Novel Helicotubeufia and Tubeufia (Tubeufiaceae, Tubeufiales) species from terrestrial habitats in Hainan Province, China

57 Novel Helicotubeufia and Tubeufia (Tubeufiaceae, Tubeufiales) species from terrestrial habitats in Hainan Province, China Ting-Hong Tan1,2 , Fan Gao1, Song Bai3, Chun-Fang Wu1, Ning-Ning Zhao1, Na Qiu1, Min Zhou1 , Jian Ma3,4 1 School of Agriculture and Forestry Engineering and Planning, Tongren University, Tongren, Guizhou 554300, China 2 Guizhou Provincial Key Laboratory for Biodiversity Conservation and Utilization in the Fanjing Mountain Region, Tongren University, Tongren, Guizhou 554300, China 3 Guizhou Industry Polytechnic College, Guiyang, Guizhou 550008, China 4 School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang, Guizhou 550003, China Corresponding authors: Ting-Hong Tan ([email protected]); Song Bai ([email protected]); Jian Ma ([email protected]) Copyright: © Ting-Hong Tan 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). Research Article Abstract Helicosporous hyphomycetes are a group of fungi, characterised by coiled or spiral conidia and are known for their potential to produce bioactive secondary metabolites. During a survey of helicosporous hyphomycetes, four isolates were obtained from decaying wood in Hainan Province, southern China. Based on phylogenetic analyses of a combined dataset (ITS, LSU, tef1-α and rpb2) and morphological characteristics, two novel species, Helicotubeufia qixianlingensis and Tubeufia diaoluoshanensis, are introduced. Comprehensive descriptions, illustrations and phylogenetic analyses supporting the taxonomic placement of these new taxa are provided. Notably, H. qixianlingensis represents the first record of Helicotubeufia from a terrestrial habitat, thereby expanding the known diversity of tropical terrestrial fungi. Key words: Asexual morph, phylogeny, saprobic fungi, taxonomy, two new species, Introduction Liu et al. (2018) introduced the new genus Helicotubeufia Y.Z. Lu & J.K. Liu to accommodate three species, H. guangxiensis, designated as the type species, alongside H. hydei and H. jonesii, based on phylogenetic analyses of combined ITS, LSU and tef1-α sequences data and morphological features. These three species were obtained from submerged decaying wood in China and Thailand (Liu et al. 2018). Recently, Ma et al. (2024) identified an additional species within the Helicotubeufia, named H. laxisporum, which was also isolated from submerged decaying wood in Hainan Province, China. Thus, the genus Helicotubeufia now encapsulates four recognised species (Lu et al. 2018b; Ma et al. 2024). The asexual morph of Helicotubeufia is characterised by hyaline, macronematous conidiophores, holoblastic, polyblastic, sympodial, hyaline conidiogenous cells and helicoid, hyaline conidia (Liu et al. 2018). The sexual morph is characterised by superficial, seated on a subiculum, scattered, subglobose to ellipsoidal-ovate ascomata, 8-spored, bitunicate asci and fusiform, hyaline ascospores (Liu et al. 2018). Academic editor: Xin-Cun Wang Received: 7 October 2025 Accepted: 12 November 2025 Published: 4 December 2025 Citation: Tan T-H, Gao F, Bai S, Wu C-F, Zhao N-N, Qiu N, Zhou M, Ma J (2025) Novel Helicotubeufia and Tubeufia (Tubeufiaceae, Tubeufiales) species from terrestrial habitats in Hainan Province, China. MycoKeys 126: 57–74. https://doi.org/10.3897/ mycokeys.126.174148 MycoKeys 126: 57–74 (2025) DOI: 10.3897/mycokeys.126.174148 58 MycoKeys 126: 57–74 (2025), DOI: 10.3897/mycokeys.126.174148 Ting-Hong Tan et al.: Introduce two new species Tubeufia was established by Penzig and Saccardo (1897) with T. javanica as the type species, based on morphological characteristics. The genus currently comprises 88 species, including 29 from freshwater habitats, 44 from terrestrial habitats and 15 occurring in both freshwater and terrestrial habitats (Ma et al. 2023, 2024; 2025; Lu et al. 2025). Tubeufia species are widely distributed, having been reported from Austria, Bermuda, Brazil, Canada, China, Colombia, Cuba, India, New Zealand, Panama, Peru, South Africa, Sri Lanka, Tanzania, Thailand, Trinidad, Uganda, Venezuela and the USA (Morgan 1892; Talbot 1956; Moore 1957; Rao and Rao 1964; Deighton and Pirozynski 1966; Munk 1966; Ellis 1971; Panwar et al. 1973; Barr 1979, 1980; Barr and Rogerson 1983; Goos 1985, 1990; Matsushima 1987, 1993; Rossman 1987; Holubová-Jechová 1988; 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; 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, 2021; Tibpromma et al. 2018; Li et al. 2022; Tian et al. 2022; Ma et al. 2023, 2024; 2025; Lu et al. 2025). The conidial morphology in Tubeufia is highly diverse, encompassing a variety of forms such as dictyosporous, muriform, dorsiventrally curved, coiled, ovate, ellipsoid to ovoid, spherical to obclavate and subreniform forms, sometimes accompanied by 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, four hyphomycete isolates were obtained from terrestrial habitats in Hainan Province, China, specifically from Diaoluoshan National Nature Reserve and Qixianling Hot Spring National Forest Park. Based on morphological observations, detailed illustrations, descriptive notes and multi-gene phylogenetic analyses, we propose two novel species: Helicotubeufia qixianlingensis and Tubeufia diaoluoshanensis. Comparative analyses with closely-related taxa further substantiate their taxonomic classification. Materials and methods Sample collection, specimen examination and isolation Decaying wood was collected from Hainan Province, south-western China. Samples were taken to the laboratory in plastic bags with the collection details, including localities and dates. 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 (Canon, China). Measurements were made by Tarosoft (R) Image Frame Work software. Photo plates were assembled using Adobe Photoshop CC 2019 (Adobe Systems, USA). Single spore isolation was performed following the methods described by Senanayake et al. (2020) and the germinated conidia were aseptically transferred to fresh PDA plates. Morphological characters of fungal mycelia, including colour, shape and size, were documented. Dried fungal specimens were deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (Herb. HKAS) in Kunming, China and the Herbarium of Guizhou Academy of Agriculture Sciences (Herb. GZAAS), Guiyang, China. Pure cultures 59 MycoKeys 126: 57–74 (2025), DOI: 10.3897/mycokeys.126.174148 Ting-Hong Tan et al.: Introduce two new species were deposited in the Guizhou Culture Collection (GZCC), Guiyang, China. 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 sterilised lancet for genomic DNA extraction. Genomic DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, China). ITS5/ITS4, LR0R/LR5, EF1-983F/ EF1-2218R and fRPB2-5F/fRPB2-7cR were employed to amplify the internal transcribed spacer (ITS; White et al. (1990)), large ribosomal subunit (LSU; Vilgalys and Hester (1990)), translation elongation factor 1-alpha (tef1-α; Rehner and Buckley (2005)) and RNA polymerase II second largest subunit (rpb2; Liu et al. (1999)) sequence fragments, respectively. DNA preparation was conducted in a 25 μl mixture, which included 1 μl DNA, 1 μl of the forward and reverse primers each and 22 μl of 1.1× T3 Super PCR Mix (including 8.5 μl distilled-deionised water; Qingke Biotech, Chongqing, 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. Genealogical concordance phylogenetic species recognition (GCPSR) analysis The pairwise homoplasy index (PHI) test of Helicotubeufia species was carried out in SplitsTree4 (Bruen et al. 2006). It indicates that there is no statistically significant evidence for recombination for the selected taxa when the P-value is above 0.05. Both the LogDet transformation and splits decomposition options were used to reveal the relationship amongst closely-related species. Phylogenetic analyses The newly-obtained sequences 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 et al. (2019)). The dataset was trimmed using trimAl v.1.2rev59 software (Capella-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) analysis was carried out using the RAxML-HPC v.8 on XSEDE (8.2.12) tool using a GTRGAMMA approximation with rapid bootstrap analysis followed by 1000 bootstrap replicates (Stamatakis 2014). The substitution model was automatically tested by the server. Bayesian Inference (BI) analysis was performed by using MrBayes on XSEDE (3.2.7a) via CIPRES (Stamatakis 2014). The aligned FASTA file was converted to a Nexus format file using AliView (Daniel et al. 2010). The best-fit evolutionary model for the individual dataset was determined using MrModelTest v. 2.3. 10 (Nylander et al. 2008). 60 MycoKeys 126: 57–74 (2025), DOI: 10.3897/mycokeys.126.174148 Ting-Hong Tan et al.: Introduce two new species Table 1. Taxa used in this study and their GenBank accession numbers of DNA sequences. Taxon Strain GenBank Accessions ITS LSU tef1-α rpb2 Acanthohelicospora aurea GZCC 16-0060 KY321323 KY321326 KY792600 MF589911 Acanthohelicospora guianensis UAMH 1699 AY916479 AY856891 N/A N/A Helicotubeufia guangxiensis MFLUCC 17-0040TMH290018 MH290023 MH290028 MH290033 Helicotubeufia hydei MFLUCC 17-1980TMH290021 MH290026 MH290031 MH290036 Helicotubeufia jonesii MFLUCC 17-0043TMH290020 MH290025 MH290030 MH290035 Helicotubeufia laxisporum CGMCC 3.25545TPP626589 PP639445 PP596346 PP596473 Helicotubeufia qixianlingensis GZCC 25-0644 PX575636 PX575659 PX512842 PX512833 Helicotubeufia qixianlingensis GZCC 25-0645TPX575635 PX575658 PX512841 PX512832 Tubeufia abundata MFLUCC 17-2024 MH558769 MH558894 MH550961 MH551095 Tubeufia acropleurogena CGMCC 3.25582TPP626645 PP639501 PP596394 PP596513 Tubeufia africana BCRC FU30906 LC371247 LC424099 N/A LC494221 Tubeufia aquatica MFLUCC 17-1794 MH558770 MH558895 MH550962 MH551096 Tubeufia bambusicola MFLUCC 17-1803TMH558771 MH558896 MH550963 MH551097 Tubeufia baomeilingensis CGMCC 3.25580TPP626648 PP639504 PP596397 PP596515 Tubeufia brevis MFLUCC 17-1799TMH558772 MH558897 MH550964 MH551098 Tubeufia brunnea MFLUCC 17-2022TMH558773 MH558898 MH550965 MH551099 Tubeufia chiangmaiensis MFLUCC 11-0514TKF301530 KF301538 KF301557 N/A Tubeufia chlamydospora MFLUCC 16-0223TMH558775 MH558900 MH550967 MH551101 Tubeufia cocois MFLUCC 22-0001TOM102541 OL985957 OM355486 OM355491 Tubeufia cylindrothecia MFLUCC 16-1283 KY320518 KY320535 KY320552 MH551143 Tubeufia denticulata CGMCC 3.25583TPP626653 PP639509 PP596402 PP596520 Tubeufia diaoluoshanensis GZCC 22-2142 PX575637 PX575660 PX512843 PX512834 Tubeufia diaoluoshanensis GZCC 25-0643TPX575638 PX575661 PX512844 PX512835 Tubeufia dictyospora MFLUCC 17-1805TMH558778 MH558903 MH550970 MH551104 Tubeufia dongfangensis GZCC 22-2125TPQ098479 PQ098516 N/A N/A Tubeufia eccentrica MFLUCC 17-1524TMH558782 MH558907 MH550974 MH551108 Tubeufia entadae MFLU 18-2102TMK347727 MK347943 N/A N/A Tubeufia fangchengensis MFLUCC 17-0047TMH558783 MH558908 MH550975 MH551109 Tubeufia filiformis MFLUCC 16-1128TN/A KY092407 KY117028 MF535284 Tubeufia formosiformis BCRC FU30757TLC193730 LC201751 N/A LC494220 Tubeufia formosiformis BCRC FU30851TLC371250 LC424102 N/A LC494218 Tubeufia freycinetiae MFLUCC 16-0252TMH275089 MH260323 MH412786 N/A Tubeufia geniculata BCRC FU30849TLC335817 N/A N/A N/A Tubeufia guangxiensis MFLUCC 17-0045TMG012025 MG012018 MG012004 MG012011 Tubeufia guttulata GZCC 23-0404TOR030841 OR030834 OR046678 OR046684 Tubeufia hainanensis GZCC 22-2015TOR030842 OR030835 OR046679 OR046685 Tubeufia hechiensis MFLUCC 17-0052TMH558785 MH558910 MH550978 MH551112 Tubeufia hyalospora MFLUCC 15-1250TMH558786 MH558911 MH550979 N/A Tubeufia inaequalis MFLUCC 17-0053TMH558789 MH558914 MH550982 MH551115 Tubeufia javanica MFLUCC 12-0545TKJ880034 KJ880036 KJ880037 N/A Tubeufia jianfenglingensis GZCC 23-0021TPQ098481 PQ098518 N/A N/A Tubeufia krabiensis MFLUCC 16-0228TMH558792 MH558917 MH550985 MH551118 Tubeufia latispora MFLUCC 16-0027TKY092417 KY092412 KY117033 MH551119 Tubeufia laxispora MFLUCC 16-0232TKY092413 KY092408 KY117029 MF535287 Tubeufia lilliputea NBRC 32664 AY916483 AY856899 N/A N/A Tubeufia liyui GZCC 22-2030TOP888466 OP888465 OP856589 OP856588 Tubeufia longihelicospora MFLUCC 16-0753TMZ538531 MZ538565 MZ567106 N/A Tubeufia longiseta MFLUCC 15-0188TKU940133 N/A N/A N/A Tubeufia machaerinae MFLUCC 17-0055 MH558795 MH558920 MH550988 MH551122 61 MycoKeys 126: 57–74 (2025), DOI: 10.3897/mycokeys.126.174148 Ting-Hong Tan et al.: Introduce two new species The GTR+G+I substitution model was selected for ITS, LSU, tef1-α and rpb2. The posterior probabilities (BYPP) were determined, based on Bayesian Markov Chain Monte Carlo (BMCMC) sampling (Huelsenbeck and Ronquist 2001). Two simultaneous Markov chains were run for 10,000,000 generations and trees were sampled every 1,000th generation. The burn-in phase was set at 25% and the remaining trees were used for calculating posterior probabilities (BYPP). Phylogenetic trees were visualised using FigTree v.1.4.4 and edited with Adobe Illustrator CC 2019 (v.23.1.0; Adobe Systems, USA). Phylogenetic results The phylogenetic placements of the newly-isolated taxa were determined by multi-locus phylogenetic analysis. A total of 70 strains, including our newly-isolated strains and two outgroups, were analysed. The concatenated sequence matrix consisted of 3,365 characters (ITS = 565 bp, LSU = 843 bp, tef1-α = 912 bp and rpb2 = 1,045 bp). Base frequencies and rates were A = 0.248200, C = 0.249485, G = 0.256312 and T = 0.246003; substitution rates were AC = 1.017042, AG = 5.690331, AT = 2.161337, CG = 0.779562, CT = 8.363479 and GT = 1.000000. The distribution shape parameter α equalled 0.179497. Based on the phylogenetic analysis (Fig. 1), our collections belong to Helicotubeufia and Tubeufia within Tubeufiaceae (Tubeufiales, Dothideomycetes). Two isolates (GZCC 25-0644 and GZCC 25-0645) formed a sister clade with Helicotubeufia laxisporum (CGMCC 3.25545), supported by 100% ML and 1.00 BYPP. Additionally, GZCC 22-2142 and GZCC 25-0643 form a sister lineage to Tubeufia fangchengensis (MFLUCC 17-0047) with 55% ML support. Taxon Strain GenBank Accessions ITS LSU tef1-α rpb2 Tubeufia mackenziei MFLUCC 16-0222TKY092415 KY092410 KY117031 MF535288 Tubeufia muriformis GZCC 22-2039TOR030843 OR030836 OR046680 OR046686 Tubeufia nigroseptum CGMCC 3.20430TMZ092716 MZ853187 OM022002 OM022001 Tubeufia pandanicola MFLUCC 16-0321TMH275091 MH260325 N/A N/A Tubeufia parvispora MFLUCC 16-0324TMH275090 MH260324 MH412787 MH412761 Tubeufia roseohelicospora MFLUCC 15-1247TKX454177 KX454178 N/A MH551144 Tubeufia rubra GZCC 16-0081TMH558801 MH558926 MH550994 MH551128 Tubeufia sahyadriensis NFCCI 4252/RAJ 99.1TMH033849 MH033850 MH033851 N/A Tubeufia sessilis MFLUCC 16-0021TMH558803 N/A MH550996 MH551130 Tubeufia subrenispora CGMCC 3.25560TPP781938 PP781939 PP785815 PP785813 Tubeufia sympodihylospora MFLUCC 17-0044TMH558806 MH558930 MH550999 MH551133 Tubeufia sympodilaxispora MFLUCC 17-0048TMH558808 MH558932 MH551001 MH551135 Tubeufia taiwanensis BCRC FU30844TLC316605 N/A N/A N/A Tubeufia tectonae MFLUCC 12-0392TKU144923 KU764706 KU872763 N/A Tubeufia tratensis MFLUCC 17-1993TMH558811 MH558935 MH551004 MH551138 Tubeufia tropica GZCC 23-0219TPP626675 PP639531 N/A PP596536 Tubeufia xylophila MFLUCC 17-1520 MH558813 MH558937 MH551006 MH551140 Tubeufia xylophila GZCC 16-0038 MH558812 MH558936 MH551005 MH551139 Tubeufia yanuodaensis GZCC 23-0488TPQ098484 PQ098521 PV768323 PV768332 Tubeufia yinggelingensis GZCC 23-0525TPQ098483 PQ098520 PV768321 PV768330 Tubeufiaceae sp. BCC 3512 AY916484 AY856905 N/A N/A Note: “T” denotes ex-type strain. Newly generated sequences are indicated in bold black. “N/A” means no data available in GenBank. 62 MycoKeys 126: 57–74 (2025), DOI: 10.3897/mycokeys.126.174148 Ting-Hong Tan et al.: Introduce two new species Figure 1. Phylogenetic tree generated from the Maximum Likelihood (ML) analysis, based on a combined dataset of ITS, LSU, tef1-α and rpb2 sequence data. Bootstrap support values from ML analyses ≥ 55% and Bayesian posterior probabilities (BYPP) ≥ 0.95 are indicated at the nodes as ML/BYPP, respectively. The Maximum Likelihood (ML) and Bayesian Inference (BYPP) analyses yielded similar tree topologies. Hyphen (“-”) indicates a value lower than 55% for ML and a posterior probability lower than 0.95 for Bayesian. The tree is rooted with Acanthohelicospora aurea GZCC 16-0060 and A. guianensis UAMH 1699. The newly-obtained strains are indicated in black bold. Ex-type strains are denoted with “T”. 63 MycoKeys 126: 57–74 (2025), DOI: 10.3897/mycokeys.126.174148 Ting-Hong Tan et al.: Introduce two new species Taxonomy Helicotubeufia qixianlingensis T.H. Tan & J. Ma, sp. nov. MycoBank No: 904431 Fig. 2 Etymology. “qixianlingensis” refers the place “Qixianling Hot Spring National Forest Park” from where the fungus was collected. Holotype. GZAAS 25-0674. Description. Saprobic on decaying wood in a terrestrial habitat. Sexual morph Undetermined. Asexual morph Hyphomycetous, helicosporous. Colonies on natural substrate superficial, effuse, gregarious, with masses of crowded, glistening conidia, white to pale brown. Mycelium partly immersed, partly superficial, composed of hyaline to pale brown, branched, septate, arising Figure 1. Continued. 64 MycoKeys 126: 57–74 (2025), DOI: 10.3897/mycokeys.126.174148 Ting-Hong Tan et al.: Introduce two new species from creeping hyaphae, guttulate, smooth hyphae. Conidiophores 13–38 × 4.5–5.5 μm (x – = 28 × 5 μm, n = 25), macronematous, mononematous, solitary or cespitose, erect, irregular cylindrical, short, flexuous, branched or unbranched, septate, pale brown, smooth-walled, thick-walled. Conidiogenous cells holoFigure 2. Helicotubeufia qixianlingensis (GZAAS 25-0674, holotype). a, b. Colonies on the host surface; c. Conidiophores and conidia; d–h. Conidiophores; i–l. Conidia; m, n. Colonies on PDA, m from above, n from below. Scale bars: 50 μm (c); 20 μm (i–l); 10 μm (d–h). 65 MycoKeys 126: 57–74 (2025), DOI: 10.3897/mycokeys.126.174148 Ting-Hong Tan et al.: Introduce two new species blastic, monoblastic or polyblastic, integrated, terminal, cylindrical, truncate at apex after conidial secession, pale brown, smooth-walled. Conidia solitary, acrogenous, helicoid, tapering towards the rounded ends, 77–101 μm diameter and conidial filament 6–7 μm wide (x – = 90 × 6.5 μm, n = 20), 483–520 μm long (x – = 499 μm, n = 20), loosely coiled 2–21/2 times, becoming loosely coiled in water, indistinctly multi-septate, subhyaline to pale brown, smooth-walled. Culture characteristics. Conidia germinated on PDA and produced germ tubes within 9 h. Colonies on PDA reached 26 mm in diameter after 38 days of incubation at 25 °C with an irregular shape, flat surface and undulate margin, pale brown to brown; the reverse was brown to black. Material examined. China • Hainan Province, Baoting Li and Miao Autonomous County, Qixianling Hot Spring National Forest Park, on decaying wood in a terrestrial habitat, 2 November 2024, Jian Ma, Q26 (GZAAS 25-0674, holotype), ex-type living culture GZCC 25-0645; • Ibid., Q28 (GZAAS 25-0673, paratype), living culture GZCC 25-0644. Notes. In the phylogenetic analysis (Fig. 1), Helicotubeufia qixianlingensis (GZCC 25-0644 and GZCC 25-0645) formed a sister lineage to H. laxisporum (CGMCC 3.25545) with 100% ML and 1.00 BYPP statistical support. Sequence comparisons revealed that our isolates (GZCC 25-0645, ex-type) differ from H. laxisporum (CGMCC 3.25545) by 17/517 bp in ITS (3.3%, including eight gaps), 2/861 bp in LSU (0.2%, no gaps), 3/914 bp in tef1-α (0.3%, no gaps) and 0/1,097 bp in rpb2 (0%, no gaps). Morphologically, H. qixianlingensis (GZAAS 25-0674) differs from H. laxisporum (HKAS 128907) by its shorter conidiophores (13–38 × 4.5–5.5 μm vs. up to 59 × 5–8.5 μm) and a greater number of conidial times (2–21/2 vs. 11/3–2). In addition, the PHI test results (Fig. 3) revealed no significant recombination relationships between Helicotubeufia qixianlingensis (GZCC 250644 and GZCC 25-0645) and its phylogenetically related taxa. Therefore, Helicotubeufia qixianlingensis is introduced here as a new species, based on morphological and phylogenetic analyses, with supporting evidence from the PHI test. Tubeufia diaoluoshanensis T.H. Tan & J. Ma, sp. nov. MycoBank No: 904432 Fig. 4 Etymology. “diaoluoshanensis” refers the place “Diaoluoshan National Nature Reserve” from where the fungus was collected. Figure 3. Results of the PHI test of Helicotubeufia qixianlingensis (GZCC 25-0644 and GZCC 25-0645) with closely-related species (combined LSU-ITS-tef1-α-rpb2) using both LogDet transformation and splits decomposition. PHI test results (Φw) < 0.05 indicate significant recombination within the dataset. New species are indicated in red bold and type strains are marked with “T”. 72 MycoKeys 126: 57–74 (2025), DOI: 10.3897/mycokeys.126.174148 Ting-Hong Tan et al.: Introduce two new species Lu B, Hyde K, Ho W, Tsui K, Taylor J, Wong K, Yanna, Zhou D (2000) Checklist of Hong Kong fungi. Fungal Diversity Press, Hong Kong. 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