Morphological and Phylogenetic Analyses Reveal Three New Species of Distoseptispora (Distoseptisporaceae, Distoseptisporales) from Yunnan, China

Abstract

Three new species of Distoseptispora, viz. D. mengsongensis, D. nabanheensis, and D. sinensis, are described and illustrated from specimens collected on dead branches of unidentified plants in Yunnan Province, China. Phylogenetic analyses of LSU, ITS, and TEF1 sequence data, using maximum-likelihood (ML) and Bayesian inference (BI), reveal the taxonomic placement of D. mengsongensis, D. nabanheensis, and D. sinensis within Distoseptispora. Both morphological observations and molecular phylogenetic analyses supported D. mengsongensis, D. nabanheensis, and D. sinensis as three new taxa. To extend our knowledge of the diversity of Distoseptispora-like taxa, a list of recognized species of Distoseptispora with major morphological features, habitat, host, and locality is also provided.

1. Introduction

The genus Distoseptispora K.D. Hyde, McKenzie & Maharachch was established by Su et al. [1] with D. fluminicola McKenzie, Hong Y. Su, Z.L. Luo & K.D. Hyde as the type species, and was mainly characterized by being macronematous, septate, unbranched, smooth, olivaceous to brown conidiophores with monoblastic, integrated, terminal, determinate, cylindrical conidiogenous cells that produce acrogenous, solitary, distoseptate conidia. Subsequently, the phylogenetic analyses motivated the inclusion of taxa with euseptate conidia (e.g., D. guttulata J. Yang & K.D. Hyde and D. suoluoensis J. Yang, Maharachch. & K.D. Hyde) [2] and polyblastic conidiogenous cells (e.g., D. palmarum S.N. Zhang, K.D. Hyde & J.K. Liu) [3]. To date, 61 epithets for Distoseptispora are listed in Index Fungorum [4]. Monkai et al. [5] provided a synopsis of relevant morphological features that distinguish 29 Distoseptispora species. Zhai et al. [6] subsequently published an expanded synopsis that includes additional nine species following the same format as Monkai et al. [5], but ignored the species D. submersa Z.L. Luo, K.D. Hyde & H.Y. Su, which was regarded as the synonym of D. tectonae Doilom & K.D. Hyde by Dong et al. [7]. Thus, Distoseptispora currently contains 60 taxa, 38 of which were found in China [1,2,3,6,8,9,10,11,12,13,14,15,16,17,18,19,20,21].

Distoseptispora is one of the Sporidesmium-like genera with high morphological similarity to the Sporidesmium Link and Ellisembia Subram. Distoseptispora species with eu- or distoseptate conidia covering the criteria of Ellisembia and Sporidesmium. Accordingly, Distoseptispora species cannot be classified based on morphology alone, as phylogenetic analysis showed that these genera are not closely related [1,2,22]. Distoseptispora also appears similar in conidial ontogeny to Aquapteridospora Jiao Yang, K.D. Hyde & Maharachch., but the latter has terminal and intercalary conidiogenous cells with circular scars, and produces 3-euseptate conidia [23,24]. Additionally, Distoseptispora formed a sister clade to Aquapteridospora, and is well separated with high support in phylogenetic trees [24]. Distoseptispora and Aquapteridospora belonged to the order Distoseptisporales Z.L. Luo, K.D. Hyde & H.Y. Su, but are now, respectively, treated in the families Distoseptisporaceae K.D. Hyde & McKenzie and Aquapteridosporaceae K.D. Hyde & Hongsanan [24,25].

Distoseptispora species are primarily found as saprobes on submerged wood, dead branches, culms, or leaves in freshwater or terrestrial habitats except for D. caricis Crous and D. palmarum S.N. Zhang, K.D. Hyde & J.K. Liu occurring on the leaves of Carex sp. and rachis of Cocos nucifera [3,6,26]. Species of the genus decompose lignocellulose in wood [22,27,28], but their ecological functions, geographical distribution, alpha-taxonomy, and teleomorph relationships are poorly known. During our continuing survey (2007–2022) of saprophytic microfungi from the forest ecosystems of southwest China, several Sporidesmium-like taxa were isolated on dead branches of unidentified perennial dicotyledonous plants from terrestrial habitats in Yunnan Province, China. Using multi-gene loci of LSU, ITS, and TEF1 sequence data, the systematic placement of these isolates represented several Distoseptispora species. Based on morphological characteristics and multi-locus phylogenetic analysis, three new species of Distoseptispora, viz. D. mengsongensis, D. nabanheensis, and D. sinensis, are proposed and described in this paper.

2. Materials and Methods

2.1. Sample Collection, Isolation, and Morphological Examination

Samples of decomposing wood and bark were collected from the forest floor in Yunnan Province, China, and brought them back to the laboratory in Ziploc™ bags. Samples were treated following the methods described by Ma et al. [29]. Colonies on the surface of dead branches were examined and visually observed with a stereomicroscope (Motic SMZ-168, Xiamen, China) from low (7.5 times) to high (50 times) magnification. Fresh colonies were picked with sterile needles at a stereomicroscope magnification of 50 times, placed on a slide with a drop of lactic acid–phenol solution (lactic acid, phenol, glycerin, sterile water; 1:1:2:1, respectively), then placed under an Olympus BX 53 light microscope fitted with an Olympus DP 27 digital camera (Olympus Optical Co., Ltd., Tokyo, Japan) for microscopic morphological characterization. The tip of a sterile toothpick dipped in sterile water was used to pick conidia from the specimen; the conidia were then streaked on the surface of potato dextrose agar (PDA; 20% potato + 2% dextrose + 2% agar, w/v) and incubated at 25 °C overnight. Single-spore isolations were made on potato dextrose agar (PDA) following Goh [30]. Colony colors were assessed according to the charts of Rayner [31]. All fungal strains were stored in 10% sterilized glycerin at 4 °C for further studies. The studied specimens and cultures were deposited in the Herbarium of Jiangxi Agricultural University, Plant Pathology, Nanchang, China (HJAUP).

2.2. DNA Extraction, PCR Amplification, and Sequencing

Fungal hyphae (500 mg) were scraped from the surface of colonies growing on PDA plates, transferred to 2 mL safe-lock microtubes, and ground with liquid nitrogen. DNA was extracted using the Solarbio Fungal Genomic DNA Extraction Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Primer sets were used for the amplification of LSU and ITS, and TEF1: ITS5/ITS4 [32], 28S1-F/28S3-R [8], and EF1-983F/EF1-2218R [33]. The final volume of the PCR reaction was 25 μL, containing 1 μL of DNA template, 1 μL of each forward and reward primer, 12.5 μL of 2 × Power Taq PCR MasterMix, and 9.5 μL of double-distilled water (ddH₂O). The PCR thermal cycling conditions of ITS and LSU were initialized at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 50 s, elongation at 72 °C for 1 min, a final extension at 72 °C for 10 min, and finally kept at 4 °C. TEF1 was initialized at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, elongation at 72 °C for 1 min, a final extension at 72 °C for 10 min, and finally kept at 4 °C. The PCR products were checked on 1% agarose gel electrophoresis stained with ethidium bromide. Purification and DNA sequencing were carried out at Beijing Tsingke Biotechnology Co., Ltd., Beijing, China.

2.3. Sequence Alignment and Phylogenetic Analyses

Sequences, including those obtained from GenBank (

Table 1. List of Distoseptispora species and GenBank accessions used in the phylogenetic analyses. New sequences are in bold.

Taxon	Voucher/Strain Number	GenBank Accession Numbers			References
		LSU	ITS	TEF1
Aquapteridospora aquatica	MFLUCC 17-2371T	MW287767	MW286493	—	[7]
A. fusiformis	MFLUCC 18-1606T	MK849798	MK828652	MN194056	[22]
A. lignicola	MFLUCC 15-0377T	KU221018	—	—	[23]
Distoseptispora adscendens	HKUCC 10820	DQ408561	—	—	[40]
D. amniculi	MFLUCC 17-2129T	MZ868761	MZ868770	—	[11]
D. appendiculata	MFLUCC 18-0259T	MN163023	MN163009	MN174866	[22]
D. aqualignicola	KUNCC 21-10729T	ON400845	OK341186	OP413480	[21]
D. aquamyces	KUNCC 21-10731T	OK341199	OK341187	OP413482	[21]
D. aquatica	MFLUCC 16-0904	MK849794	MK828649	—	[22]
D. aquatica	S-965	MK849792	MK828647	MN194051	[22]
D. aquisubtropica	GZCC 22-0075T	ON527941	ON527933	ON533677	[20]
D. atroviridis	GZCC 20-0511T	MZ868763	MZ868772	MZ892978	[11]
D. bambusae	MFLUCC 20-0091T	MT232718	MT232713	MT232880	[15]
D. bambusae	MFLUCC 14-0583	MT232717	MT232712	—	[15]
D. bangkokensis	MFLUCC 18-0262T	MZ518206	MZ518205	—	[17]
D. cangshanensis	MFLUCC 16-0970T	MG979761	MG979754	MG988419	[9]
D. caricis	CPC 36498T	MN567632	NR_166325	—	[26]
D. chinensis	GZCC 21-0665T	MZ474867	MZ474871	MZ501609	[12]
D. clematidis	MFLUCC 17-2145T	MT214617	MT310661	—	[41]
D. crassispora	KUMCC 21-10726T	OK341196	OK310698	OP413479	[21]
D. curvularia	KUMCC 21-10725T	OK341195	OK310697	OP413478	[21]
D. cylindricospora	DLUCC 1906T	OK513523	OK491122	OK524220	[19]
D. dehongensis	KUMCC 18-0090T	MK079662	MK085061	MK087659	[3]
D. effusa	GZCC 19-0532T	MZ227224	MW133916	MZ206156	[11]
D. euseptata	MFLUCC 20–0154T	MW081544	MW081539	—	[16]
D. euseptata	DLUCC S2024	MW081545	MW081540	MW084994	[16]
D. fasciculata	KUMCC 19-0081T	MW287775	MW286501	MW396656	[7]
D. fluminicola	MFLUCC 15-0417T	KU376270	MF077553	—	[9]
D. fusiformis	GZCC 20-0512T	MZ868764	MZ868773	MZ892979	[11]
D. guizhounesis	GZCC 21-0666T	MZ474869	MZ474868	MZ501610	[12]
D. guttulata	MFLUCC 16-0183T	MF077554	MF077543	MF135651	[2]
D. hyalina	MFLUCC 17-2128T	MZ868760	MZ868769	MZ892976	[11]
D. hydei	MFLUCC 20-0481T	MT742830	MT734661	—	[5]
D. lancangjiangensis	KUN-HKAS 112712T	MW879522	MW723055	—	[17]
D. leonensis	HKUCC 10822T	DQ408566	—	—	[40]
D. lignicola	MFLUCC 18-0198T	MK849797	MK828651	—	[22]
D. longispora	HFJAU 0705T	MH555357	MH555359	—	[14]
D. martinii	CGMCC 3.18651T	KX033566	KU999975	—	[8]
D. meilingensis	JAUCC 4727T	OK562396	OK562390	OK562408	[6]
D. mengsongensis	HJAUP C2126T	OP787874	OP787876	OP961937	This study
D. multiseptata	MFLUCC 15-0609T	KX710140	KX710145	MF135659	[42]
D. multiseptata	MFLUCC 16-1044	MF077555	MF077544	MF135652	[2]
D. nabanheensis	HJAUP C2003T	OP787877	OP787873	OP961935	This study
D. neorostrata	MFLUCC 18-0376T	MN163017	MN163008	—	[22]
D. nonrostrata	KUNCC 21-10730T	OK341198	OK310699	OP413481	[21]
D. obclavata	MFLUCC 18-0329T	MN163010	MN163012	—	[22]
D. obpyriformis	DLUCC 0867	MG979765	MG979757	MG988423	[9]
D. pachyconidia	KUMCC 21-10724T	OK341194	OK310696	OP413477	[21]
D. palmarum	MFLUCC 18-1446T	MK079663	MK085062	MK087660	[3]
D. phangngaensis	MFLUCC 16-0857T	MF077556	MF077545	MF135653	[2]
D. rayongensis	MFLUCC 18-0415T	MH457137	MH457172	MH463253	[28]
D. rostrata	MFLUCC 16-0969T	MG979766	MG979758	MG988424	[9]
D. rostrata	DLUCC 0885	MG979767	MG979759	MG988425	[9]
D. saprophytica	MFLUCC 18-1238T	MW287780	MW286506	MW396651	[7]
D. septate	GZCC 22-0078T	ON527947	ON527939	ON533683	[20]
D. songkhlaensis	MFLUCC 18-1234T	MW287755	MW286482	MW396642	[7]
D. sinensis	HJAUP C2044T	OP787875	OP787878	OP961936	This study
D. suoluoensis	MFLUCC 17-0224T	MF077557	MF077546	MF135654	[2]
D. suoluoensis	MFLUCC 17-1305	MF077558	MF077547	—	[2]
D. tectonae	MFLUCC 12-0291T	KX751713	KX751711	KX751710	[42]
D. tectonae	MFLUCC 16-0946	MG979768	MG979760	MG988426	[9]
D. tectonigena	MFLUCC 12-0292T	KX751714	KX751712	—	[42]
D. thailandica	MFLUCC 16-0270T	MH260292	MH275060	MH412767	[10]
D. thysanolaenae	KUN-HKAS 112,710	MW879524	MW723057	MW729783	[17]
D. thysanolaenae	KUN-HKAS 102247T	MK064091	MK045851	MK086031	[13]
D. tropica	GZCC 22-0076T	ON527943	ON527935	ON533679	[20]
D. verrucosa	GZCC 20-0434T	MZ868762	MZ868771	MZ892977	[11]
D. wuzhishanensis	GZCC 22-0077T	ON527946	ON527938	ON533682	[20]
D. xishuangbannaensis	KUMCC 17-0290T	MH260293	MH275061	MH412768	[10]
D. yongxiuensis	JAUCC 4725T	OK562394	OK562388	OK562406	[6]
D. yunjushanensis	JAUCC 4723T	OK562398	OK562392	OK562410	[6]
D. yunnanensis	MFLUCC 20-0153T	MW081546	MW081541	MW084995	[16]

Notes: The ex-type cultures are indicated using “^T” after strain numbers; “—” stands for no sequence data in GenBank.

), were initially aligned using MAFFTv.7 [34] on the online server (http://maffTh.cbrc.jp/alignment/server/, accessed on 1 February 2023) and optimized manually when needed. The LSU, ITS, and TEF1 sequence data were concatenated by using Phylosuite software v1.2.1 [35], and absent sequence data in the alignments were treated with the question mark and “-” as missing data. The phylogenetic tree was constructed using Phylosuite software v1.2.1 [35] based on the combined data of LSU, ITS, and TEF1 sequence (Supplementary Materials). The concatenated aligned dataset was analyzed separately using maximum likelihood (ML) and Bayesian inference (BI). Maximum-likelihood phylogenies were inferred using IQ-TREE [36] under edge-linked partition model for 10,000 ultrafast [37] bootstraps. The final tree was selected among suboptimal trees from each run by comparing the likelihood scores using the TNe+I+G4 for ITS, TNe+R3 for LSU, and TN+F+I+G4 for TEF1 substitution model. Bayesian inference phylogenies were inferred using MrBayes 3.2.6 [38] under partition model (2 parallel runs, 2,000,000 generations), in which the initial 25% of sampled data were discarded by burning. The best-fit model was GTR+F+I+G4 for ITS+TEF1, and GTR+F+I+G4 for LSU. ModelFinder was used to select the best-fit partition model (edge-linked) using BIC criterion [39]. The trees were viewed in FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree, accessed on 1 February 2023), and further edited in Adobe Illustrator 2021. Sequences generated in this study were deposited in GenBank (Table 1).

3. Results

3.1. Molecular Phylogeny

The combined sequence alignment comprised 72 strains representing 64 species (Table 1), 2248 total characters (ITS:1–493, LSU:494–1329, TEF1:1330–2248), including 891 distinct patterns, 537 parsimony-informative, 275 singleton sites, and 1436 constant sites), and used Aquapteridospora aquatica (MFLUCC 17-2371), A. fusiformis (MFLUCC 18-1606) and A. lignicola (MFLUCC 15-0377) as outgroup. Maximum-likelihood (ML) and Bayesian inference (BI) analyses of the combined dataset resulted in phylogenetic reconstructions with largely similar topologies, and the best-scoring ML tree (lnL = −16,099.937) is shown in Figure 1. Maximum-likelihood bootstrap support (MLBS) values above 75% and Bayesian posterior probabilities (BPP) greater than 0.90 are given above the nodes. Our three isolates in this lineage formed distinct clades with good support value, and can be recognized as three new phylogenetic species, Distoseptispora mengsongensis, D. nabanheensis, and D. sinensis. Phylogenetic analyses suggested sister group relatedness of D. nabanheensis (HJAUP C2003) and D. clematidis (MFLUCC 17-2145) (MLBS/BPP = 100/0.97); D. mengsongensis (HJAUP C2126) and D. fasciculata (KUMCC 19-0081) (MLBS/BPP = 99/0.92); and D. sinensis (HJAUP C2044), D. tectonae (MFLUCC 12-0291, MFLUCC 16-0946), and D. tectonigena (MFLUCC 12-0292) (MLBS/BPP = 85/0.99).

3.2. Taxonomy

Distoseptispora mengsongensis Jing W. Liu, X.G. Zhang & Jian Ma, sp. nov., Figure 2.

Index Fungorum number: IF900033.

Etymology: In reference to the locality, Mengsong Township, where the fungus was collected.

Holotype: HJAUP M2126.

Description: Saprobic on dead branches in terrestrial habitats. Teleomorph: Undetermined. Anamorph (Figure 2): Hyphomycetes. Colonies on natural substratum effuse, brown, hairy. Mycelium is superficial and immersed, composed of branched, septate, pale brown to brown, smooth-walled hyphae. Conidiophores macronematous, mononematous, cylindrical, 1–5-septate, erect, unbranched, smooth, straight or slightly flexuous, brown to dark brown, 17–54 × 4.5–7 μm ($\overline{\mathrm{x}}$ = 33.5 × 5.5 μm, n = 20). Conidiogenous cells monoblastic, integrated, terminal, cylindrical, flat at the conidiogenous loco, determinate, pale brown to brown, smooth, 8.5–14.2 × 3–5.7 μm ($\overline{\mathrm{x}}$ = 11.3 × 4.1 μm, n = 20). Conidial secession schizolytic. Conidia acrogenous, solitary, obclavate, 15–31-distoseptate, sometimes constricted at the septa, especially in proximal parts, straight or slightly curved, smooth, brown to dark brown, sometimes with percurrent regeneration forming a secondary conidium from the conidial apex, 86–200 × 6–13 μm ($\overline{\mathrm{x}}$ = 141 × 9.7 μm, n = 25), base truncate and 3–5.5 μm wide, apex rounded, 2.9–8.6 μm wide.

Culture characteristics: Colony on PDA reached 81–86 mm diam. after 2 weeks in an incubator under dark conditions at 25 °C, irregularly rounded, surface velvety, with dense, brown mycelium, margin entire, dark brown to black; the reverse is black.

Material examined: China, Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, Menghai County, Mengsong Township, on dead branches of an unidentified broadleaf tree, 12 July 2021, J.W. Liu, HJAUP M2126 (holotype), ex-type culture permanently preserved in a metabolically inactive state, HJAUP C2126.

Notes: Phylogenetic analyses showed that D. mengsongensis (HJAUP C2126) clusters with D. fasciculata (KUMCC 19-0081). BLASTn analysis of D. mengsongensis (HJAUP C2126) and D. fasciculata (KUMCC 19-0081) showed 99% identity (544/551, no gaps) using ITS, 99% identity (565/567, two gaps) using LSU, and 100% identity (927/927, no gaps) using TEF1. Moreover, D. mengsongensis morphologically differs from D. fasciculata W. Dong, H. Zhang & K.D. Hyde, which occurs in freshwater habitats and has smaller conidiophores (12–16 × 5–6 μm) and wider conidia (10–16.5 μm wide) [7]. Distoseptispora mengsongensis is morphologically similar to D. xishuangbannaensis Tibpromma & K.D. Hyde, but the latter differs by occurring on dead leaf sheath and not on wood, and further differs by its shorter and narrower conidiophores (12–17 × 2–5 μm) and bigger conidia (160–305 × 8–15 μm) with up to 40 distosepta [10].

Distoseptispora nabanheensis Jing W. Liu, X.G. Zhang & Jian Ma, sp. nov., Figure 3.

Index Fungorum number: IF900054.

Etymology: In reference to the locality, Nabanhe Nature Reserve, in which the fungus was collected.

Holotype: HJAUP M2003.

Description: Saprobic on dead branches in terrestrial habitats. Teleomorph: Undetermined. Anamorph (Figure 3): Hyphomycetes. Colonies on natural substratum effuse, brown, hairy. Mycelium is superficial and immersed, composed of branched, septate, pale brown to brown, smooth-walled hyphae. Conidiophores macronematous, mononematous, cylindrical, 3–8-septate, erect, unbranched, solitary, smooth, straight or slightly flexuous, brown to dark brown, 29–42 × 8–10 μm ($\overline{\mathrm{x}}$ = 37.5 × 9.2 μm, n = 9). Conidiogenous cells monoblastic, integrated, terminal, cylindrical, flat at the conidiogenous loco, determinate, brown to pale brown, smooth, 4–6 × 4–5 μm ($\overline{\mathrm{x}}$ = 5.2 × 4.6 μm, n = 9). Conidial secession schizolytic. Conidia acrogenous, solitary, obclavate, 18–31-distoseptate, slightly constricted at the septa, smooth, brown to dark brown, 102–214.5 × (7–)11–14.5 μm ($\overline{\mathrm{x}}$ = 171 × 11.3 μm, n = 20), base truncate and 3.5–7.5 μm wide, apex rounded, 3.2–8 μm wide.

Culture characteristics: Colony on PDA reached 83–88 mm diam. after 2 weeks in an incubator under dark conditions at 25 °C, circular, surface velvety, with dense, gray mycelium on the surface along the entire margin; the reverse is dark brown to black.

Material examined: China, Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, the Nabanhe National Nature Reserve, on dead branches of an unidentified broadleaf tree, 12 July 2021, J.W. Liu, HJAUP M2003 (holotype), ex-type culture permanently preserved in a metabolically inactive state, HJAUP C2003.

Notes: Phylogenetic analyses showed that D. nabanheensis (HJAUP C2003) clusters with D. clematidis (MFLUCC 17-2145). BLASTn analysis of D. nabanheensis (HJAUP C2003) and D. clematidis (MFLUCC 17-2145) shows 99% identity (531/533, no gaps) using ITS, 99% identity (471/477, two gaps) using LSU. Moreover, D. nabanheensis morphologically differs from D. clematidis Phukhams., M.V. de Bult & K.D. Hyde, which has smaller conidiophores (22–40 × 4–10 μm) and wider conidia (12–20 μm wide) with 28–35 distosepta [41]. Distoseptispora nabanheensis is morphologically similar to D. chinensis X. Tang, Jayaward., J.C. Kang & K.D. Hyde, and D. tectonigena Doilom & K.D. Hyde, but D. chinensis lives in freshwater habitats and differs by its narrower conidiophores (5.5–9 μm wide) and bigger conidia (81–283 × 10–19 μm), with up to 40 distosepta [12]; D. tectonigena differs by its longer conidiophores (up to 110 μm long) and longer conidia (83–360 μm long), with 20–46 distosepta [42].

Distoseptispora sinensis Jing W. Liu, X.G. Zhang & Jian Ma, sp. nov., Figure 4.

Index Fungorum number: IF900055.

Etymology: In reference to the country “China” in which the fungus was collected.

Holotype: HJAUP M2044.

Description: Saprobic on dead branches in terrestrial habitats. Teleomorph: Undetermined. Anamorph: Hyphomycetes. Colonies on natural substratum effuse, brown, hairy. Mycelium is superficial and immersed, composed of branched, septate, pale brown to brown, smooth-walled hyphae. Conidiophores macronematous, mononematous, cylindrical, 2–5-septate, erect, unbranched, solitary, smooth, straight or slightly flexuous, brown to dark brown, 23.5–56.5 × 3.5–7 μm ($\overline{\mathrm{x}}$ = 39.6 × 5 μm, n = 20). Conidiogenous cells monoblastic, integrated, terminal, cylindrical, flat at the conidiogenous loco, determinate, pale brown, smooth, 6.5–10 × 3.3–3.6 μm ($\overline{\mathrm{x}}$ = 8.3 × 3.5 μm, n = 20). Conidial secession schizolytic. Conidia acrogenous, solitary, obclavate, straight or slightly curved, smooth, 10–25-distoseptate, brown to dark brown, apical cell paler, 40–107(–137) × 10–12 μm ($\overline{\mathrm{x}}$ = 78.8 × 10.5 μm, n = 30), base truncate and 3–3.5 μm wide, apex rounded, 3.5–10 μm wide.

Culture characteristics: Colony on PDA reaching 64–69 mm diam. after 2 weeks in an incubator under dark conditions at 25 °C, irregularly rounded, surface velvety, with dense, gray mycelium, black at the entire margin; the reverse is dark brown to black.

Material examined: China, Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, Jinghong City, Gasa Township, on dead branches of an unidentified broadleaf tree, 12 July 2021, J.W. Liu, HJAUP M2044 (holotype), ex-type culture permanently preserved in a metabolically inactive state, HJAUP C2044.

Notes: Phylogenetic analyses showed that D. sinensis (HJAUP C2044) clusters with D. tectonae (MFLUCC 12–0291^T, MFLUCC 16–0946) and D. tectonigena (MFLUCC 12–0292). BLASTn analysis of D. sinensis (HJAUP C2044) and D. tectonae (MFLUCC 12-0291^T) shows 98% identity (559/569, five gaps) using ITS, 98% identity (574/583, five gaps) using LSU, and 99% identity (926/930, no gaps) using TEF1; BLASTn analysis of D. sinensis (HJAUP C2044) and D. tectonigena (MFLUCC 12–0292) shows 96% identity (549/570, seven gaps) using ITS and 99% identity (575/583, five gaps) using LSU. Moreover, D. sinensis morphologically differs from D. tectonae, which has smaller conidiophores (up to 40 × 4–6 μm) and larger conidia (90–170 × 11–16 μm) with 20–28 distosepta [42], as well as from D. tectonigena, which has larger conidiophores (up to 110 × 5–11 μm) and larger conidia (83–360 × 10–13 μm), with 20–46 distosepta [42].

4. Discussion

Sporidesmium-like taxa have undergone convergent evolution, and the morphological characteristics used to delimit Sporidesmium-like genera are shown to be insignificant in a phylogenetic context. Responding to the heterogeneity of Sporidesmium, the genus Distoseptispora was introduced by Su et al. [1] based on multi-locus phylogenies together with morphology. In recent years, the number of Distoseptispora species steadily increased and currently reached 63 species, including D. mengsongensis, D. nabanheensis, and D. sinensis. In the phylogenetic tree (Figure 1), some Distoseptispora species form sister clades, but they show different morphological characteristics, such as how D. mengsongensis and D. fasciculata are clustered, but the conidia of D. mengsongensis are obclavate, constricted at the septa, especially in proximal parts, sometimes with percurrent regeneration forming a secondary conidium from the conidial apex, with an average conidial length/width ratio of 14.54, while the conidia of D. fasciculata are subcylindrical to obclavate, with an average conidial length/width ratio of 8.44. Distoseptispora nabanheensis and D. clematidis have a close phylogenetic relationship, but D. nabanheensis has obclavate, slightly constricted at the septa, brown to dark brown conidia, with an average conidial length/width ratio of 15.13, while D. clematidis has oblong, obclavate, cylindrical or rostrate, brown with green tinge conidia, with an average conidial length/width ratio of 10.29. Distoseptispora sinensis, D. tectonigena, and D. tectonae have different morphology of conidiophores and conidia, and comparisons of nucleotides between D. tectonae (MFLUCC 12-0291^T) and our isolate (HJAUP C2044) showed 10 and 9 (2%, including five gaps) nucleotide differences in ITS and LSU regions, respectively; D. tectonigena (MFLUCC 12–0292^T) and our isolate (HJAUP C2044) showed 21 and 8 (4%, including seven gaps; 1.2%, including five gaps) nucleotide differences in ITS and LSU regions, respectively. Considering this scenario, additional molecular data and morphological characteristics are required for verification and expansion.

To date, all Distoseptispora species were identified by morphological and phylogenetic analyses, which led to a better evaluation of their phylogenetic relationships and taxonomic placements. However, studies conducted on Distoseptispora have no universally accepted standards with respect to barcode selection for phylogenetic analyses. For instance, Su et al. [1] established the genus Distoseptispora, but the initial species had only LSU sequences. Tibpromma et al. [10] introduced D. thailandica Tibpromma & K.D. Hyde and D. xishuangbannaensis using LSU and ITS. Monkai et al. [5] introduced D. hydei Monkai & Phookamsak using LSU, ITS, and RPB2. Hyde et al. [12] introduced D. chinensis X. Tang, Jayaward., J.C. Kang & K.D. Hyde and D. guizhouensis X. Tang, Jayaward., J.C. Kang & K.D. Hyde using ITS, LSU, SSU, TEF1, and RPB2. Zhai et al. [6] introduced D. meilingensis Z.J. Zhai & D.M. Hu, D. yongxiuensis Z.J. Zhai & D.M. Hu, and D. yunjushanensis Z.J. Zhai & D.M. Hu using ITS, LSU, SSU, and TEF1. Ma et al. [20] and Zhang et al. [21] introduced 10 Distoseptispora species using LSU, ITS, TEF1, and RPB2. The recent studies also indicated that the use of only LSU and ITS sequences might be problematic in resolving the phylogeny of Distoseptisporaceae, as RPB2 and/or TEF1 usually increased phylogenetic resolution significantly. In our study, we conducted phylogenetic analyses based on combined LSU, ITS, and TEF1 sequences, and obtained good phylogenetic support. Our three species, viz. D. mengsongensis, D. nabanheensis, and D. sinensis, are considerably distinct from all other described Distoseptispora species by morphological characteristics and multi-locus phylogenetic analysis, and thus we are convinced that the newly introduced species are new to science.

Studies conducted to date on Distoseptispora are mainly focused on their alpha-taxonomy, and most species of this genus are known from dead parts of plants as saprobic fungi in aquatic and terrestrial habitats [5,11,12,17,19,20,21,43], except D. caricis and D. palmarum which are reported, respectively, on leaves of Carex sp. and rachis of Cocos nucifera on [3,6,26], whereas we have little attention on their roles in ecosystem function. They total 63 valid species (

Table 2. Synopsis of morphological characteristics, habitat, host, and locality compared across Distoseptispora anamorph species.

Species	Conidiophores (μm)	Conida		Habitat	Host/Locality	References
		Size (µm)	Morphology
Distoseptispora adscendens	20–50 × 7–10	110–375 × 14–20	Cylindrical to obclavate, rostrate, brown to dark brown, paler towards the apex, 16–62-distoseptate	Terrestrial	Rotten wood and dead branches of many woody plant species, China	[21,45]
D. amniculi	90–180 × 3–4.5	85–167 × 9–11.8	Obclavate, rostrate, olivaceous-brown, grayish-brown or mid-brown, sometimes with a secondary conidium, (7–)12–24-distoseptate	Freshwater	Unidentified submerged wood, Thailand	[11]
D. appendiculata	62–86 × 4.5–5.5	67–89 × 10–16	Obpyriform or obclavate, olivaceous or dark brown, with a gelatinous sheath around tip, 13–17-distoseptate	Freshwater	Unidentified submerged wood, Thailand	[22]
D. aqualignicola	90–190(–240) × 5–8	41–94(–104) × 10.5–12.5	Obclavate, rostrate, brown at the base, subhyaline to pale-brown at the apex, 4–8-euseptate	Freshwater	Unidentified submerged wood, China	[21]
D. aquamyces	(78–)91–198 × 4–7	30–95 × 7–12	Obclavate to obpyriform, mostly rostrate, pale-brown to brown at the base, subhyaline to pale-brown at the apex, 4–10-euseptate, verrucose	Freshwater	Unidentified submerged wood, China	[21]
D. aquatica	29–41 × 7–9	110–157 × 13.5–16.5	Obclavate, dark brown with bluish-green to malachite green tinge, paler towards the apex, 15–28-distoseptate	Freshwater	Unidentified submerged wood, China	[1]
D. aquisubtropica	16–83 × 5–11	43–278 × 11–19	Obclavate or lanceolate, rostrate, pale brown or dark brown, olivaceous,16–31-distoseptate, verrucose	Freshwater	Unidentified submerged wood, China	[20]
D. atroviridis	100–167 × 2.7–4	31–43 × 13–20	Ellipsoidal to obovoid, dark green, subhyaline at the basal cell, 6-distoseptate	Freshwater	Unidentified submerged wood, China	[11]
D. bambusae	40–96 × 4–5.5	45–74 × 5.5–9.5	Obclavate, olivaceous or brown, 5–10-distoseptate	Terrestrial	Dead bamboo culms, China	[15]
D.bambusicola	64–116 × 4–7	72–193 × 7.5–14.5	Obclavate or lanceolate, rostrate, pale brown, up to 16-distoseptate	Freshwater	Unidentified submerged decaying bamboo culms, China	[18]
D. bangkokensis	37–55 × 3–4	400–568 × 13–16	Obclavate, rostrate, dark olivaceous to dark brown, conidia percurrent proliferation, which forms another conidium at the apex, multi-distoseptate	Freshwater	Unidentified submerged wood, Thailand	[17]
D. cangshanensis	44–68 × 4–8	58–166(–287) × 10–14	Obclavate or lanceolate, rostrate, olivaceous or brown, multi-distoseptate	Freshwater	Unidentified submerged wood, China	[9]
D. caricis	35–90 × 6–7	(55–)65–85(–100) × 15–16(–17)	Obclavate, brown, septa with central pore, basal cell pale brown, 5–10-distoseptate	Terrestrial	Leaves of Carex sp., Thailand	[26]
D. chinensis	16.5–44 × 5.5–9	81–283 × 10–19	Obclavate or lanceolate, rostrate, olivaceous to dark brown, up to 40-distoseptate	Freshwater	Unidentified submerged wood, China	[12]
D. clematidis	22–40 × 4–10	120–210 × 12–20	Oblong, obclavate, cylindrical or rostrate, brown with green tinge, lighter towards the apex, 28–35-distoseptate	Terrestrial	Dried stem of Clematis sikkimensis, Thailand	[41]
D. crassispora	14–27 × 6–10	95–197(–214) × 13–24	Obclavate, rostrate, brown with a green tinge, 15–36(–41)-distoseptate	Freshwater	Unidentified submerged wood, China	[21]
D. curvularia	11–28 × 5–9	(60–)100–200(–314) × 12–19	Obclavate, rostrate, brown with a green tinge, (9–)16–48(–59)-distoseptate	Freshwater	Unidentified submerged wood, China	[21]
D. cylindricospora	105–157 × 6.5–8.5	136.5–278 × 8.5–11	Cylindrical to elongated, greenish-brown to dark brown, hyaline at the apex, 20–65-distoseptate	Freshwater	Unidentified submerged wood, China	[19]
D. dehongensis	45–80 × 4–5	17–30 × 7.5–10	Obpyriform to obclavate, broad cylindrical or irregular, olivaceous, 3–5-distoseptate	Freshwater	Unidentified submerged wood, China	[3]
D. effusa	72–171 × 5–6.5	35.5–113 × 7–12.5	Obclavate, rostrate, olivaceous-brown to dark brown, sometimes slightly paler at the apex, 4–9-distoseptate	Freshwater	Unidentified submerged wood, China	[11]
D. euseptata	19–28 × 4–5	37–54 × 8–9	Obpyriform to obclavate, olivaceous, becoming paler towards the apex, 4–7-euseptate	Freshwater	Unidentified submerged wood, China	[16]
D. fasciculata	12–16 × 5–6	46–200 × 10–16.5	Subcylindrical to obclavate, olivaceous when young, dark brown when mature, 10–40-distoseptate	Freshwater	Unidentified submerged wood, Thailand	[7]
D. fluminicola	21–33 × 5.5–6.5	125–250 × 13–15	Oblong, obclavate, cylindrical or rostrate, brown with green tinge, paler towards the apex, 17–34-distoseptate	Freshwater	Unidentified submerged wood, China	[1]
D. fusiformis	40–110 × 3.5–5.8	35–52 × 13.5–22	Ellipsoidal to fusiform, dark olivaceous-brown to dark brown, pale brown at both ends, 6–8-distoseptate	Freshwater	Unidentified submerged wood, China	[11]
D. guizhouensis	21–50 × 4–9	90–273 × 15–21	Obclavate, brown to dark brown, usually paler towards apex, 10–38-distoseptate	Terrestrial	Unidentified decaying wood, China	[12]
D. guttulata	55–90(–145) × 3.5–5.5	75–130(–165) × 7–11	Obclavate or lanceolate, rostrate, mid- to dark brown or olivaceous, 11–14(–20)-euseptate	Freshwater	Unidentified submerged wood, Thailand	[2]
D. hydei	87–145 × 3–7	32–58 × 10–15	Obpyriform to fusiform, olivaceous to brown, with a hyaline, globose, gelatinous sheath around tip, 7–9-distoseptate	Terrestrial	Decaying bamboo culms, Thailand	[5]
D. lancangjiangensis	144–204 × 5–6	64–84 × 9–10	Narrowly obclavate or obspathulate, brown to dark brown, becoming paler or hyaline towards apex, 3–10-euseptate	Freshwater	Unidentified submerged wood, China	[17]
D. leonensis	110–130 × 6–8	50–75 × 15–18	Obclavate, fusiform or ellipsoidal, rostrate, medium brown, becoming pale brown towards the apex, 8–10-distoseptate	Terrestrial	Dead culms of grasses or dead branches, China	[21,45]
D. lignicola	84–124 × 4–5	60–108 × 7–9	Obclavate, solitary or catenate, brown, 5–9-euseptate	Freshwater	Unidentified submerged wood, Thailand	[22]
D. longispora	17–37 × 6–10	189–297 × 16–23	Obclavate, elongated, brown to yellowish-brown, 31–56-distoseptate	Freshwater	Unidentified submerged wood, China	[14]
D. martinii	50–110 × 3.5–4.5	15–20 × 11–16	Transversal ellipsoid, oblate or subglobose, muriform, pale brown to brown	Terrestrial	Unidentified dead branches, China	[8]
D. meilingensis	69–192 × 4–7	32–64.5 × (7–)9–12.5	Obclavate, mostly bright brown when mature, 5–7-distoseptate	Freshwater	Decaying bamboo culms, China	[6]
D. mengsongensis	17–54 × 4.5–7	86–200 × 6–13	Obclavate, brown to dark brown, sometimes with percurrent proliferation and forming another conidium from the conidial apex, 15–31-distoseptate	Terrestrial	Unidentified dead branches, China	This study
D. multiseptata	23–65 × 4.5–8.5	95–290 × 11–20	Obclavate, rostrate, dark olivaceous-green, multi-distoseptate	Freshwater	Unidentified submerged wood, Thailand	[42]
D. nabanheensis	29–42 × 8–10	102–214.5 × (7–)11–14.5	Obclavate, brown to dark brown, 18–31-distoseptate	Terrestrial	Unidentified dead branches, China	This study
D. neorostrata	93–117 × 5.5–6.5	109–147 × 13–15	Obclavate, rostrate, dark olivaceous to mid- or dark brown, pale brown towards apex, multi-distoseptate	Freshwater	Unidentified submerged wood, Thailand	[22]
D. nonrostrata	105–160 × 4.5–7	22–51 × 8–14	Oblong, obclavate or narrowly obpyriform, mostly non-rostrate, rarely rostrate, pale-olivaceous or pale-brown, 4–10-distoseptate	Freshwater	Unidentified submerged wood, China	[21]
D. obclavata	117.5–162.5 × 5–7	46–66 × 9–11	Obclavate, olivaceous to pale or dark brown, 9-11-distoseptate	Freshwater	Unidentified submerged wood, Thailand	[22]
D. obpyriformis	97–119 × 5–7	53–71 × 12–16	Obpyriform, olivaceous to pale or dark brown, 9–11-distoseptate	Freshwater	Unidentified submerged wood, China	[9]
D. pachyconidia	11–27 × 4–9	42–136 × 14–22	Obclavate, lanceolate, rostrate or not, pale-brown with a green tinge, 8–21-distoseptate	Freshwater	Unidentified submerged wood, China	[21]
D. palmarum	90–165 × 4–7	35–180 × 7–11	Oblong, obclavate, cylindrical or rostrate, greenish-black to brown, paler towards the apex, 7–27-distoseptate	Terrestrial	Rachis of Cocos nucifera, Thailand	[3]
D. phangngaensis	18–30(–40) × 4.3–6.5	165–350 × 14–19	Obclavate, rostrate, dark olivaceous to mid- or dark brown, multi-distoseptate	Freshwater	Unidentified submerged wood, Thailand	[2]
D. rayongensis	75–125 × 3.5–5.5	(36–)60–106(–120) × 9–14.5	Obclavate or obspathulate, rostrate, pale brown or pale olivaceous, becoming paler or hyaline towards the apex, sometimes with percurrent proliferation and forming another conidium from the conidial apex, mostly 9–13-euseptate, rarely 14–15-septate	Freshwater	Unidentified submerged wood, Thailand	[28]
D. rostrata	82–126 × 5–7	115–155 × 9–11	Obclavate or lanceolate, rostrate, olivaceous to pale brown, (15–)18–23-distoseptate	Freshwater	Unidentified submerged wood, China	[9]
D. saprophytica	50–140 × 3.2–4.2	14.5–30 × 4.5–7.5	Subcylindrical to obclavate, olivaceous to brown, solitary or occasionally catenate, 2–6-distoseptate	Freshwater	Unidentified submerged wood, Thailand	[7]
D. septata	23–86 × 3–7	22–179 × 10–16	Obclavate, rostrate, pale brown or dark brown, olivaceous-green, usually paler towards apex, verrucose, up to 25-distoseptate	Freshwater	Unidentified submerged wood, China	[20]
D. sinensis	23.5–56.5 × 3.5–7	40–107(–137) × 10–12	Obclavate, brown to dark brown, apical cell paler, 10–25-distoseptate	Terrestrial	Unidentified dead branches, China	This study
D. songkhlaensis	70–90 × 4–5.5	44–125 × 9–14.5	Obclavate, olivaceous to brown, 9–16-distoseptate	Freshwater	Unidentified submerged wood, Thailand	[7]
D. suoluoensis	80–250 × 4.5–5.8	(65–)80–125(–145) × 8–13	Narrowly obclavate or obspathulate, yellowish-brown or dark olivaceous, becoming paler or hyaline towards the apex, verrucose, sometimes with percurrent proliferation, which forms another conidium from the conidial apex, 8–10-euseptate	Freshwater	Unidentified submerged wood, China	[2]
D. tectonae	Up to 40 × 4–6	(90–)130–140(–170) × (11–)13–14(–16)	Cylindric-obclavate, dark reddish-brown, slightly paler towards the apex, verruculose, 20–28-distoseptate	Terrestrial	Dead twig of Tectona grandis, Thailand	[42]
D. tectonigena	Up to 110 × 5–11	(83–)148–225(–360) × (10–)11–12(–13)	Cylindric-obclavate, dark reddish-brown and slightly paler towards the apex, verruculose, 20–46-distoseptate	Terrestrial	Dead twig of Tectona grandis, Thailand	[42]
D. thailandica	15–26 × 3–6	130–230 × 13.5–17	Oblong, obclavate, cylindrical or rostrate, reddish-brown to brown, pale brown towards the apex, 35–52-distoseptate	Terrestrial	Dead leaves of Pandanus sp., Thailand	[10]
D. thysanolaenae	30–80 × 3.5–5.5	21.5–80 × 6.5–12.8	Narrow and elongated obclavate, light to dark brown, paler at the apex, 8–14-distoseptate	Terrestrial	Dead culms of Thysanolaena maxima, China	[13]
D. tropica	60–151 × 3.5–7	39–75 × 7.5–10.5	Obclavate, rostrate, olivaceous-brown or dark brown, verrucose, 5–7 distoseptate	Terrestrial	Unidentified dead wood, China	[20]
D. verrucosa	92–250 × 4.7–6.3	41–63 × 8.8–12.6	Obclavate, rostrate, olivaceous-brown, becoming paler at the apex, verrucose, 6–8-euseptate	Freshwater	Unidentified submerged wood, China	[11]
D. wuzhishanensis	16–56 × 5–7	76–143 × 11–17	Obclavate, rostrate, pale brown or dark brown, olivaceous-green and yellow, usually paler towards apex, verrucose, up to 22-distoseptate	Freshwater	Unidentified submerged wood, China	[20]
D. xishuangbannaensis	12–17 × 2–5	160–305 × 8–15	Cylindrical-obclavate, green brown to brown, up to 40-distoseptate	Terrestrial	Decaying leaves of Pandanus utilis, China	[10]
D. yongxiuensis	112–253 × 4–9	46–74(–86) × 10–13(–16)	Obclavate or obspathulate, olivaceous to yellowish-brown or brown, becoming paler or hyaline towards the apex, 6–9-euseptate	Freshwater	Decaying bamboo culms, China	[6]
D. yunjushanensis	100–175 × 5.5–10	39–67.5(–77) × (7–)9.5–13.5(–16.5)	Obpyriform or obclavate, olivaceous when young, dark brown when mature, sometimes with the percurrent proliferation which forms another conidium from the conidial apex, 7–13-distoseptate	Freshwater	Decaying bamboo culms, China	[6]
D. yunnanensis	131–175 × 6–7	58–108 × 8–10	Obclavate, rostrate, mid-olivaceous to brown, becoming paler towards the apex, 6–10-euseptate	Freshwater	Unidentified submerged wood, China	[16]

Notes: All conidia are smooth except where indicated.

and

Table 3. Synopsis of morphological characteristics, habitat, host, and locality compared across Distoseptispora teleomorph species.

Species	Asci (μm)	Ascospores			Habitat	Host/Locality	References
		Size (μm)	Septation	Characteristics
Distoseptispora hyalina	145–190 × 8–11	(19.5–)23–26(–28.5) × 4.5–7	0–3	Fusiform, hyaline, guttulate, and with a mucilaginous sheath	Freshwater	Unidentified submerged wood, Thailand	[11]
D. licualae	120–210 × 8–15	20–30 × 5–10	0	Inequilateral to fusiform, hyaline, guttulate, and with a thick mucilaginous sheath	Terrestrial	Dead leaves of Licuala glabra, Thailand	[43]

), 44 of which are from freshwater, and 19 are from terrestrial habitats. Most Distoseptispora species are described based on their anamorph alone, and only two species, D. hyalina and D. licualae (Table 3), are reported as sexual morphs based on molecular DNA data, but the connection of teleomorph and anamorph has not been proved by pure culture or sequence data. The genus Distoseptispora has mainly been reported in China (41 species) and Thailand (22 species) [4], and only a small amount of published information is recorded in other regions (e.g., Hungary, Malaysia, Puerto Rico, Sierra Leone) [4,6,44]. Thus, it is unclear whether it has a close relationship with geographic regions, but we expect that large-scale surveys of Distoseptispora in aquatic and terrestrial habitats within different geographic regions, ecological environments, and climatic conditions are needed. This will contribute to a comprehensive knowledge of the species diversity of this genus, and further evaluate their phylogenetic relationships and taxonomic placements by molecular methods.