Morphological and phylogenetic analyses reveal two new species of Rhodoveronaea (Rhamphoriaceae, Rhamphoriales) from China

105 Morphological and phylogenetic analyses reveal two new species of Rhodoveronaea (Rhamphoriaceae, Rhamphoriales) from China Hong Zhang1,2,3 , Ting-Hong Tan4,5,6 , Jian Ma7,8 1 School of Food Engineering, Moutai Institute, Renhuai, Guizhou 564507, China 2 Guizhou Engineering Research Center for Health-Functional Liquor Brewing Technology, Moutai Institute, Renhuai, Guizhou 564507, China 3 Special Food Resources Utilization Talent Base of Moutai Institute, Moutai Institute, Renhuai, Guizhou 564507, China 4 School of Agriculture and Forestry Engineering and Planning, Tongren University, Tongren, Guizhou 554300, China 5 Guizhou Provincial Key Laboratory for Biodiversity Conservation and Utilization in the Fanjing Mountain Region, Tongren University, Tongren, Guizhou 554300, China 6 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand 7 Guizhou Industry Polytechnic College, Guiyang, Guizhou 550008, China 8 School of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang, Guizhou 550003, China Corresponding authors: Ting-Hong Tan ([email protected]); Jian Ma ([email protected]) Copyright: © Hong Zhang 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 During a recent survey of freshwater fungi, four isolates were obtained from decaying wood in Chishui City, Guizhou Province, southern China. Based on phylogenetic analyses of a combined dataset (LSU, ITS, SSU, tef1-α, and rpb2) and detailed morphological comparisons, two novel species, Rhodoveronaea aquisubtropica and R. guizhouensis, are introduced. Comprehensive descriptions, illustrations, and results of the phylogenetic analyses supporting the taxonomic placement of these new taxa are provided. Additionally, a checklist of currently accepted Rhodoveronaea species supported by molecular data is included. Notably, R. aquisubtropica and R. guizhouensis represent the first records of Rhodoveronaea in the Chishui River Basin, thereby enriching the known diversity of subtropical freshwater habitats. Key words: Asexual morph, phylogeny, Sordariomycetes, taxonomy, two new species Introduction Freshwater fungi represent a taxonomically and ecologically diverse group of organisms that are widely distributed across various geographical regions, particularly in China (Guizhou, Hainan, Hong Kong, Taiwan, and Yunnan) and northern Thailand (Chiang Mai and Chiang Rai) (Zhang et al. 2011; Hyde et al. 2016, 2021; Su et al. 2016; Bao et al. 2018, 2019, 2020, 2021, 2023; Luo et al. 2019; Dong et al. 2020, 2021a, b; Fryar and Catcheside 2023; Fryar et al. 2023; Shen et al. 2024). They play a crucial role in the decomposition of organic matter and nutrient cycling within freshwater ecosystems (Luo et al. 2004; Hyde et al. 2016; Calabon et al. 2022, 2023). These fungi typically colonize fully or partially submerged woody substrates in a variety of freshwater habitats, including ponds, lakes, rivers, streams, and swamps, as well as artificial environments such as pools, reservoirs, dams, drainage ditches, and water-cooling towers (Lu et al. 2018; Xiao et al. 2023; Yang et al. 2023; Ma et al. 2024; Wang et al. 2024a, b; Xu et al. 2025). Academic editor: Xinlei Fan Received: 6 August 2025 Accepted: 24 September 2025 Published: 10 October 2025 Citation: Zhang H, Tan T-H, Ma J (2025) Morphological and phylogenetic analyses reveal two new species of Rhodoveronaea (Rhamphoriaceae, Rhamphoriales) from China. MycoKeys 123: 105–119. https://doi.org/10.3897/ mycokeys.123.167930 MycoKeys 123: 105–119 (2025) DOI: 10.3897/mycokeys.123.167930 106 MycoKeys 123: 105–119 (2025), DOI: 10.3897/mycokeys.123.167930 Hong Zhang et al.: Introduce two new species Rhodoveronaea was established by Arzanlou et al. (2007) with R. varioseptata as the type species. Rhodoveronaea species are primarily known from their asexual morphs, which are characterized by reddish-brown, septate, straight or flexuous conidiophores with inflated basal cells, terminally integrated conidiogenous cells with crowded, slightly conspicuous conidium-bearing denticles, pale brown, ellipsoidal to obovoidal and septate conidia with a protruding base and a marginal basal frill (Arzanlou et al. 2007; Réblová 2009; Luo et al. 2019; Crous et al. 2021, 2023; Hyde et al. 2023; Chen et al. 2024; Yang et al. 2025). The first sexual morph characterized by immersed ascomata with subglobose to conical venter bearing conical neck, filamentous, septate paraphyses longer than asci, unitunicate, cylindrical asci with long-stipitate and fusiform, septate, hyalina ascospores (Réblová 2009). Currently, eight species are accepted within the genus Rhodoveronaea, which occur in both freshwater and terrestrial habitats (Chen et al. 2024; Yang et al. 2025). In this study, four hyphomycetes isolates were obtained from freshwater habitats in Chishui River, Guizhou Province, China. Based on morphological characteristics, illustrations, and multi-gene phylogenetic analyses, two novel species are introduced, namely, Rhodoveronaea aquisubtropica and R. guizhouensis. Materials and methods Sample collection, examination, and isolation Samples of submerged decaying wood pieces were collected from the Chishui River, Chishui City, Guizhou Province in southern China. Samples were taken to the laboratory in plastic bags with the collection details, including localities and dates (Rathnayaka et al. 2024). Fresh specimens were incubated in ziplock 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. Measurements were made using Tarosoft (R) Image Frame Work software. Photo-plates were assembled using Adobe Photoshop CC 2019 (Adobe Systems, USA). Single spore isolations were performed on PDA plates following the methods described by Chomnunti et al. (2014) and Senanayake et al. (2020), and the germinated conidia were aseptically transferred to fresh PDA plates. Morphological characters of fungal mycelium, including color, shape, and size, were documented. Dried fungal specimens were deposited in the Herbarium of Guizhou Academy of Agriculture Sciences (Herb. GZAAS), Guiyang, China. Pure cultures were deposited in the Guizhou Culture Collection, China (GZCC), Guiyang, China. The MycoBank numbers were obtained as described in 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 genomic DNA extraction. Genomic DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, China). LR0R/LR5,ITS5/ITS4, NS1/NS4, EF1-983F/EF1-2218R, and fRPB2-5F/fRPB2-7cR were employed to amplify large ribosomal subunit (LSU; Vilgalys and Hester 1990), the internal transcribed 107 MycoKeys 123: 105–119 (2025), DOI: 10.3897/mycokeys.123.167930 Hong Zhang et al.: Introduce two new species spacer (ITS; White et al. 1990), small subunit of the nuclear ribosomal DNA (SSU, Rehner and Samuels 1994), translation elongation factor 1-α (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 50 μL mixture, which included 2 μL ofDNA, 2 μL of each forward and reverse primer, and 44 μL of 1.1 × T3 Supper PCR Mix (Qingke Biotech, Chongqing, China). The conditions for the polymerase chain reaction (PCR) correspond to those reported by Chen et al. (2024). The PCR products were purified and sequenced with the same primers at Beijing Tsingke Biotechnology Co., Ltd. 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-Gutiérrezet et al. 2009). A combined sequence dataset was created using SequenceMatrix-Windows-1.7.8 software (Vaidya et al. 2011). Maximum likelihood (ML) analysis was carried out using the IQ Tree online website (http://iqtree.cibiv.univie.ac.at/) based on Bayesian Information Criteria (BIC) (Nguyen et al. 2015). 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 the nexus format file by using AliView (Daniel et al. 2010). The best-fit evolutionary model for individual dataset was determined using MrModeltest v. 2.3. 10 (Nylander et al. 2008). The GTR+G+I substitution model was selected for LSU, tef1-α, and rpb2, whereas the SYM+I+G and HKY+G models were applied to ITS and SSU, respectively.The posterior probabilities (BYPP) were determined based on Bayesian Markov chain Monte Carlo (BMCMC) sampling (Huelsenbeck and Ronquist 2001). Five 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). Trees were visualized using FigTree v.1.4.4 and edited with Adobe Illustrator CC 2019 (v.23.1.0; Adobe Systems, USA). Results The phylogenetic placements of the four new strains were determined by multi-locus phylogenetic analysis. The concatenated sequence matrix comprised 4,318 characters (LSU: 1–860,ITS: 861–1,402, SSU: 1,403–2,409, tef1-α: 2,410–3,309, and rpb2: 3,310–4,318) across 29 taxa. Base frequencies and rates were A = 0.251521, C = 0.250418, G = 0.274699, and T = 0.223362; substitution rates were AC = 1.428534, AG = 2.806428, AT = 1.229261, CG = 1.263192, CT = 7.729689, and GT = 1.000000. The distribution shape parameter α equaled 0.144696. Fig. 1 presents the best-scoring ML tree, which had a final log-likelihood value of -16,381.027. 108 MycoKeys 123: 105–119 (2025), DOI: 10.3897/mycokeys.123.167930 Hong Zhang et al.: Introduce two new species Based on the multi-gene phylogenetic tree (Fig. 1), our collections represent two species of Rhodoveronaea within the family Rhamphoriaceae. Our isolates, GZCC 24-0170 and GZCC 25-0635, group together and this clade forms a distinct lineage with Rhodoveronaea aquatica (GZCC 20-0447 and MFLUCC 181339) with 76% ML support. In addition, GZCC 25-0633 and GZCC 25-0634 form a clade together and are sister to Rhodoveronaea lignicola (GZCC 23-0624) with 92% ML and 1.00BYPP support. Taxonomy Rhodoveronaea aquisubtropica H. Zhang & J. Ma, sp. nov. MycoBank No: 904169 Fig. 2 Etymology. “aqui-’’ refers to aquatic habitat of this fungus, and ‘‘-subtropica’’ means the climate type where the fungus was collected. Holotype. GZAAS 25-0665. Table 1. Taxa used in this study and their GenBank accession numbers. Taxon Strain GenBank Accessions LSU ITS SSU tef1-α rpb2 Myrmecridium schulzeri CBS 100.54 EU041826 EU041769 --- Myrmecridium sorbicola CBS 143433TMH107948 MH107901 --- Rhamphoria pyriformis CBS 139033 KT991665 KT991677 MG600406 -KT991656 Rhamphoria pyriformis CBS 139024 MG600397 MG600392 MG600405 -MG600401 Rhamphoriopsis aquimicrospora GZCC 20-0515TOP377911 OP377812 OP377996 OP472992 OP473085 Rhamphoriopsis hyalospora MFLU 19-2849TMN846342 MN846344 --- Rhamphoriopsis muriformis CBS 127683 MG600395 MG600389 MG600403 -MG600400 Rhamphoriopsis muriformis CBS 131269TMG600396 -MG600404 -MG600399 Rhamphoriopsis sympodialis HKAS 105172TMT079191 MT079187 --- Rhodoveronaea aquatica MFLUCC 18-1339TMK849785 MK828641 MK828310 MN194046 - Rhodoveronaea aquatica GZCC 20-0447 OP377947 OP377862 OP378027 OP473041 OP473107 Rhodoveronaea aquisubtropica GZCC 25-0635TPX062375 PX062369 PX062379 PX072387 PX072391 Rhodoveronaea aquisubtropica GZCC 24-0170 PX062376 PX062370 PX062380 PX072388 PX072392 Rhodoveronaea everniae CBS 148309TOK663776 OK664737 - - OK651172 Rhodoveronaea guizhouensis GZCC 25-0633TPX062373 PX062367 PX062377 PX072385 PX072389 Rhodoveronaea guizhouensis GZCC 25-0634 PX062374 PX062368 PX062378 PX072386 PX072390 Rhodoveronaea hainanensis GZAAS 22-2020TOP748932 OP748935 --- Rhodoveronaea hyalina GZCC 23-0622TPP102207 PP102206 PP102214 PP259403 PP259399 Rhodoveronaea lignicola GZCC 23-0624TPP102209 PP102212 PP102216 PP259405 PP259401 Rhodoveronaea nieuwwulvenica CBS 149447TOQ629048 OQ628466 -OQ627955 OQ627935 Rhodoveronaea querci HKAS 145561TPV097144 PQ932528 PV097142 PV164602 - Rhodoveronaea sp. XX-2025aT-PV053196 PV053387 - - Rhodoveronaea varioseptata CBS 431.88TMH873827 MH862134 --- Rhodoveronaea varioseptata CBS 123472 FJ617559 MG600393 MG600408 -JX066701 Rhodoveronaea varioseptata CBS 123473 FJ617560 KT991676 JX066710 -JX066700 Xylolentia aseptata GZCC 20-0424TOP377944 OP377859 OP378024 OP473038 OP473104 Xylolentia aseptata GZCC 20-0426 OP377945 OP377860 OP378025 OP473039 OP473105 Xylolentia brunneola PRA 13611TMG600398 MG600394 MG600407 -MG600402 Xylolentia reniformis MFLU 19-0210TMK547648 MK547646 --- Note: “T” indicates ex-type strains.Newly generated sequences are inbold. “-” indicates the unavailable data in GenBank. 109 MycoKeys 123: 105–119 (2025), DOI: 10.3897/mycokeys.123.167930 Hong Zhang et al.: Introduce two new species Figure 1. Phylogenetic tree generated from the RAxML analysis based on the combined LSU, ITS, SSU, tef1-α, and rpb2 sequence data. Bootstrap support values of RAxML (ML) equal to or greater than 75%, and Bayesian posterior probabilities (PP) equal to or greater than 0.95 are given near the nodes as ML/BYPP, respectively. The Maximum Likelihood (ML) and Bayesian Inference (BI) analyses yielded similar tree topologies. Hyphen (“-”) indicates a posterior probability lower than 0.95 for Bayesian.Myrmecridium schulzeri (CBS 100.54) and M. sorbicola (CBS 143433) were selected as outgroups. Ex-type strains are denoted with “T” and newly obtained isolates are in bold black fonts. 110 MycoKeys 123: 105–119 (2025), DOI: 10.3897/mycokeys.123.167930 Hong Zhang et al.: Introduce two new species Figure 2. Rhodoveronaea aquisubtropica (GZAAS 25-0665, holotype). a, b. Colonies on the host surface; c–f. Conidiophores, conidiogenous cells and conidia; g, h. Conidiogenous cells and conidia; i–l. Conidia; m. Germinated conidium; n, o. Colonies on PDA from above and below after 36 days of incubation at room temperature. Scale bars: 50 μm (c–f); 20 μm (g, h); 5 μm (i–m). 111 MycoKeys 123: 105–119 (2025), DOI: 10.3897/mycokeys.123.167930 Hong Zhang et al.: Introduce two new species Description. Saprobic on decaying wood in a freshwater habitat. Sexual morph Undetermined. Asexual morph Hyphomycetous. Colonies on wood effuse, hairy, scattered or aggregated, brown. Mycelium partly superficial, partly immersed, composed of branched, septate, guttulate, smooth-walled, hyalina to brown hyphae. Conidiophores 204–270 × 4.9–7 μm (x – = 241.5 × 6 μm, n = 20), macronematous, mononematous, erect, flexuous, solitary, cylindrical, smoothwalled, septate, unbranched, black brown, mid brown, paler towards the apex. Conidiogenous cells polyblastic, integrated, terminal, determinate, sympodial, forming a rachis with subdenticulate loci, flexuous, pale brown to subhyalina, pigmented, with inconspicuous denticles. Conidia 10.5–13.5 × 4–5.5 μm (x – = 12.5 × 4.7 μm, n = 25), acropleurogenous, aggregated in slimy masses, ellipsoidal to narrowly obovoid, 1–3-septate, mostly 3-septate, smooth-walled, pale yellowish brown, guttulate, thin-walled, sometimes slightly constricted at the septa. Culture characteristics. Conidia germinating on PDA within 12 hours, producing germ tubes from the conidial body. Colonies on PDA are circular with a raised surface and entire margin, reaching 30 mm in diameter after 36 days at room temperature (approximately 25 °C), and are brown to reddish brown in color on both the surface and reverse sides. Material examined. China • Guizhou Province, Chishui City, Chishui River, on decaying submerged wood in a freshwater habitat, 10 October 2024, Hong Zhang & Jian Ma, CS12 (GZAAS 25-0665, holotype), ex-type living cultures GZCC 25-0635; Ibid., CS18 (GZAAS 24-0083, paratype), living culture GZCC 24-0170. Notes. In our phylogenetic tree (Fig. 1), our isolates (GZCC 25-0635 and GZCC 24-0170) formed a sister clade to Rhodoveronaea aquatica (GZCC 20-0447 and MFLUCC 18-1339) with 76% ML support. A comparison of LSU, ITS, SSU, and tef1-α sequences between Rhodoveronaea aquatica (MFLUCC 18-1339) and R. aquisubtropica (GZCC 25-0635) and reveals their nucleotide differences of 3/774 bp (0.4%, without gap),20/502 bp (4%, including 4 gaps), 5/852 bp (0.6%, including 3 gaps), and 29/882 bp (3.3%, without gap), respectively, indicating that they are distinct species. Morphologically, Rhodoveronaea aquisubtropica (GZAAS 25-0665) differs from R. aquatica (MFLU 18-1593, ex-type) by its narrower conidiophores (204–270 × 4.9–7 μmvs.182–310 × 9–13 μm), and smaller conidia (10.5–13.5 × 4–5.5 μm vs. 23–27 × 9–11 μm) (Luo et al.2019). Therefore, we introduce Rhodoveronaea aquisubtropica as a novel species based on the multi-gene phylogenetic analysis and morphological differences. Rhodoveronaea guizhouensis H. Zhang & J. Ma, sp. nov. MycoBank No: 904170 Fig. 3 Etymology.The specific epithet ‘guizhouensis’ refers to the locality “Guizhou Province”, from where the holotype was collected. Holotype. GZAAS 25-0663. Description. Saprobic on decaying submerged wood in a freshwater habitat. Sexual morph Undetermined. Asexual morph Hyphomycetous. Colonies on wood effuse, hairy, scattered or aggregated, brown. Mycelium partly superficial, partly immersed, composed of branched, septate, guttulate, smooth-walled, hyalina to brown hyphae. Conidiophores 211–268 × 4.5–6.3 μm (x – = 234 × 5.2 μm, n = 25), 112 MycoKeys 123: 105–119 (2025), DOI: 10.3897/mycokeys.123.167930 Hong Zhang et al.: Introduce two new species Figure 3. Rhodoveronaeaguizhouensis (GZAAS 25-0663, holotype). a, b. Colonies on the host surface; c–f. Conidiophores, conidiogenous cells, and conidia; g–i. Conidiogenous cells and conidia; j. A germinated conidium; k–o. Conidia; p, q. Colonies on PDA from above and below after 32 days of incubation at room temperature. Scale bars: 60 μm (c–f); 30 μm (g, h); 20 μm (i); 10 μm (j); 5 μm (k–o). 113 MycoKeys 123: 105–119 (2025), DOI: 10.3897/mycokeys.123.167930 Hong Zhang et al.: Introduce two new species macronematous, mononematous, erect, flexuous, solitary, cylindrical, smoothwalled, septate, unbranched, black brown, mid brown, paler towards the apex. Conidiogenous cells polyblastic, integrated, terminal, determinate, sympodial, forming a rachis with subdenticulate loci, flexuous, pale brown to subhyalina, pigmented, with inconspicuous denticles. Conidia 11.5–17 × 4.3–6 μm (x – = 13.8 × 5.2 μm, n = 25), acropleurogenous, aggregated in slimy masses, ellipsoidal to narrowly obovoid, 1–3-septate, mostly 3-septate, smooth-walled, pale yellowish brown, guttulate, thin-walled, sometimes slightly constricted at the septa. Culture characteristics. Conidia germinating on PDA within 11 hours, producing germ tubes from the conidial body. Colonies on PDA are circular with a raised surface and entire margin, reaching 27 mm in diameter after 32 days at room temperature (approximately 25 °C), and are white, reddish brown to black in color on both the surface and reverse sides. Material examined. China • Guizhou Province, Chishui City, Chishui River, on decaying submerged wood in a freshwater habitat, 10 October 2024, Hong Zhang &Jian Ma, CSF2 (GZAAS 25-0663, holotype), ex-type living cultures GZCC 25-0633; Ibid., CSF9 (GZAAS 25-0664, paratype), living culture GZCC 25-0634. Notes. In our phylogenetic tree (Fig. 1), our isolates (GZCC 25-0633 and GZCC 25-0634) formed a sister clade to Rhodoveronaea lignicola (GZCC 23-0624) with 92% ML and 1.00BYPP support. Rhodoveronaea guizhouensis (GZAAS 25-0663) can be distinguished from R. lignicola (GZAAS 23-0612) by its longer conidiophores (211–268 μmvs.75–125 μm), longer conidia (up to 17 μmvs.9–13.5 μm) (Chen et al. 2024). Moreover, base pair comparison of Rhodoveronaea guizhouensis (GZCC 25-0633) and R. lignicola (GZCC 23-0624) shows 30/528 bp differences in ITS (5.7%, gaps 5 bp), 7/862 bp differences in LSU (0.8%, gaps 2 bp), 3/910 bp differences in SSU (0.3%, without gap), 39/887 bp differences in tef1-α (4.4%, without gap), and 69/803 bp differences in rpb2 (8.6%, without gap). Therefore, based on DNA molecular data and morphological characteristics, we introduce Rhodoveronaea guizhouensis as a novel species. Discussion The genus Rhodoveronaea currently comprises ten species, including the two new species described in the present study (Arzanlou et al. 2007; Luo et al. 2019; Crous et al. 2021, 2023; Hyde et al. 2023; Chen et al. 2024; Yang et al. 2025). Among them, four species are found in freshwater habitats and six in terrestrial habitats (Table 2), with primary distributions in Guizhou, Yunnan, and Hainan provinces of China.Rhodoveronaea species are distributed in China, Czech Republic, France, Germany, Netherlands, and Sweden (Arzanlou et al. 2007; Réblová 2009; Luo et al. 2019; Crous et al. 2021, 2023; Hyde et al. 2023; Chen et al. 2024; Yang et al. 2025). They occur as saprobes on bamboo stick, Bertia moriformis, Carpinus betulus, Evernia prunastri, and decaying wood in both freshwater and terrestrial habitats (Arzanlou et al. 2007; Réblová 2009; Luo et al. 2019; Crous et al. 2021, 2023; Hyde et al. 2023; Chen et al. 2024; Yang et al. 2025). Based on multi-gene phylogenetic analyses, considerable morphological variation can occur even within the same Rhodoveronaea species. For example, two collections (MFLU 18-1593 from decaying submerged wood in Yunnan Province and HKAS 112574 from a similar substrate in Guizhou Province, China) have both been identified as the same species, namely Rhodoveronaea aquatica