP639531 - PP596536 MFLUCC 17-1520 MH558813 | MH558937 | MH551006 | MH551140 GZCC 16-0038 MH558812 | MH558936 | MH551005 | MH551139 GZCC 23-0488" PQ098484 PQ098521 PV768323 PV768332 GZCC 24-0165 PV730408 PV730412 PV768324 PV768333 GZCC 23-0525" PQ098483 PQ098520 PV768321 PV768330 GZCC 24-0166 PV730407 PV730411 PV768322 PV768331 BCC 3512 AY916484 AY856905 - - n Note: T denotes ex-type strain. Newly generated sequences are indicated in bold black. “-” means no data available in GenBank. Description. Saprobic on submerged decaying wood in a freshwater habitat. Asexual morph Hyphomycetous, helicosporous. Colonies on natural substrate superficial, effuse, white to pale brown. Mycelium partly superficial, composed of hyaline to pale brown, branched, septate, smooth hyphae. Conidiophores 64-83 x 6-7 um (xX = 71.5 x 6.5 um, n = 20), macronematous, mononematous, erect, cy- lindrical, straight or slightly flexuous, branched, septate, subhyaline to pale brown, thick-walled. Conidiogenous cells 13.5-16 x 3-5 um (x = 14.5 x 4 um, n = 20), holoblastic, monoblastic, integrated, subcylindrical, terminal, truncate at apex af- ter conidial secession, subhyaline to pale brown, smooth-walled. Conidia solitary, acrogenous, helicoid, 35.5-40 um diam., and conidial filament 4.5-—5 um wide MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 97 Xiao-Yan Ma et al.: Introducing two new Tubeufia species oto Lubeufia dictvospora MFLUCC 16-0220 Tubeufia dictyospora MFLUCC 17-1805 LO0/1.00- Tubeufia machaerinae MFLUCC 17-0055 oer Hi Tubeufia chlamydospora MFLUCC 16-0223" Tubeufia fangchengensis MFLUCC 17-0047" eainniees 99/1.00 Tubeufia baomeilingensis CGMCC 3.25580" 93/0.99 Tubeufia hainanensis GZCC 22-2015" — Tubeufia acropleurogena CGMCC 3.25582! Tubeufiaceae sp. BCC 3512 Tubeufia baoshanensis ZHKUCC 23-0682" 1 00/1.00 Tubeufia coffeae MFLUCC 25-0190" Tubeufia chiangraiensis MFLUCC 25-0189" 1oo/1.oo7 LUbeufia parvispora MFLUCC 16-0324" Tubeufia parvispora MFLUCC 17-2009 ee 100/1.00| 2#beufia yanuodaensis GZCC 23-0488" 100/1.00 Tubeufia yanuodaensis GZ.CC 24-0165 74/- 100/1.00| /“beufia yinggelingensis GZCC 23-0525" Et pith | Tubeufia yinggelingensis GZ.CC 24-0166 Tubeufia sympodilaxispora GZCC 16-0058 Tubeufia sympodilaxispora MFLUCC 17-00487 9911.00 100/1.00| /“beufia chiangmaiensis MFLUCC 11-0514" 83/- Tubeufia chiangmaiensis MFLUCC 17-1801 WEEE Tubeufia entadae MFLU 18-2102? 100/1.00 Tubeufia denticulata CGMCC 3.25583" Tubeufia pandanicola MFLUCC 16-0321" gg/.| Lubeufia sympodihylospora GZCC 16-0051 a 75/-1+ Tubeufia sympodihylospora GZCC 16-0049 28 OO ubeufia sympodihylospora MFLUCC 17-0044" es Tubeufia xylophila GZCC 16-0038 100/1.00.| | Tybeufia xylophila MFLUCC 17-1520 100/1.00 | Tubeufia eccentrica GZCC 16-0084 ate Tubeufia eccentrica MFLUCC 17-1524" Tubeufia guangxiensis MFLUCC 17-0045" ice ubeufia guangxiensis MFLUCC 17-0046 100/1.00 Tubeufia tratensis MFLUCC 17-1993T aT od/1. of Lubeufia inaequalis MFLUCC 17-0053" Tubeufia inaequalis MFLUCC 17-1989 Tubeufia rubra GZCC 16-0081" emcee be ubeufia rubra GZCC 16-0083 100/1.00| Lubeufia cylindrothecia MFLUCC 16-1283 98/1.00 $b /1.00 Tubeufia cylindrothecia MFLUCC 17-1792 ool1.oof Lubeufia lilliputea NBRC 32664 Tubeufia mackenziei MFLUCC 16-0222" Tubeufia liyui GZCC 22-2030 100/1.00 Figure 1. Phylogenetic tree generated from the ML analysis based on a combined dataset of LSU, ITS, tef1-a, and rpb2 sequence data. Bootstrap support values from ML analyses = 75% and Bayesian posterior probabilities (BYPP) = 0.95 are indicated at the nodes as ML/BYPP. respectively. The ML and BI analyses yielded similar tree topologies. The tree is rooted with Acanthohelicospora aurea GZCC 16-0060 and A. guianensis UAMH 1699. The newly obtained strains are indicated in bold black. Ex-type strains are denoted with “"”. MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 98 Xiao-Yan Ma et al.: Introducing two new Tubeufia species Tubeufia brevis MFLUCC 17-1799" Tubeufia bambusicola MFLUCC 17-1803" 94/1.00!'— Tubeufia tropica GZCC 23-0219" Tubeufia muriformis GZCC 22-2039° Tubeufia longiseta MFLUCC 15-0188" 100/1.00 Tubeufia sessilis MFLUCC 16-00217 Tubeufia latispora MFLUCC 16-0027" Tubeufia subrenispora CGMCC 3.25560! Tubeufia javanica MFLUCC 12-0545" Tubeufia filiformis MFLUCC 16-1135 Tubeufia filiformis MFLUCC 16-1128? 100/007 Tubeufia krabiensis MFLUCC 16-0228" Tubeufia hyalospora MFLUCC 15-1250° Tubeufia geniculata BCRC FU30849" Le Tubeufia geniculata NCYU-U2-1B Tubeufia abundata MFLUCC 17-2024 Tubeufia roseohelicospora MFLUCC 15-1247" go/1 oot Lubeufia roseohelicospora MFLUCC 17-1797 Tubeufia Tubeufia tectonae MFLUCC 12-0392? 96/1.00 Lubeufia tectonae MFLUCC 17-1985 Tubeufia freycinetiae MFLUCC 16-0252? 96/1.00 Tubeufia africana BCRC FU30840 99/1.00} Tubeufia africana BCRC FU30867 Tubeufia africana BCRC FU30906 Tubeufia formosiformis BCRC FU30757' iaore ner Tubeufia formosiformis BCRC FU30850 Tubeufia formosiformis BCRC FU30851' Tubeufia sahyadriensis NFCCI 4252 RAJ/99.17 Lubeufia sahyadriensis NFCCI RAJ 99.2 93). Lubeufia hechiensis MFLUCC 17-0052" 100/1.00|"- Tubeufia taiwanensis BCRC FU30844' 100/1.00 Tubeufia brunnea MFLUCC 17-2022" Tubeufia longihelicospora MFLUCC 16-0753? oan 98/0.98, Ue ubeufia longihelicospora MFLUCC 21-0151 Lubeufia nigroseptum CGMCC 3.20430! 100/1.00| 1ubeufia laxispora MFLUCC 16-0219 Tubeufia laxispora MFLUCC 16-0232" 100/1.00 1oo/1.oo7 Lubeufia aquatica MFLUCC 16-1249 100/1.00 Tubeufia aquatica MFLUCC 17-1794 Tubeufia cocois MFLUCC 22-0001* 85/- 100/1.00 100/1.00) 97/1.00 99/1.00 95/- 100/1.00 98/1 .00 7 ay ubeufia guttulata GZCC 23-0404! Neohelicomyces xiayadongensis MUCL 15702 Neohelicomyces 100/1.00 Acanthohelicospora aurea GZCC 16-0060 Outerotn 0.04 Acanthohelicospora guianensis UAMH 1699 Figure 1. Continued. MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 99 Xiao-Yan Ma et al.: Introducing two new Tubeufia species 7 at or . - } “a. we i c ' Tit? 1 "/ 4 = ° , - af : . } “ 2 A : a i eX af \' 7 - * 1 2 bod 2. i >* a - *. “ ee he es ge) le win _ 5 ; 7 "? « 5 -i we Ll f Figure 2. Tubeufia yanuodaensis (HKAS 128924, holotype). a, b. Colonies on the host surface; c. Conidiophores and conidiogenous cells; d—h. Conidia; i. Germinated conidium; j, k. Colonies on PDA, j from above, k from below. Scale bars: 40 um (c, d); 30 um (e); 20 um (f-i). (X = 37.5 x 4.8 um, n = 20), 252-338 um long (X = 291 um, n = 25), tightly coiled 3'/,-34/, times, becoming loosely coiled in water, multi-septate, slightly constrict- ed at septa, guttulate, hyaline, smooth-walled. Sexual morph Undetermined. MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 100 Xiao-Yan Ma et al.: Introducing two new Tubeufia species Culture characteristics. Conidia germinated on PDA and produced germ tubes within 9 h. Colonies on PDA are irregular with a flat surface and undu- lating margin, reaching 2.5 cm in diameter after 30 days at room temperature (approximately 25 °C), and are pale brown to dark brown. Material examined. CHINA + Hainan Province, Baoting Li and Miao Auton- omous County, Yanuoda Rainforest Cultural Tourist District, on submerged decaying wood in a freshwater stream, 23 October 2021, Jian Ma, Y2 (HKAS 128924, holotype), ex-type living culture GZCC 23-0488; Ibid., Y2-2 (GZAAS 24- 0079, paratype), living culture GZCC 24-0165. Notes. In the present phylogenetic analysis, Tubeufia yanuodaensis (GZCC 23-0488 and GZCC 24-0165) formed a sister lineage to T. yinggelin- gensis (GZCC 23-0525 and GZCC 24-0166) with 100% ML and 1.00 BYPP statistical support (Fig. 1). Based on the molecular sequence compari- son, our isolate (GZCC 23-0488, ex-type) differs from T. yinggelingensis (GZCC 23-0525, ex-type) by 71/857 bp for ITS (8.3%, gaps 42 bp), 7/833 bp (0.8%, gap one bp) for LSU, 20/892 bp (2.2%, without gaps) for tef1-a, and 45/767 bp (5.9%, gaps 3 bp) for rpb2. Morphologically, Tubeufia yanuo- daensis (HKAS 128924) can be distinguished from T. yinggelingensis (HKAS 128856) by its larger conidia (35.5-40 um diameter and 252-338 um long vs. 22-31 um diameter and 148-170 um long). Therefore, Tubeufia yanuo- daensis is introduced here as a new species based on molecular evidence and morphological comparison. Tubeufia yinggelingensis X.Y. Ma, J. Ma & Y.Z. Lu, sp. nov. MycoBank No: 904048 Figad Etymology. “yinggelingensis’ refers to the type locality, “Yinggeling National Na- ture Reserve”. Holotype. HKAS 128856. Description. Saprobic on submerged decaying wood in a freshwater hab- itat. Asexual morph Hyphomycetous, helicosporous. Colonies on natural substrate superficial, effuse, gregarious, with crowded, glistening conid- ia, white. Mycelium partly superficial, composed of hyaline to pale brown, branched, septate, guttulate, smooth hyphae. Conidiophores 21.5-143 x 4.5-7 um (X = 67.5 x 5.5 um, n = 25), macronematous, mononematous, erect, cylindrical, flexuous, branched, septate, pale brown to brown, thick- walled. Conidiogenous cells 9-15 x 4-5 um (x = 11.5 x 4.5 um, n = 20), holoblastic, monoblastic, integrated, terminal, cylindrical, with denticles, pale brown to brown, smooth-walled. Conidia solitary, acrogenous, heli- coid, rounded at apex, developing on tooth-like protrusion, 22-31 um diam. and conidial filament 4-6 um wide (x = 27 x 5 um, n = 20), 148-170 um long (X = 157.5 um, n = 20), tightly coiled 2°/,-3'/, times, not becoming loose in water, indistinctly multi-septate, guttulate, hyaline, smooth-walled. Sexual morph Undetermined. Culture characteristics. Conidia germinated on PDA and formed germ tubes after 11 hours of incubation. Colonies on PDA are irregular with a flat surface MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 101 Xiao-Yan Ma et al.: Introducing two new Tubeufia species Figure 3. Tubeufia yinggelingensis (HKAS 128856, holotype). a, b. Colonies on the host surface; c—f. Conidiophores and conidiogenous cells; g-i. Conidiogenous cells and conidia; k-p. Conidia; j. Germinated conidium; q, r. Colonies on PDA, q from above, r from below. Scale bars: 50 um (c, d); 40 um (e, f); 10 um (g-p). MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 102 Xiao-Yan Ma et al.: Introducing two new Tubeufia species and undulating margin, reaching 3.2 cm in diameter after 39 days at room tem- perature (approximately 25 °C), and are brown to dark brown in color. Material examined. CHINA * Hainan Province, Yinggeling National Nature Reserve, on rotting wood in a freshwater stream, 12 July 2022, Jian Ma, YG5 (HKAS 128856, holotype), ex-type living culture GZCC 23-0525; Ibid., YG5-2 (GZAAS 24-0080, paratype), living culture GZCC 24-0166. Notes. Morphologically, Tubeufia yinggelingensis (HKAS 128856) closely re- sembles T. denticulata (HKAS 131090) in having macronematous, mononem- atous, erect, cylindrical, branched, septate conidiophores; holoblastic, mono- blastic, integrated, cylindrical conidiogenous cells; and solitary, hyaline, guttulate, helicoid conidia (Ma et al. 2024). However, Tubeufia yinggelingensis (HKAS 128856) differs from T. denticulata (HKAS 131090) by its unique con- idiogenous cells (terminal vs. intercalary) and larger conidia (22-31 um diam. and conidial filament 4-6 um wide, 148-170 um long vs. 19-21 um diam. and conidial filament 2-4 um wide, 73.5-142.5 um long) (Ma et al. 2024). Based on molecular phylogenetic analyses, Tubeufia yinggelingensis (GZCC 23-0525 and GZCC 24-0166) formed a sister clade with T. yanuodaensis (GZCC 23- 0488 and GZCC 24-0165), which is phylogenetically distinct from T. dentic- ulata (CGMCC 3.25583) (Fig. 1). Thus, based on morphological comparison and multigene phylogenetic analysis, we identified GZCC 23-0525 and GZCC 24-0166 as anew species, Tubeufia yinggelingensis. Discussion Based on DNA sequence data and/or morphological characteristics, the ge- nus Tubeufia currently comprises 88 species, including our newly described species, T. yanuodaensis and T. yinggelingensis (Karandikar and Patwardhan 1992; Lu et al. 2000; Chang 2001; Ho et al. 2002; Tsui and Berbee 2006; Tsui 2007; Zhao et al. 2007; Pande 2008; Boonmee et al. 2011, 2014, 2021; Hyde et al. 2016; Singh and Singh 2016; Li et al. 2022; Tian et al. 2022; Ma et al. 2023, 2024; Lu et al. 2025). Among them, 57 species are helicosporous fungi (Ma et al. 2024; this study). Currently, molecular data are unavailable for 30 Tubeufia species, and the sexual and asexual morphs have been linked in only six spe- cies (Lu et al. 2017, 2018a, 2018b, 2022, 2023; Luo et al. 2017; Kuo and Goh 2018a, 2018b, 2021; Tibpromma et al. 2018; Lu and Kang 2020; Li et al. 2022; Tian et al. 2022; Ma et al. 2023, 2024; Lu et al. 2025). The genus Tubeufia exhibits diverse asexual and sexual morphologies. However, morphologically similar species within this genus typically form stable and distinct clades in multi-gene phylogenetic analyses (Fig. 1) (Zhao et al. 2007; Rajeshkumar et al. 2019; Kuo and Goh 2021; Ma et al. 2023, 2024; Lu et al. 2025). For instance, Tubeufia africana and T. formosiformis possess ellipsoidal or ovoid conidia (Kuo and Goh 2021; Ma et al. 2024), whereas T. sahyadriensis features maize corncob-like, dictyoseptate conidia (Rajeshkumar et al. 2019). Additionally, Tubeufia longiseta, T. muriformis, T. sessilis, and T. tropica are characterized by helicoid or curved, muriform conidia, occasionally bearing a single globose secondary conidium (Zhao et al. 2007; Ma et al. 2023, 2024), while Tubeufia subrenispora exhibits curved to subreniform, multicelled muriform conidia (Zhao et al. 2007; Ma et al. 2024). Moreover, Tubeufia baoshanensis, T. coffeae, and T. chiangraiensis— MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 103 Xiao-Yan Ma et al.: Introducing two new Tubeufia species which were collected from decaying branches of Coffea sp.—produce broad- ly fusiform, cylindrical, aseptate, hyaline ascospores (Lu et al. 2025). To date, five asexual species of Tubeufia with distinct conidial charac- teristics have been reported, primarily from China and Thailand (Zhao et al. 2007; Rajeshkumar et al. 2019; Kuo and Goh 2021; Ma et al. 2023, 2024). Among them, Tubeufia sahyadriensis—characterized by maize corncob-like, dictyoseptate conidia—is the only species recorded from India (Rajeshku- mar et al. 2019; Ma et al. 2024). Acknowledgments We would like to thank Dr. Shaun Pennycook (Manaaki Whenua Landcare Re- search, New Zealand) for his advice on new Latin names. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of Al No use of Al was reported. Funding This work was funded by the Guizhou Institute of Technology High-level Talent Scien- tific Research Start-up Fund (grant number 2023GCC065), the Guizhou Provincial High- er Education Undergraduate Teaching Content and Curriculum System Reform Project (grant number GZJG2024209), the Zunyi Technology and Big Data Bureau-Moutai Insti- tute Joint Science and Technology Research and Development Project (grant number ZunShiJiaoHe HZ zi [2023]110), the Science and Technology Foundation of Guizhou Province (Qian Ke He Pingtai ZSYS [2025]029), and the Innovation and Entrepreneurship Training Program for College Students (S2024144400956x). Author contributions Methodology: JFF, MMW, HHS. Writing - review and editing: JM, XYM, FT. Author ORCIDs Xiao-Yan Ma ® https://orcid.org/0000-0001-5874-9979 Fu Tian © https://orcid.org/0000-0002-5503-4350 Jiang-Fen Feng ® https://orcid.org/0009-0009-2467-891X Min-Min Wang ® hittps://orcid.org/0009-0003-6106-9992 Huan-Huan Shi © https://orcid.org/0009-0001-6486-1315 Jian Ma © https://orcid.org/0009-0008-1291-640X Data availability All of the data that support the findings of this study are available in the main text. MycoKeys 121: 93-109 (2025), DOI: 10.3897/mycokeys.121.158724 104 Xiao-Yan Ma et al.: Introducing two new Tubeufia species References Barr ME (1979) A classification of Loculoascomycetes. Mycologia 75: 935-957. https:// doi.org/10.1080/00275514.1979.12021099 Barr ME (1980) On the family Tubeufiaceae (Pleosporales). Mycotaxon 12: 137-167. Barr ME, Rogerson CT (1983) Two new species of Loculoascomycetes. 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