«83 MycoKeys MycoKeys 106: 117-132 (2024) DOI: 10.3897/mycokeys.106.123279 Research Article Multi-gene phylogenetic analyses revealed two novel species and one new record of Trichobotrys (Pleosporales, Dictyosporiaceae) from China Wen-Jing Zhang™, Gui-Ping Xu2, Yu Liu’, Yang Gao", Hai-Yan Song‘, Hai-Jing Hu’, Jian-Ping Zhou", Ming-Hui Chen"4, Deng-Mei Fan®, Dian-Ming Hu“®, Zhi-Jun Zhai'4© 1 College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, 1107 Zhimin Road, Nanchang, 330045, China 2 School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China 3 Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Ocean Science and Technology, Dalian University of Technology, Panjin Campus, China 4 Jiangxi Key Laboratory for Excavation and Utilization of Agricultural Microorganisms, Jiangxi Agricultural University, Nanchang, 1101 Zhimin Road, Nanchang, 330045, China 5 School of Agricultural Sciences, Jiangxi Agricultural University, Nanchang, 1101 Zhimin Road, Nanchang, 330045, China Corresponding authors: Dian-Ming Hu (hudianming1@163.com); Zhi-Jun Zhai (zhjzh002@163.com) OPEN Qaceess Academic editor: Huzefa Raja Received: 19 March 2024 Accepted: 29 May 2024 Published: 20 June 2024 Citation: Zhang W-J, Xu G-P, Liu Y, Gao Y, Song H-Y, Hu H-J, Zhou J-P, Chen M-H, Fan D-M, Hu D-M, Zhai Z-J (2024) Multi-gene phylogenetic analyses revealed two novel species and one new record of Trichobotrys (Pleosporales, Dictyosporiaceae) from China. MycoKeys 106: 117-132. https://doi.org/10.3897/ mycokeys.106.123279 Copyright: © Wen-Jing 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). Abstract The rotting wood in freshwater is a unique eco-environment favoring various fungi. During our investigation of freshwater fungi on decaying wood, three hyphomycetes were col- lected from Jiangxi and Guangxi Provinces, China. Based on the morphological observa- tions and phylogenetic analysis of a combined DNA data containing ITS, LSU, SSU and tef1-a sequences, two new Trichobotrys species, T. meilingensis and T. yunjushanensis, as well as a new record of T. effusa, were introduced. Additionally, a comprehensive de- scription of the genus with both morphological and molecular data was first provided. Key words: Freshwater hyphomycetes, phylogenetic analysis, taxonomy, Trichobotrys Introduction Trichobotrys Penzig & Saccardo is a genus introduced with the discovery of the type species Trichobotrys effusa (Berk. & Br.) Petch from Sri Lanka, which was placed in Pleosporales genera incertae sedis (Pleosporales, Dothideomycetes, Ascomycota) (Petch 1924; Morgan-Jones et al. 1987). Trichobotrys effusa is known for producing compounds which can exhibit significant growth-inhibito- ry activities against the A549 lung cancer cell line (Chen et al. 2014). In addition, the bioactive compounds obtained from the deep-sea-derived fungus T. effusa DEFSCS021 could strongly inhibit the larvae settlement of Bugula neritina and Balanus amphitrite larvae (Sun et al. 2016). Trichobotrys encompasses fungi characterised by their mononematous con- idiophores producing catenate, dark brown, spherical and echinulate conidia on fertile, smooth, short, lateral branches with polyblastic conidiogenous cells. So far, only five species are recognised in this genus (http://www.indexfungorum. org/Names/Names.asp), namely T. effusa, T. ipomoeae, T. pannosa, T. ramosa 117 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China and T. trechispora. However, T. pannosa has been treated as a synonym of T. effusa (Morgan-Jones et al. 1987; D'Souza and Bhat 2001). Therefore, Tricho- botrys is supposed to comprise four saprobic species, of which one (T. effusa) is from aquatic habitats and three (T. ipomoeae, T. ramosa and T. trechispora) are from terrestrial habitats (Petch 1917, 1924; Sawada 1959; Morgan-Jones et al. 1987; D'Souza and Bhat 2001). To date, the phylogenetic positions of representatives of Trichobotrys within the Ascomycota have not yet been inves- tigated, as T. effusa has only ITS sequence and there are no molecular data for T. ipomoeae, T. ramosa and T. trechispora. In the current study, we attempt to clarify the classification status of Tricho- botrys through further identified materials and a more appropriate multi-gene genealogy. During our investigation of the freshwater hyphomycetes from decaying wood in Jiangxi and Guangxi provinces of China, two novel species named T. meilingensis and T. yunjushanensis, as well as a new record of T. effu- sa, are described according to morphological examination and multi-loci phy- logenetic evidence. Materials and methods Samples collection, morphological observation and isolation Samples of dead wood submerged in freshwater streams were collected from Jiangxi and Guangxi Provinces, China and were brought to the laboratory in plastic bags. Observations for fungi on natural substrates were made using a Nikon SMZ-1270 microscope (Nikon Corporation, Japan). With a syringe needle, the fungal structures were gathered and transferred to a small drop of distilled water on a clean slide, which was covered with a cover slide (Yang et al. 2018a). Micro-morphological characters were observed by a Nikon ECLIPSE Ni-U com- pound microscope (Nikon Corporation, Japan) and photographed by a Nikon DS-Fi3 camera. All measurements of the fungal structures were performed with PhotoRuler v. 1.1 software (The Genus Inocybe, Hyogo, Japan) and figures were made with Adobe Photoshop CC 2017 software (Adobe Systems, USA). Pure cultures of the fungi were obtained by the single spore isolation method (Chom- nunti et al. 2014). Germinating conidia were transferred to fresh potato dextrose agar (PDA, from Beijing Bridge Technology Co., Ltd., Beijing, China) supplement- ed with two types of antibiotics (100 ug/mL penicillin, 50 ug/mL streptomycin), and then incubated at 25 °C for 2-3 weeks. Pure cultures were deposited at the Jiangxi Agricultural University Culture Collection (JAUCC) and specimens were stored in the Herbarium of Fungi, Jiangxi Agricultural University (HFJAU). DNA extraction, PCR amplification and sequencing Fresh mycelia of each strain, scraped from the growing culture with a sterile scal- pel, were ground to a fine powder with liquid nitrogen to break the cells for DNA extraction. Subsequently, total genomic DNA was extracted following the modi- fied CTAB method (Doyle and Doyle (1987). Four primer pairs, ITS1/ITS4 (White et al. 1990), LROR/LR7 (Hopple and Vilgalys 1999), NS1/NS4 (White et al. 1990) and EF1-983F/EF1-2218R (Rehner and Buckley 2005), were used to amplify ITS, LSU, SSU and tef1-a gene regions, respectively. Polymerase chain reaction (PCR) MycoKeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 118 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China was performed in a final volume of 25 ul, containing 9.5 ul double distilled water (ddH,O), 12.5 pl 2 x Taq PCR MasterMix (Qingke, Changsha, China), 1 pl each prim- er (10 uM) and 1 ul genomic DNA extract. Amplification conditions for ITS, LSU, SSU and tef7-a gene regions followed Zhai et al. (2022). The PCR products were sent to be sequenced by the commercial company QingKe Biotechnology Co. (Changsha, China). All sequences were edited with SeqMan v. 7.1.0 (DNASTAR, Inc, Madison, WI) and were deposited in the NCBI GenBank database (Table 1). Data analyses Based on ITS, LSU, SSU and tef1-a sequence comparison with the GenBank database, similar species in Dictyosporiaceae were found. The sequences of 37 relevant species according to the blasting result and recent publications (Tanaka and Harada 2003; Chen et al. 2014; Tanaka et al. 2015; Boonmee et al. 2016; Liu et al. 2017; Yang et al. 2018a; Chen et al. 2020) were chosen for phy- logenetic analyses (Table 1) and were downloaded from GenBank. Four gene regions (ITS, LSU, SSU and tef7-a) were individually aligned using the online ser- vice of MAFFT v. 7 (Madeira et al. 2019) and concatenated using PhyloSuite v. 1.2.2 (Zhang et al. 2020). The alignments were checked visually and improved manually using BioEdit (Hall 1999; Liu et al. 2017). Maximum Likelihood (ML) and Bayesian Inference (BI) were used to assess phylogenetic relationships. Maximum Likelihood (ML) analysis was conducted with RAXML v. 7.2.6 (Stamatakis and Alachiotis 2010) using the default sub- stitution model GTR-GAMMA with rapid bootstrap analysis followed by 1000 bootstrap replicates to estimate ML bootstrap (BS) values. Bayesian Inference (BI) analysis was carried out with MrBayes v. 3.2 under partitioned models (Ronquist et al. 2012). The best-fit models of nucleotide substitutions were selected according to the Akaike information criterion (AIC) implemented in jModelTest v. 2.1.1 (Darriba et al. 2012) on XSEDE in the CIPRES web portal (Miller et al. 2010). The models for ITS, LSU, SSU and tef1-a datasets used for phylogenetic analysis are TIM2+I+G model (-InL = 5321.6598), TIM2+I+G model (-InL = 3199.3778), TIM2+I+G model (-InL = 3481.7971) and GTR+I+G model (-InL = 4762.6993), respectively. The data sets were run for 10,000,000 generations, with four chains, sampling trees every 1,000 generations. The first 10% trees were discarded as burn-in. Phylogenetic trees were visualized with FigTree v. 1.4.4 (Rambaut 2018), edited and beautified using Adobe Illustrator 2020 (Adobe Systems Inc., USA). Results Molecular phylogenetic results According to sequence alignment analysis, the ITS sequences of the new record Trichobotrys effusa (JAUCC 6359 and JAUCC 6826) have only two different loci from that of T. effusa FS524 and three loci from that of T. effusa YMJ1179. The aligned sequence matrix for the combined analysis consists of ITS (574 bp), LSU (1259 bp), SSU (1459 bp) and tef1-a (962 bp) with a total of 4254 characters including gaps. The combined dataset shows the new species T. meilingensis and T. yunjushanensis share 98.61% (59 different loci), 98.40% (68 different loci) MycoKeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 119 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China Table 1. Sequences used in this study. Species Aquadictyospora clematidis Aquadictyospora lignicola Dendryphiella paravinosa Dendryphiella vinosa Dictyocheirospora aquatica Dictyocheirospora bannica Dictyocheirospora bannica Dictyocheirospora garethjonesii Dictyocheirospora garethjonesii Dictyocheirospora pseudomusae Dictyocheirospora pseudomusae Dictyocheirospora heptaspora Dictyocheirospora rotunda Dictyocheirospora rotunda Dictyosporium alatum Dictyosporium bulbosum Dictyosporium digitatum Dictyosporium digitatum Dictyosporium digitatum Dictyosporium elegans Dictyosporium hughesii Dictyosporium meiosporum Dictyosporium nigroapice Dictyosporium olivaceosporum Dictyosporium pandanicola Dictyosporium stellatum Dictyosporium strelitziae Dictyosporium tetrasporum Dictyosporium thailandicum Dictyosporium tratense Digitodesmium bambusicola Gregarithecium curvisporum Jalapriya pulchra Jalapriya toruloides Periconia igniaria Periconia igniaria Pseudocoleophoma calamagrostidis Pseudocoleophoma flavescens Pseudocoleophoma polygonicola Pseudocoleophoma zingiberacearum Pseudodictyosporium elegans Pseudodictyosporium thailandica Pseudodictyosporium wauense Trichobotrys effusa Trichobotrys effusa Trichobotrys effusa* Trichobotrys effusa* Trichobotrys meilingensis* Trichobotrys meilingensis* Trichobotrys yunjushanensis* Trichobotrys yunjushanensis* Ex-type strains or type materials are marked in bold. Newly generated sequences are indicated with Mycokeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 Isolate MFLU 172080 MFLUCC 17-1318 CPC 26176 MFLU 200444 KUMCC 15-0305 KH 332 MFLU 18-1040 MFLUCC 16-0909 DLUCC 0848 yone 234 KH 412 DLUCC 1992 MFLUCC 14-0293 MFLUCC 17-0222 ATCC 34953 yone 221 KT 2660 KH 401 yone 280 NBRC 32502 KT 1847 MFLUCC 10-0131 MFLUCC 17-2053 KH 375 MFLUCC 18-0331 CCFC 241241 CBS 123359 KT 2865 MFLUCC 13-0773 MFLUCC 17-2052 CBS 110279 KT 922 MFLU 17-1683 CBS 209.65 CBS 379.86 CBS 845.96 KT 3284 CBS 178.93 KT 731 NCYUCC 190054 CBS 688.93 MFLUCC 16-0029 CBS 126094 FS524 YMJ1179 JAUCC 6359 JAUCC 6826 JAUCC 4985 JAUCC 4986 JAUCC 4987 JAUCC 4988 ITS MT310592 MF948621 KX228257 MT907477 KY320508 LC014543 MH381765 KY320509 MF948623 LC014550 LC014549 MT 756244 KU179099 MH381764 NR-077171 LC014544 LC014546 LC014545 LC014547 DQ018087 LC014548 KP710944 MH381768 LC014542 MZ490792 NR-154608 NR-156216 LC014551 KP716706 MH381767 DQ018091 AB809644 MF948628 DQ018093 LC014585 LC014586 LC014592 AB809634 MN615941 MH862454 KX259520 MH864014 MN545626 KJ630313 PP406377 PP830649 PP406380 PP406381 PP406378 PP406379 GenBank accession number LSU MT214545 MF948629 KX228309 MT907480 KY320513 AB807513 MH381774 KY320514 MF948631 AB807520 AB807516 MT 756243 KU179100 MH381773 DQO018101 AB807511 AB807518 AB807515 AB807512 DQ018100 AB807517 KP710945 MH381777 AB807514 MZ490776 JF951177 FJ839653 AB807519 KP716/707 MH381776 DQ018103 AB80754 MF948636 DQ018104 AB807566 AB807567 LC014609 GU238075 AB807546 MN616755 MH874101 KX259522 MH875472 PP407503 PP830650 PP407504 PP407505 PP407506 PP407507 A a“ an” SSU MT226664 AB787223 MH381759 AB797230 AB7/97226 KU179101 MH381758 DQ018080 AB7/97221 AB797228 AB797225 AB7/97222 DQ018079 AB7/97227 KP710946 MH381762 AB797224 AB797229 MH381761 AB797257 DQ018081 AB7/97276 AB/97277 LC014604 GU238216 AB797256 DQ018084 KX259524 PP407508 PP830652 PP407509 PP407510 PP407511 PP407512 tef1-a MT394727 MF953164 AB808489 MF953166 AB808496 AB808492 MT776563 MH388818 AB808487 AB808491 AB808488 AB808493 MH388821 AB808490 MZ501208 AB808495 MF388820 AB808523 MF953171 AB808542 AB808543 LC014614 AB808522 MN629283 KX259526 PP405621 PP845300 PP405623 PP405625 PP405622 PP405624 , the sequence is unavailable. 120 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China sequence similarity with T. effusa (JAUCC 6359 and JAUCC6826), respectively, but are less similar to Gregarithecium curvisporum [95.75% (181 different loci) and 95.53% (190 different loci), respectively]. In addition, there are 57 different loci between the sequences of the two new species. The topologies of the phylogenetic trees produced by ML and BI are congru- ent, and the best RAXML tree with BS and PP is shown in Fig. 1. Phylogenetic analyses indicate that the new Trichobotrys effusa isolates (JAUCC 6359 and JAUCC 6826) cluster with other T. effusa collections (FS524 and YMJ1179) in a strongly-supported monophyletic clade (BS/PP = 100/1). Moreover, T. yun- jushanensis is sister to the T. effusa clade, but only with low ML bootstrap sup- port values (BS = 43) and Bayesian posterior probabilities (PP = 0.67). How- ever, these two species and T. meilingensis form a well- supported clade (BS/ PP = 100/1), which is phylogenetically close to Gregarithecium curvisporum (BS/PP = 100/1). Dictyosporium U a Dictyocheirospora O “” © eo) Digitodesmium m= Jalapriya 2 Aquadictyospora © Pseudodictyosporium ® Paraconiothyrium Dendryphiella 100/1 Trichobotrys Gregarithecium 100/1 Outgroup Figure 1. Phylogenetic tree of Dictyosporiaceae inferred from the combined regions (ITS-LSU-SSU-tef7-a) using Max- imum Likelihood (ML) analysis. The Periconia igniaria clade was used as the outgroup. PP = 0.95 and BS = 75% were indicated around the branches. The new sequences generated in this study are given in red and type strains are in bold. MycoKeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 121 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China Taxonomy Trichobotrys meilingensis G. P. Xu & Z. J. Zhai, sp. nov. MycoBank No: 852617 Fige2 Etymology. Referring to the collection site of the Meiling Mountain in Jiangxi Province, China. Holotype. HFJAU10042. Description. Saprobic on the stems of bamboo in freshwater habitats. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies ef- fuse, white to yellow, hairy. Mycelium partly superficial, partly immersed, gre- garious and creeping, composed of septate, branched, pale brown hyphae. Conidiophores 2.5-4.5 um wide (x = 3.5 um, n = 20), up to 510 um long, mononematous, variously curved, dichotomously branched in the conidio- phore, septate, thick-walled, verruculose, echinulate, brown to dark brown. Conidiophore branches 15-39 x 3-4 um (x = 24.5 x 3.4 um, n = 15), fertile, 0—1(—2)-septate, verruculose, pale to dark brown. Conidiogenous cells 7-12 x 3-5 um (x= 9.0 x 4.0 um, n = 10), polyblastic, integrated, erect or curved, widely distributed in the fertile branches, denticulate, hyaline to brown. Conid- ia 7-13 um diam (x = 9.8 um, n = 30), catenate, usually in branched, acropetal chains, aseptate, globose, verruculose, echinulate, sometimes guttulate, yel- low brown to dark brown. Cultural characteristics. Conidia germinating on PDA within 24 h. Colonies incubated on PDA media at 25 °C attaining 30.5 mm diam after 9 days, in nat- ural light, circular, white, slightly cottony, yellow at the margin part, with white dense aerial mycelium; reverse yellow, white at the entire margin. Material examined. CHINA. Jiangxi Province: Nanchang City, Meiling Mountain, on decaying bamboo culms submerged in a freshwater stream, alt. 305 m, near 28.79°N, 115.72°E, 16 August 2021, G. P. Xu, Y. Liu and Z. J. Zhai, SLT-32 (HFJAU10042, holotype), ex-type living culture, JAUCC 4985 = JAUCC 4986. Notes. Trichobotrys meilingensis is similar to other species of Tricho- botrys in having monomatous conidiophores, spherical and echinulate co- nidia, and polyblastic conidiogenous cells. Trichobotrys meilingensis is easily distinguished from T. effusa, T. ipomoeae and T. trechispora by its dichoto- mously branched conidiophores and its conidial size (7-13 um vs. 3-4 um, 13-15.5 um and 3-5 um, respectively) (Petch 1917, 1924; Sawada 1959). Trichobotrys meilingensis is morphologically most similar to 7; ramosa and shares some characteristics, such as dichotomously branched conidiophores and catenate conidia. However, T. meilingensis has larger conidia (7-13 um vs. 3-5 um) and thinner conidiophores (2.5-4.5 um vs. 8-18 pm) (D’Souza and Bhat 2001). Therefore, T. meilingensis can be distinguished from T. ramo- sa based on morphological characters in spite of the unavailable molecular data of the latter species. Thus, it should be identified as an independent tax- on in Trichobotrys. A comparison of morphological features of Trichobotrys species is provided in Table 2. MycoKeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 122 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China me! Figure 2. Trichobotrys meilingensis (HFJAU10042, holotype) a, b colonies on bamboo culms ce conidiophores with con- idiogenous cells f portion of conidiophore with fertile lateral branches g, h conidiogenous cells i-n conidia o germinating conidium p, q culture on PDA from above (p) and reverse (q). Scale bars: 100 pm (a, b); 20 pm (c); 5 um (d-o); 25 mm (p, q). MycoKeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China Trichobotrys yunjushanensis W. J. Zhang & Z. J. Zhai, sp. nov. MycoBank No: 852618 Fig. 3 Etymology. Referring to the collection site of the Yunjushan Mountain in Jiangxi Province, China. Holotype. HFJAU 10044. Description. Saprobic on decaying bamboo culms. Sexual morph: Undeter- mined. Asexual morph: Hyphomycetous. Colonies effuse, white, yellow to oliva- ceous, velvety. Mycelium mostly superficial, creeping and twining, composed of Figure 3. Trichobotrys yunjushanensis (HFJAU 10044, holotype) a, b colonies on bamboo culm e, d conidiophores with conidiogenous cells e, f conidiogenous cell with conidia g portion of conidiophore with fertile lateral branches h-k co- nidia | germinating conidium m, n culture on PDA from above (m) and reverse (n). Scale bars: 100 um (a, b); 20 um (c); 5 um (d-l); 25 mm (m, n). Mycokeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 124 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China septate, brown to olivaceous, branched hyphae. Conidiophores 3-4 um wide (x = 3.4 um, n = 20), up to 1150 um long, mononematous, erect, straight or flexous, septate, with fertile dichotomously branched, pale brown to olivaceous, verrucu- lose, echinulate, thick-walled. Conidiophores branches 18-48 x 3—4 um (x = 29.1 x 3.6 um, n = 15), sometimes long, fertile, O—1(—2)-septate, verruculose, rough, pale brown. Conidiogenous cells 6-11 x3-5 um (x = 8.5 x 4.0 um, n = 10), integrated, polyblastic, terminal to subterminal on fertile branches, with several denticulate conidiogenous loci, hyaline to dark brown. Conidia 7-12 um diam (x = 9.3 um, n = 30), catenate, usually acrogenous or lateral, aseptate, spherical, verrucose, echi- nulate, sometimes guttulate, yellowish brown to dark brown when mature. Cultural characteristics. Conidia germinating on PDA within 24 h. Colonies in- cubated on PDA media at 25 °C grow rapidly, reaching 21 mm diam after 6 days, in natural light, circular, pale on the margin, yellow at the centre, with white dense aerial mycelium; reverse yellow white to dark green. Hyphae hypline, superficial, septate but not obvious, with a layer of yellow pigment, 1.9-3.7 um wide. Material examined. CHINA. Jiangxi Province: Jiujiang City, Yongxiu County, Yunjushan Mountain, on decaying bamboo culms submerged in a freshwater stream, alt. 672.5 m, 29.23°N, 115.59°E, 28 April 2020, G. P. Xu, Y. Liu and Z. J. Zhai, YJS112 (HFJAU10044, holotype), ex-type living culture, JAUCC 4987 = JAUCC 4988. Notes. In the multi-gene phylogenetic tree, Trichobotrys yunjushanensis groups with T. effusa clade with low support (BS/PP = 43/0.67), but they form a monophyletic group when including T. meilingensis (Fig. 1). Morphological- ly, 7. yunjushanensis is distinct from the holotype of T. effusa by its conidial size (7-12 um vs. 3-4 um) and longer conidiophores (up to 1150 um vs. up to 200 um) (Petch 1924). Trichobotrys yunjushanensis is mostly similar to T. meilingensis and T. ramosa in having dichotomously branched and rough co- nidiophores. However, T. yunjushanensis can be easily distinguished from T. ramosa by its larger conidia (7-12 um vs. 3-5 um) (D’Souza and Bhat 2001). Furthermore, T. yunjushanensis differs from T. meilingensis in having longer co- nidiophores (up to 1150 um vs. up to 510 um) and is phylogenetically distinct from the latter. Therefore, both morphological characters and phylogenetic analyses supported T. yunjushanensis as a new taxon within Trichobotrys. Trichobotrys effusa (Berk. & Br.) Petch, Ann. R. bot. Gdns Peradeniya 9: 169 (1924) Fig. 4 Description. Saprobic on the stems of decaying wood in freshwater habitat. Sex- ual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies effuse, grayish to nut brown, velvety. Mycelium mostly superficial, creeping and twining, composed of septate, branched, subhyaline to pale brown hyphae. Conidiophores 2-4 um wide (xX = 2.7 um, n = 20), up to 650 um long, mononematous, erect, straight or somewhat curving, columniform, moderately branched, verruculose, septate, thick-walled, echinulate, light brown to nut brown, gradually attenuated distally to an infertile, setiform apex. Conidiophore branches 7-26 x 2-4 um (x = 14.0 x 3.2 um, n = 16), fertile, O0-1(-2)-septate, verruculose, light brown to dark brown, individual cells typically have a slight swelling. Conidiogenous cells MycoKeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 125 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China Figure 4. Trichobotrys effusa (HFJAU10296, HFJAU10372) a colonies on the substrate b conidiophores with conidia c portion of conidiophore with fertile lateral branches d-f conidiogenous cell with conidia g, h conidia i germinating conidia j, k culture on PDA from above (j) and reverse (k). Scale bars: 100 um (a); 20 um (b, c); 5 um (d-i); 5 mm (j, k). MycoKeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 126 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China 3-10.5 x 2.5-6.5 um (x = 6.6 x 4.0 um, n = 10), monoblastic or polyblastic, inte- grated and terminal on lateral branches, apical or lateral; columniform or cannu- late, erect or slightly curved, with several seriated conidiogenous locations, light brown to dark brown. Conidia 3.5—5 um diam (x = 4.4 um, n = 30), catenulate, simple or branched apical chains, aseptate, spherical, verruculose, echinulate, sometimes guttulate, transparent to dark brown or red brown. Cultural characteristics. Conidia germinating on PDA within 24 h. Colonies incubated on PDA media at 25 °C attaining 11.5 mm diam after 11 days, in natural light, circular, white, cottony, with white dense aerial mycelium; reverse yellow, white at the margin part. Material examined. CHINA. Guangxi Province: Guigang City, Pingtianshan National Forest Park, on decaying wood submerged in a freshwater stream, alt. 980.84 m, near 23.19°N, 109.51°E, 11 March 2023 and 16 May 2024, Wan Hu and Z. J. Zhai, HG13 and HG13-1 (HFJAU10296, HFJAU10372), ex-type living culture, JAUCC 6359 = JAUCC 6826. Notes. According to phylogenetic analysis (Fig. 1), we can find that our new isolates cluster with Trichobotrys effusa FS524 and T. effusa YMJ1179 with high support (BS/PP = 100/1). Morphologically, our new collections are similar to the holotype of T. effusa except for the slightly larger conidia (3.5-5 um vs. 3-4 um), longer conidiophores (up to 650 um vs. up to 200 um), and slightly dif- ferent colors in mycelium (grayish to nut-brown vs. dark purple-brown) (Petch 1924). The difference in color might be due to the discrepancy in incubation time and the exposure to light or different observation angles under the mi- croscope. The differences in the size of conidiophores and conidia are also occurring in another record of T. effusa, in which the conidiophores and conidia are described as being up to 1000 um long and 5-7um in diameter, respec- tively (Morgan-Jones et al. 1987). The differences among the holotype and our new collections suggest that factors such as habitat and incubation time may influence the size of conidia and conidiophores. Similar observations have also been discovered in the asexual morph of other fungal species (Yang et al. 2018b; Zhang et al. 2022; Shen et al. 2024). Owing to the unavailable molecular sequences in the holotype of T. effusa and the deficiency of morphological de- scriptions about T: effusa FS524 and T. effusa YMJ1179, the possibility cannot be excluded that our new isolates are a different species to T. effusa. However, there are no significant morphological differences between our collections and the holotype. Therefore, we propose to identify the new collections as T. effusa until more strains have been examined. The new collection was collected from submerged, decaying wood in Guangxi Province, which is a new discovery in freshwater habitat in China. Discussion The new isolates Trichobotrys effusa (JAUCC 6359 and JAUCC 6826) group well with two strains (FS524 and YMJ1179) of T. effusa (BS/PP = 100/1). The high molecular support and morphological similarities among them indicate that they are conspecific and the two isolates (JAUCC 6359 and JAUCC 6826) are identified as a new record of T. effusa. Although four-loci data for T. effusa FS524 and T. effusa YMJ1179 were lacking and they were sequenced only by ITS, our result should be convincing because the fungal ITS marker generally MycoKeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 127 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China produces considerably more sequence variability, and thus can provide high res- olution for species delimitation (Nilsson et al. 2008; Szczepanska et al. 2021). The holotype of T. effusa was discovered on dead bamboo from Sri Lanka (Berkeley and Broome 1873; Petch 1924). Subsequently, a series of T. effusa strains have been found but were mostly isolated from marine sediment sam- ples collected in the South China Sea (Chen et al. 2014; Sun et al. 2015, 2016; Chen et al. 2020; Liu et al. 2020; Huang et al. 2023), and they were identified as T. effusa almost only based on ITS region sequence comparison with the Gen- Bank database. This study is the first report of collection of T. effusa from the freshwater habitat and provides both molecular phylogenetical and morpholog- ical description for this species. Two new Species, T. meilingensis and T. yunjushanensis, were proposed as members of Trichobotrys based on four-loci (ITS, LSU, SSU and tef7-a) phyloge- netic analyses in combination with morphological characteristics. However, the relationship between T. yunjushanensis and the T. effusa clade was unresolved due to low support value. At present, the clade including T. meilingensis, T. yun- jushanensis and T. effusa is paraphyletic, therefore, the phylogeny relationships within this clade will become clearer with more new closely related species discovered. Besides, D'Souza and Bhat (2001) described T. ramosa from the forest of southern India, but no molecular data of these species are available, so it is difficult to clarify the phylogenetic relationship between this species and other taxa in Trichobotrys. However, T. meilingensis and T. yunjushanensis can be distinguished from T. ramosa by morphological characteristics. Detailed information about their morphological comparison can be obtained from the notes and Table 2 in this paper. Trichobotrys appears as sister to Gregarithecium with high molecular support and is hence assigned to the family Dictyosporiaceae. The asexual morphs of Trichobotrys also mostly resemble other members of Dictyosporiaceae in pos- sessing brown, cheirosporous conidia, produced from holoblastic conidioge- nous cells, on micronematous conidiophores (Boonmee et al. 2016). Although we consider that species of Trichobotrys are closely related to Gregarithecium, the position of Trichobotrys in Dictyosporiaceae and relationship between the two genera are still doubtful due to the long branches between Gregarithecium and Trichobotrys clade and the lack of asexual morph of Gregarithecium. Hence, more samples closely related to Gregarithecium and Trichobotrys are required to be discovered to clarify the position of Trichobotrys in Dictyosporiaceae. It has been widely reported that Trichobotrys effusa as the type species of Trichobotrys has the ability to produce diverse secondary metabolites (Chen et al. 2014; Chen et al. 2020; Huang et al. 2023; Liu et al. 2020; Sun et al. 2015, 2016). For example, Chen et al. (2014) obtained four novel aliphatic phenolic ethers with growth-inhibitory activity against the A549 lung cancer cell and Sun et al. (2016) received three new macrodiolides with antifouling activity. In this research, we introduce two novel species, T. meilingensis and T. yunjushanen- sis, which are both morphologically and phylogenetically similar to T. effusa. Furthermore, these two species both can produce yellow pigments and might have the ability to generate secondary metabolites like T. effusa. Therefore, fu- ture pharmacological evaluation of the two new species might be worth study- ing to confirm if they are similar to T. effusa in having similar bioactive constit- uents and function in secondary metabolites. Mycokeys 106: 117-132 (2024), DOI: 10.3897/mycokeys.106.123279 128 Wen-Jing Zhang et al.: Two novel species and one new record of Trichobotrys from China Apnys siy, (ZL6L) YOI8d (L002) ‘Je J8 eznos,q Apnys siy. 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Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding This study was supported by the National Natural Science Foundation of China (NSFC 32070023 and NSFC 32060014), the Natural Science Foundation of Jiangxi Province (20151BAB214002) and Science and Technology Plan Project of Jiangxi Province (GJJ160417). Author contributions Gui-Ping Xu, Yu Liu and Zhi-Jun Zhai collected samples. Wen-Jing Zhang, Gui-Ping Xu and Yu Liu performed morphological identification, photo-plates, DNA isolation and PCR amplifcation. Wen-Jing Zhang, Gui-Ping Xu, Deng-Mei Fan and Zhi-Jun Zhai analyzed data and wrote the original draft. Yang Gao, Hai-Yan Song, Hai-Jing Hu, Jian-Ping Zhou and Ming-Hui Chen reviewed the paper. Zhi-Jun Zhai and Dian-Ming Hu designed the research and revised the manuscript. All authors approved the final manuscript version. 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