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Research Article
Multi-gene phylogenetic analyses revealed two novel species and one new record of Trichobotrys (Pleosporales, Dictyosporiaceae) from China
expand article infoWen-Jing Zhang‡, Gui-Ping Xu§, Yu Liu|, Yang Gao‡, Hai-Yan Song‡, Hai-Jing Hu‡, Jian-Ping Zhou‡, Ming-Hui Chen‡, Deng-Mei Fan‡, Dian-Ming Hu‡, Zhi-Jun Zhai‡
‡ Jiangxi Agricultural University, Nanchang, China
§ South China University of Technology, Guangzhou, China
| Dalian University of Technology, Panjin, China

Corresponding author: Dian-Ming Hu ( hudianming1@163.com )

Corresponding author: Zhi-Jun Zhai ( zhjzh002@163.com )

Academic editor: Huzefa Raja
© 2024 Wen-Jing Zhang, Gui-Ping Xu, Yu Liu, Yang Gao, Hai-Yan Song, Hai-Jing Hu, Jian-Ping Zhou, Ming-Hui Chen, Deng-Mei Fan, Dian-Ming Hu, Zhi-Jun Zhai.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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
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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 collected from Jiangxi and Guangxi Provinces, China. Based on the morphological observations and phylogenetic analysis of a combined DNA data containing ITS, LSU, SSU and tef1-α sequences, two new Trichobotrys species, T. meilingensis and T. yunjushanensis, as well as a new record of T. effusa, were introduced. Additionally, a comprehensive description 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-inhibitory 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 conidiophores 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 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, Trichobotrys 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 investigated, 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 Trichobotrys 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. effusa, are described according to morphological examination and multi-loci phylogenetic 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 compound 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 (Chomnunti et al. 2014). Germinating conidia were transferred to fresh potato dextrose agar (PDA, from Beijing Bridge Technology Co., Ltd., Beijing, China) supplemented with two types of antibiotics (100 μg/mL penicillin, 50 μg/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 scalpel, were ground to a fine powder with liquid nitrogen to break the cells for DNA extraction. Subsequently, total genomic DNA was extracted following the modified CTAB method (Doyle and Doyle (1987). Four primer pairs, ITS1/ITS4 (White et al. 1990), LR0R/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-α gene regions, respectively. Polymerase chain reaction (PCR) was performed in a final volume of 25 μl, containing 9.5 μl double distilled water (ddH2O), 12.5 μl 2 × Taq PCR MasterMix (Qingke, Changsha, China), 1 μl each primer (10 μM) and 1 μl genomic DNA extract. Amplification conditions for ITS, LSU, SSU and tef1-α 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, lnc, Madison, WI) and were deposited in the NCBI GenBank database (Table 1).

Table 1.
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Sequences used in this study.

Species Isolate GenBank accession number
ITS LSU SSU tef1-α
Aquadictyospora clematidis MFLU 172080 MT310592 MT214545 MT226664 MT394727
Aquadictyospora lignicola MFLUCC 17-1318 MF948621 MF948629 – MF953164
Dendryphiella paravinosa CPC 26176 KX228257 KX228309 – –
Dendryphiella vinosa MFLU 200444 MT907477 MT907480 – –
Dictyocheirospora aquatica KUMCC 15-0305 KY320508 KY320513 – –
Dictyocheirospora bannica KH 332 LC014543 AB807513 AB787223 AB808489
Dictyocheirospora bannica MFLU 18-1040 MH381765 MH381774 MH381759 –
Dictyocheirospora garethjonesii MFLUCC 16-0909 KY320509 KY320514 – –
Dictyocheirospora garethjonesii DLUCC 0848 MF948623 MF948631 – MF953166
Dictyocheirospora pseudomusae yone 234 LC014550 AB807520 AB797230 AB808496
Dictyocheirospora pseudomusae KH 412 LC014549 AB807516 AB797226 AB808492
Dictyocheirospora heptaspora DLUCC 1992 MT756244 MT756243 – MT776563
Dictyocheirospora rotunda MFLUCC 14-0293 KU179099 KU179100 KU179101 –
Dictyocheirospora rotunda MFLUCC 17-0222 MH381764 MH381773 MH381758 MH388818
Dictyosporium alatum ATCC 34953 NR–077171 DQ018101 DQ018080 –
Dictyosporium bulbosum yone 221 LC014544 AB807511 AB797221 AB808487
Dictyosporium digitatum KT 2660 LC014546 AB807518 AB797228 –
Dictyosporium digitatum KH 401 LC014545 AB807515 AB797225 AB808491
Dictyosporium digitatum yone 280 LC014547 AB807512 AB797222 AB808488
Dictyosporium elegans NBRC 32502 DQ018087 DQ018100 DQ018079 –
Dictyosporium hughesii KT 1847 LC014548 AB807517 AB797227 AB808493
Dictyosporium meiosporum MFLUCC 10-0131 KP710944 KP710945 KP710946 –
Dictyosporium nigroapice MFLUCC 17-2053 MH381768 MH381777 MH381762 MH388821
Dictyosporium olivaceosporum KH 375 LC014542 AB807514 AB797224 AB808490
Dictyosporium pandanicola MFLUCC 18-0331 MZ490792 MZ490776 – MZ501208
Dictyosporium stellatum CCFC 241241 NR–154608 JF951177 – –
Dictyosporium strelitziae CBS 123359 NR–156216 FJ839653 – –
Dictyosporium tetrasporum KT 2865 LC014551 AB807519 AB797229 AB808495
Dictyosporium thailandicum MFLUCC 13-0773 KP716706 KP716707 – –
Dictyosporium tratense MFLUCC 17-2052 MH381767 MH381776 MH381761 MF388820
Digitodesmium bambusicola CBS 110279 DQ018091 DQ018103 – –
Gregarithecium curvisporum KT 922 AB809644 AB80754 AB797257 AB808523
Jalapriya pulchra MFLU 17-1683 MF948628 MF948636 – MF953171
Jalapriya toruloides CBS 209.65 DQ018093 DQ018104 DQ018081 –
Periconia igniaria CBS 379.86 LC014585 AB807566 AB797276 AB808542
Periconia igniaria CBS 845.96 LC014586 AB807567 AB797277 AB808543
Pseudocoleophoma calamagrostidis KT 3284 LC014592 LC014609 LC014604 LC014614
Pseudocoleophoma flavescens CBS 178.93 – GU238075 GU238216 –
Pseudocoleophoma polygonicola KT 731 AB809634 AB807546 AB797256 AB808522
Pseudocoleophoma zingiberacearum NCYUCC 190054 MN615941 MN616755 – MN629283
Pseudodictyosporium elegans CBS 688.93 MH862454 MH874101 DQ018084 –
Pseudodictyosporium thailandica MFLUCC 16-0029 KX259520 KX259522 KX259524 KX259526
Pseudodictyosporium wauense CBS 126094 MH864014 MH875472 – –
Trichobotrys effusa FS524 MN545626 – – –
Trichobotrys effusaa YMJ1179 KJ630313 – – –
Trichobotrys effusa* JAUCC 6359 PP406377 PP407503 PP407508 PP405621
Trichobotrys effusa* JAUCC 6826 PP830649 PP830650 PP830652 PP845300
Trichobotrys meilingensis * JAUCC 4985 PP406380 PP407504 PP407509 PP405623
Trichobotrys meilingensis* JAUCC 4986 PP406381 PP407505 PP407510 PP405625
Trichobotrys yunjushanensis * JAUCC 4987 PP406378 PP407506 PP407511 PP405622
Trichobotrys yunjushanensis* JAUCC 4988 PP406379 PP407507 PP407512 PP405624

Ex-type strains or type materials are marked in bold. Newly generated sequences are indicated with “*”. “–”, the sequence is unavailable.

Data analyses

Based on ITS, LSU, SSU and tef1-α 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 phylogenetic analyses (Table 1) and were downloaded from GenBank. Four gene regions (ITS, LSU, SSU and tef1-α) were individually aligned using the online service 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 substitution 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-α datasets used for phylogenetic analysis are TIM2+I+G model (-lnL = 5321.6598), TIM2+I+G model (-lnL = 3199.3778), TIM2+I+G model (-lnL = 3481.7971) and GTR+I+G model (-lnL = 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-α (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) 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 congruent, 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. yunjushanensis is sister to the T. effusa clade, but only with low ML bootstrap support values (BS = 43) and Bayesian posterior probabilities (PP = 0.67). However, 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).

Figure 1. 

Phylogenetic tree of Dictyosporiaceae inferred from the combined regions (ITS-LSU-SSU-tef1-α) using Maximum 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.

Taxonomy

Trichobotrys meilingensis G. P. Xu & Z. J. Zhai, sp. nov.

MycoBank No: 852617
Fig. 2

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 effuse, white to yellow, hairy. Mycelium partly superficial, partly immersed, gregarious and creeping, composed of septate, branched, pale brown hyphae. Conidiophores 2.5–4.5 μm wide (x̄ = 3.5 μm, n = 20), up to 510 μm long, mononematous, variously curved, dichotomously branched in the conidiophore, septate, thick-walled, verruculose, echinulate, brown to dark brown. Conidiophore branches 15–39 × 3–4 μm (x̄ = 24.5 × 3.4 μm, n = 15), fertile, 0‒1(‒2)-septate, verruculose, pale to dark brown. Conidiogenous cells 7–12 × 3–5 μm (x̄= 9.0 × 4.0 μm, n = 10), polyblastic, integrated, erect or curved, widely distributed in the fertile branches, denticulate, hyaline to brown. Conidia 7‒13 μm diam (x̄ = 9.8 μm, n = 30), catenate, usually in branched, acropetal chains, aseptate, globose, verruculose, echinulate, sometimes guttulate, yellow brown to dark brown.

Figure 2. 

Trichobotrys meilingensis (HFJAU10042, holotype) a, b colonies on bamboo culms c–e conidiophores with conidiogenous 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 µm (a, b); 20 µm (c); 5 µm (d‒o); 25 mm (p, q).

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 natural 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 Trichobotrys in having monomatous conidiophores, spherical and echinulate conidia, and polyblastic conidiogenous cells. Trichobotrys meilingensis is easily distinguished from T. effusa, T. ipomoeae and T. trechispora by its dichotomously branched conidiophores and its conidial size (7‒13 μm vs. 3‒4 μm, 13‒15.5 μm and 3‒5 μm, respectively) (Petch 1917, 1924; Sawada 1959). Trichobotrys meilingensis is morphologically most similar to T. ramosa and shares some characteristics, such as dichotomously branched conidiophores and catenate conidia. However, T. meilingensis has larger conidia (7–13 μm vs. 3‒5 μm) and thinner conidiophores (2.5‒4.5 μm vs. 8‒18 μm) (D’Souza and Bhat 2001). Therefore, T. meilingensis can be distinguished from T. ramosa based on morphological characters in spite of the unavailable molecular data of the latter species. Thus, it should be identified as an independent taxon in Trichobotrys. A comparison of morphological features of Trichobotrys species is provided in Table 2.

Table 2.
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Synopsis of morphological characteristics, habitats, hosts and district compared across Trichobotrys species.

Species Conidiophores (μm) Conidia (μm) Conidiophores characteristics Conidia characteristics Habitat Host District References
Trichobotrys effusa Up to 200 × 3–4 or up to 1000 × 4–6 3–4 or 5–7 Equal, septate, with short lateral branches, thick walled, minutely verrucose Globose, red-brown or brown, minutely verrucose Freshwater On fallen leaves of dead bamboo or decorticated wood Sri Lanka and South Africa Berkeley and Broome (1873); Petch (1924); Morgan-Jones et al. (1987)
T. effusa Up to 650 × 2–4 3.5–5 Mononematous, erect, with short lateral branches, verruculose, septate, thick-walled, light brown to nut brown Spherical, verruculose, echinulate, transparent to dark brown or red brown Freshwater On Dead wood China, Guangxi This study
T. ipomoeae 195–440 × 13–16 13–15.5 Simple, cylindrical, 2–3 septate, dark brown Spherical, verrucose, brown Terrestrial On the leaves of ipomoea pescaprae China, Taiwan Sawada (1959)
T. meilingensis Up to 510 × 2.5–4.5 7–13 Mononematous, dichotomously branched in the conidiophore, septate, echinulate, brown to dark brown Aseptate, globose, verruculose, echinulate, yellow brown to dark brown Freshwater On submerged bamboo culms China, Jiangxi This study
T. ramosa 330‒600 × 8–18 3–5 Mononematous, erect, straight or flexous, septate, dichotomously branched in the above half, dark to reddish brown, verruculose Dry, catenate, usually in branched, acropetal chains, spherical, dark brown, verruculose, aseptate Terrestrial On dead leaves of Dendrocalamus strictus India, Goa D’souza et al. (2001)
T. trechispora Up to 1500 × 8–12 5 × 3 (oval) or 4 (spherical) Erect, olivaceous, septate, everwhere minutely spinulose Oval or spherical, ornamented with sharp, raised, broken ridges Terrestrial On dead wood Sri Lanka, Peradeniya Petch (1917)
T. yunjushanensis Up to 1150 × 3–4 7–12 Mononematous, dichotomously branched in the conidiophore, septate, echinulate, pale brown to olivaceous Aseptate, spherical, verrucose, echinulate, yellowish brown to dark brown when mature Freshwater On submerged bamboo culms China, Jiangxi This study

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: Undetermined. Asexual morph: Hyphomycetous. Colonies effuse, white, yellow to olivaceous, velvety. Mycelium mostly superficial, creeping and twining, composed of septate, brown to olivaceous, branched hyphae. Conidiophores 3‒4 μm wide (x̄ = 3.4 μm, n = 20), up to 1150 μm long, mononematous, erect, straight or flexous, septate, with fertile dichotomously branched, pale brown to olivaceous, verruculose, echinulate, thick-walled. Conidiophores branches 18–48 × 3–4 μm (x̄ = 29.1 × 3.6 μm, n = 15), sometimes long, fertile, 0‒1(‒2)-septate, verruculose, rough, pale brown. Conidiogenous cells 6–11 ×3–5 μm (x̄ = 8.5 × 4.0 μm, n = 10), integrated, polyblastic, terminal to subterminal on fertile branches, with several denticulate conidiogenous loci, hyaline to dark brown. Conidia 7‒12 μm diam (x̄ = 9.3 μm, n = 30), catenate, usually acrogenous or lateral, aseptate, spherical, verrucose, echinulate, sometimes guttulate, yellowish brown to dark brown when mature.

Figure 3. 

Trichobotrys yunjushanensis (HFJAU 10044, holotype) a, b colonies on bamboo culm c, d conidiophores with conidiogenous cells e, f conidiogenous cell with conidia g portion of conidiophore with fertile lateral branches h–k conidia l germinating conidium m, n culture on PDA from above (m) and reverse (n). Scale bars: 100 µm (a, b); 20 µm (c); 5 µm (d‒l); 25 mm (m, n).

Cultural characteristics

Conidia germinating on PDA within 24 h. Colonies incubated 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 μm 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). Morphologically, T. yunjushanensis is distinct from the holotype of T. effusa by its conidial size (7‒12 μm vs. 3‒4 μm) and longer conidiophores (up to 1150 μm vs. up to 200 μm) (Petch 1924). Trichobotrys yunjushanensis is mostly similar to T. meilingensis and T. ramosa in having dichotomously branched and rough conidiophores. However, T. yunjushanensis can be easily distinguished from T. ramosa by its larger conidia (7‒12 μm vs. 3‒5 μm) (D’Souza and Bhat 2001). Furthermore, T. yunjushanensis differs from T. meilingensis in having longer conidiophores (up to 1150 μm vs. up to 510 μm) 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. Sexual 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 μm wide (x̄ = 2.7 μm, n = 20), up to 650 μm 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 × 2–4 μm (x̄ = 14.0 × 3.2 μm, n = 16), fertile, 0–1(–2)-septate, verruculose, light brown to dark brown, individual cells typically have a slight swelling. Conidiogenous cells 3–10.5 × 2.5–6.5 μm (x̄ = 6.6 × 4.0 μm, n = 10), monoblastic or polyblastic, integrated and terminal on lateral branches, apical or lateral; columniform or cannulate, erect or slightly curved, with several seriated conidiogenous locations, light brown to dark brown. Conidia 3.5–5 μm diam (x̄ = 4.4 μm, n = 30), catenulate, simple or branched apical chains, aseptate, spherical, verruculose, echinulate, sometimes guttulate, transparent to dark brown or red brown.

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 µm (a); 20 µm (b, c); 5 µm (d–i); 5 mm (j, k).

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 μm vs. 3–4 μm), longer conidiophores (up to 650 μm vs. up to 200 μm), and slightly different 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 microscope. 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 μm long and 5–7μm in diameter, respectively (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 descriptions 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 produces considerably more sequence variability, and thus can provide high resolution for species delimitation (Nilsson et al. 2008; Szczepańska 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 samples 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 GenBank database. This study is the first report of collection of T. effusa from the freshwater habitat and provides both molecular phylogenetical and morphological 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 tef1-α) phylogenetic 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. yunjushanensis 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 possessing brown, cheirosporous conidia, produced from holoblastic conidiogenous 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. yunjushanensis, 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, future pharmacological evaluation of the two new species might be worth studying to confirm if they are similar to T. effusa in having similar bioactive constituents and function in secondary metabolites.

Acknowledgements

We are grateful to Wan Hu and Yi Yang (School of Agricultural Sciences, Jiangxi Agricultural University) for the valuable advice in the context of this study.

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.

Author ORCIDs

Wen-Jing Zhang https://orcid.org/0009-0001-2962-498X

Deng-Mei Fan https://orcid.org/0000-0001-9825-8605

Dian-Ming Hu https://orcid.org/0000-0002-4750-2871

Zhi-Jun Zhai https://orcid.org/0009-0008-7562-9707

Data availability

All of the data that support the findings of this study are available in the main text.

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