Oxydothis ailaoshanensis sp. nov. (Oxydothidaceae, Xylariales) from dead bamboo culms in Yunnan Province, China

Introduction

China is recognized as the most diverse country for bamboo, with 43 genera and 728 species^[1]. Southwest China, particularly Yunnan Province is the richest area of bamboo diversity in the country, accounting for 50% of all bamboo species diversity^[2]. Yunnan has three types of bamboo forests viz. cold-temperate, temperate, and tropical bamboo forests^[3]. Since 2017, many studies of bambusicolous fungi have been conducted in Yunnan^[2,4−10]. However, studies on bambusicolous fungi in this region remain limited compared to other regions such as Hong Kong and Taiwan^[2,11]. Among these studies, Xylariales has been relatively under-researched in Yunnan (comprising 14% of known species), compared to Pleosporales (39% of known species). Nonetheless, Sordariomycetes has emerged as the largest group of Ascomycota found on bamboo^[2].

The genus Oxydothis was introduced by Penzig & Saccardo^[12] to initially accommodate three species (viz. O. grisea, O. maculosa, and O. nigricans) within the Amphisphaeriaceae, with O. grisea designated as the type species. The taxonomic placement of Oxydothis has been subject to extensive historical discussion^[13−21]. A comprehensive study of Oxydothis was carried out by Konta et al.^[20] who introduced Oxydothidaceae to accommodate the genus Oxydothis within Xylariales. Species of Oxydothis are characterized by solitary or aggregated ascomata that form in large groups, appearing as darkened, raised regions or dots on the host surface, and cylindrical asci with a J+ (rarely J-) subapical apparatus. The ascospores are filiform to fusiform, hyaline, 1-septate, and have pointed or blunt ends. In some species, ascospores produce appressoria upon germination^[20,22]. The asexual morph of Oxydothis has been linked to Selenosporella species by Samuels & Rossman^[23], although the sexual-asexual connection between Oxydothis and Selenosporella remains unproven. Furthermore, Samuels & Rossman^[23] reported that the asexual morph of O. selenosporellae sporulated in vitro, producing black stromatic masses with perithecia developing throughout the colony. This morph displays macronematous, mononematous conidiophores that are (1–)2–3 septate, unbranched or branched, brown to olivaceous, with finely denticulate conidiogenous cells. A minute refractive scar remains after the conical dehiscence, and the conidia are arcuate, hyaline, and unicellular.

Oxydothis is a common genus mainly found on monocotyledons, such as bamboo (Poaceae), palms (Arecaceae), and Pandanus (Pandanaceae) where it exists mainly as a saprobe^{[18−20,22,24−29]}. A few species have been reported as phytopathogens^[30] or as endophytes^[31]. Hyde et al.^[22] suggested that the Oxydothis species may serve as biological control agents against plant pathogens. Species in this genus with appressorium formation are involved in protein and amino acid degradation, as well as secondary metabolite synthesis i.e. melanin biosynthesis^[32]. Neoxylaria, Oxydothis, and Xylaria species collected from palms produce appressoria indicating they may have the ability to produce secondary metabolites especially when they live as endophytes^[24,32]. Ninety-one epithets are currently listed under the Oxydothis in Index Fungorum (https://indexfungorum.org/Names/Names.asp; accessed on 11 November 2024), though more than half of the described species lack molecular data to clarify their phylogenetic relationships. Among those that do have sequence data, most species are represented only by ITS, LSU, and SSU sequences. Recently, Zhang et al.^[29] introduced five new Oxydothis species (O. caryotae, O. foliata, O. palmae, O. pyriforme, and O. sinensis) and improved the phylogenetic resolution of the genus using multigene phylogeny based on a concatenated dataset of ITS, LSU, SSU, tef1-α, and rpb2 sequences. Unfortunately, only a few protein coding genes (i.e. rpb2 and tef1-α) are currently available for Oxydothis species in GenBank, resulting in incomplete classification of the phylogenetic relationships within the genus.

During ongoing studies of Xylariomycetidae in Southwest China^[33−37], numerous new species have been reported. As part of this continuing effort, the new species Oxydothis ailaoshanensis was discovered in the Ailaoshan rain forest of Yunnan Province, China on dead bamboo culms. This is the second report of an Oxydothis species associated with a bamboo host in China. The new species is described based on its morphological characteristics and supported by multi-gene phylogenetic analyses.

Materials and methods

Sample collection and morphological examination

Dead bamboo culms were collected from the Ailaoshan subtropical evergreen broad-leaved forest (24.536944° N, 101.019444° E, 2,500 m), in Yunnan Province, China during the rainy season (April 2023). Specimens were processed according to the methods outlined by Senanayake et al.^[38]. Observation and photography of the morphological features followed the procedure described in Dissanayake et al.^[37]. Melzer's reagent was used to examine the apical apparatus of the asci. Measurements were taken using the Tarosoft (R) Image Framework software and photo plates representing the morphology were edited with Adobe Photoshop CS6 software (Adobe Systems, USA). Type specimens (holotype and isotype) were deposited in the Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS), Kunming, China. The new taxon was registered in MycoBank (www.mycobank.org) and Facesoffungi^[39].

DNA extraction, PCR amplification, and sequencing

Fungal DNA was extracted from 15–20 fresh fruiting bodies of the fungus as described in Wanasinghe et al.^[40] using Forensic DNA Kit (Omega®, Norcross, GA, USA). LSU, SSU, ITS, and tef1-α gene were amplified with primer pairs LR0R/LR5^[41], NS1/NS4^[42], ITS5/ITS4^[42], and EF1-983F/EF1-2218R^[43] respectively. The thermal cycling program was followed by Konta et al.^[20] and Hu et al.^[26]. The amplified PCR fragments were sent to a commercial sequencing provider (BGI, Ltd Shenzhen, PR China). All the sequences generated in this study were deposited in GenBank (Table 1).

Table 1.  Names, strain numbers, and GenBank accession numbers of the taxa used in the phylogenetic analyses.

Species name	Strain no.	GenBank accession no.
		ITS	LSU	SSU	tef1-α
Oxydothis ailaoshanensis	HKAS 130464T	PQ635200	PQ587528	PQ587530	PQ584438
O. ailaoshanensis	HKAS 130465IST	PQ635201	PQ587529	PQ587531	PQ584439
O. calamicola	MFLUCC 14-1165T	NA	KY206761	KY206767	NA
O. caryotae	HKAS 115712T	PP592449	PP621075	PP639207	PP761002
O. chinensis	ZHKUCC 22-0134T	OR164912	OR164957	NA	NA
O. coperniciae	CMUB 40043T	PP278359	PP278849	PP278850	NA
O. cyrtostachicola	MRC0007T	DQ660334	DQ660337	NA	NA
O. daemonoropsicola	MRC 0005	DQ660335	DQ660338	NA	NA
O. dehongensis	ZHKU 23-0986T	PP580831	PP002130	PP002127	PP001172
O. dehongensis	ZHKU 23-0987	PP580832	PP002131	PP002128	PP001173
O. dehongensis	ZHKU 23-0988	NA	PP002132	PP002129	PP001174
O. foliata	MFLU 24-0165T	PP592450	PP621076	PP639208	PP761003
O. fortunei	GMBC0315T	NR_187011	NG_228961	NA	NA
O. fortunei	GMBC0389	ON510944	ON510945	NA	NA
O. frondicola	HKUCC 3173/Mt14	AF009803	AY083835	AY083818	NA
O. garethjonesii	MFLUCC 15-0287T	KY206773	KY206762	KY206768	KY206777
O. hohnelli	HKUCC 3854	NA	DQ810227	DQ810259	NA
O. inaequalis	MRC0004T	DQ660336	DQ660339	NA	NA
O. metroxylonicola	MFLUCC 15-0281T	KY206774	KY206763	KY206769	NA
O. metroxylonis	MFLUCC 15-0283T	KY206775	KY206764	KY206770	KY206779
O. narathiwatensis	MFLUCC 24-0085T	PP824654	PP824658	PP824659	NA
O. palmae	HKAS 115711T	PP592451	PP621077	PP639209	PP761004
O. palmicola	MFLUCC 15-0806T	KY206776	KY206765	KY206771	NA
O. phoenicis	MFLUCC 18-0269T	MK088065	MK088061	MK088063	MK087667
O. phoenicis	MFLUCC 18-0270IS	MK088066	MK088062	MK088064	MK087668
O. pyriforme	HKAS 115710T	PP592452	PP621078	PP639210	PP761005
O. rhapidicola	MFLUCC 14-0616T	NA	KY206766	KY206772	NA
O. sinensis	GZCC21-0240T	PP592453	PP621079	PP639211	PP761006
Oxydothis sp.	JHGB17 3A	MH268015	NA	NA	NA
Oxydothis sp.	IFO 32218	NA	DQ810225	DQ810261	NA
Oxydothis sp.	E04B-2	PP592454	PP621080	PP639212	NA
O. yunnanensis	GZUCC 0127T	ON176681	ON176684	NA	NA
Vialaea mangiferae	MFLUCC 12-0808T	KF724974	KF724975	NA	NA
V. minutella	BRIP 56959	KC181926	KC181924	NA	NA
Superscripts 'T' and 'IST' represent the type and isotype strains. Newly generated sequences are indicated in bold. 'NA' sequences are unavailable.

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Phylogenetic analysis

Newly generated sequences were subjected to BLAST search in the NCBI GenBank database and sequences of closely related taxa were downloaded. Phylogenetic analysis was performed using ITS, LSU, SSU, and tef1-α sequences (Table 1). Multiple alignments, including both consensus sequences and reference sequences, were generated using MAFFT v. 7^[44] and manually refined using BioEdit v. 7.0.5.2^[45]. The individual datasets were combined into a concatenated dataset and further refined with BioEdit. Combined and individual datasets were subjected to maximum likelihood (ML) and Bayesian inference (BI) analyses. The best-fit substitution models were evaluated using MrModeltest v. 2.3^[46] with the Akaike Information Criterion (AIC) as the selection criteria executed in PAUP v. 4.0b10^[47]. ML and BI analyses were performed on the CIPRES Science Gateway platform^[48]. For ML analyses, RAxML-HPC2 on XSEDE v. 8.2.10^[49,50] was used, applying the GTR + I + G model with 1000 bootstrap repetitions. The BI analysis was executed with MrBayes on XSEDE v.3.2.7a^[51−53] under the GTR + I + G, with one million generations and sampling every 100 generations. The analysis stopped automatically once the standard deviation of split frequencies fell below 0.01, with a burn-in fraction of 0.25. Phylogenetic results were considered significant if ML bootstrap values (MLB) ≥ 60% and Bayesian posterior (BYPP) ≥ 0.95, which were displayed above each node in the resulting tree. The phylogram was visualized using the FigTree v1.4.0 program^[54], and final reorganization was done in Microsoft PowerPoint (2019).

Results

Phylogenetic analysis

The combined ITS, LSU, SSU, and tef1-α matrix comprised 34 strains, including Vialaea mangiferae (MFLUCC 12-0808) and V. minutella (BRIP 56959) as outgroup taxa. The concatenated alignment consisted of 3,421 characters (ITS: 1–680 bp, LSU: 681–1,510 bp, SSU: 1,511–2,512 bp, tef1-α: 2,513–3,421 bp), including gaps. The species-level relationships within Oxydothis in both ML and BI trees were similar in topology. The best-scoring RAxML tree was selected to represent the taxa relationship with a final likelihood value of –15,291.651757 (Fig. 1). The phylogenetic tree obtained from ML analysis in Fig. 1 showed a topology consistent with previous work^[28,29]. The matrix had 990 distinct alignment patterns with 33.64% of characters being undetermined or gaps. The proportion of invariable sites I = 0.453933, the gamma distribution shape parameter alpha = 0.576006 and the Tree-Length = 1.338729. The Bayesian analysis ran for one million generations, with the average standard deviation of split frequencies reaching below 0.01 (0.009728). This analysis generated 1,922 trees, from which 721 were sampled after discarding the 25% as burn-in. The alignment contained a total of 995 unique site patterns.

Figure 1.  RAxML tree based on a combined dataset of analyzed ITS, LSU, SSU, and tef1-α sequences. Bootstrap support values for ML equal to or greater than 60%, Bayesian posterior probabilities (BYPP) equal to or greater than 0.95 are shown as MLB/BYPP above the nodes. The new isolate is in red; ex-type strains are in bold. The scale bar represents the expected number of nucleotide substitutions per site.

DownLoad: Full-Size Img PowerPoint

Based on the results of multigene phylogeny, two strains of the new collection (Oxydothis ailaoshanensis sp. nov.) formed a robust subclade within the monophyletic clade of Oxydothis (60% MLB, 0.99 BYPP, Fig. 1). Oxydothis ailaoshanensis (HKAS 130464 and HKAS 130465) formed an independent lineage that is basal to O. calamicola (MFLUCC 14-1165), O. coperniciae (CMUB 40043), O. caryota (HKAS 115712), O. cyrtostachicola (MRC-007), O. metroxylonicola (MFLUCC 15-0281), O. metroxylonis (MFLUCC 15-0283), O. palmicola (MFLUCC 15-0806), O. palmae (HKAS 115711), O. phoenicis (MFLUCC 18-0269, MFLUCC 18-0270), and O. rhapidicola (MFLUCC 14-0616).

Taxonomy

Oxydothis ailaoshanensis L.S. Dissan., Phookamsak & Wanas. sp. nov. (Fig. 2)

Figure 2.  Oxydothis ailaoshanensis (HKAS 130464, holotype). (a) Substrate. (b), (c) Ascomata on the host surface. (d) Section of an ascoma. (e) Close up of ostiole. (f) Peridium. (g) Paraphyses. (h)–(j) Asci (j = Asci in Melzer's reagent showing J+, apical apparatus, arrow showing short pedicel). (k)–(o) Ascospores. (o = Ascospore in Congo Red). Scale bars: (c) 200 μm, (e) 50 μm, (f, g) 10 μm, (h–j) 50 μm, (k–o) 20 μm.

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MycoBank: MB856009; Facesoffungi number: FoF 14917

Etymology − The specific epithet is derived from the locality, Ailaoshan, where the holotype was collected

Holotype − HKAS 130464

Saprobic on dead bamboo culms (Poaceae). Sexual morph: Ascomata 450–550 μm high × 130–150 μm diameter ($\overline{\rm x}$ = 508 × 140 μm, n = 5), solitary or aggregated in groups, immersed in host's exodermis, becoming raised, superficial, visible as black, shiny knobbed, long axis horizontal to flat on the host, hemispherical (dome-shaped) to subconical, with flattened, wedge-shaped base, uniloculate, somewhat clustered, forming pseudostromatic, with 3–6 in groups of ascomata, glabrous, ostiolate, papillate. Ostiole 50–100 μm long × 40–50 μm diameter ($\overline{\rm x}$ = 75 × 140 μm, n = 5), central, broad neck, open-ended. Peridium 15–20 μm wide, composed of several layers of flattened, inside thin cell layers, composed, hyaline, textura prismatica, outside, thin, light brown to dark brown, textura prismatica, merged with host tissues. Paraphyses are cylindrical, fragmented, hyaline, branched, or non-branched. Asci 100–120 × 10–15 μm ($\overline{\rm x}$ = 110 × 13 μm, n = 15), 8-spored, unitunicate, cylindrical to elongated fusiform, with blunt apex, slightly tapering towards both ends, mostly straight, short pedicillate, with a J+, apical apparatus. Ascospores 55–75 × 3–5 μm ($\overline{\rm x}$ = 65 × 4 μm, n = 15), overlapping 1–3-seriate, elongated fusiform, with acute ends, hyaline, obliquely 1-septate, tapering gradually from the center to the ends, with multi-guttules in each cell, pointed processes. Asexual morph: Undetermined.

Material examined – CHINA, Yunnan Province, Ailaoshan Forest Mountain (24.536944° N, 101.019444° E, 2,500 m), on dead culms of bamboo, 7 April 2023, L.S. Dissanayake, ALF23-10 (HKAS 130464, holotype), ibid., ALF23-10A (HKAS 130465, isotype).

Note: The multigene phylogenetic analyses indicate that Oxydothis ailaoshanensis is closely related to O. metroxylonis (MFLUCC 15-0283), with 60% MLB, 0.99 BYPP statistical support (Fig. 1). The nucleotide difference between O. ailaoshanensis and O. metroxylonis in ITS, LSU, SSU, and tef1-α are 124/555 bp (22.3%), 37/826 bp (4.5%), 13/981 bp (1.3%), and 95/906 bp (10.5%) respectively. Oxydothis ailaoshanensis differs from O. metroxylonis in having smaller, immersed ascomata (450–550 × 130–150 μm) that become raised and superficial with the long axis horizontal to the host surface, shorter asci (100–120 × 10–15 μm) that are mostly straight, and longer ascospores (55–75 × 3–5 μm) that are elongated and fusiform. In contrast, O. metroxylonis has larger ascomata (716–1,580 μm diam), with an axis oblique or perpendicular to the host surface, longer asci (165–181 × 9–15μm) with a cylindrical-clavate shape, and shorter ascospores (47–57 × 4–6 μm) with central curve^[20]. Morphologically, O. ailaoshanensis is similar to O. bambusicola sharing characteristics such as solitary or aggregated ascomata with a central papilla, 8-spored, unitunicate, cylindrical asci with a J+, apical apparatus and elongated fusiform, 1-septate, hyaline ascospores^[18]. Both species were isolated from bamboo hosts^[18]. However, O. ailaoshanensis can be distinguished from O. bambusicola by its larger ascomata (450–550 × 130–150 μm vs 130–375 × 90–160 μm), shorter asci (100–120 × 10–15 μm vs 240 × 23 μm), and shorter ascospores (55–75 × 3–5 μm vs 93 × 7 μm) which taper gradually from the center to the pointed ends. In contrast, O. bambusicola ascospores are gradually tapering to the rounded apices and are covered with small amounts of mucilage. Based on the phylogenetic evidence and morphological differences, we describe our new collection as a distinct species, O. ailaoshanensis.

Discussion

This study listed 89 accepted Oxydothis species in Table 2 with their hosts. Among them nearly all host species for Oxydothis belong to Arecaceae, except Oxydothis aequalis, O. bambusicola, and O. miscanthicola, recorded from Poaceae^[18,31,55]. Notably, O. aequalis and O. bambusicola were recorded on bamboo hosts in the Philippines and the Hong Kong region in China respectively. In the current study, O. ailaoshanensis is introduced also from a bamboo host in China marking a new record for Poaceae hosts in Yunnan Province. Other Oxydothis species, i.e. Oxydothis caryotae, O. chinensis, O. fortunei, O. palmae, O. pyriforme, and O. sinensis were introduced from an Arecaceae host in China, Guangdong Province and Guizhou Province^[26,27,29]. Based on the findings in Table 2, Oxydothis appears to exhibit host specificity primarily within Arecaceae and Poaceae with a distribution across both temperate and tropical regions.

Table 2.  Host occurrences and distribution of all known Oxydothis species.

Species name	Host	Family	Country	Ref.
Oxydothis acutata	Orania spp.	Arecaceae	Philippines	[31]
O. aequalis	Bamboo, Calamus sp.	Arecaceae, Poaceae	Australia, Brunei, Malaysia, Philippines	[31,57]
O. alexandrarum	Archontophoenix alexandrae	Arecaceae	Australia	[31,58]
O. angustispora	Licuala ramsayi	Arecaceae	Australia, Brunei, Thailand	[57−59]
O. asiatica	Calamus flabellatus, Daemonorops sparsiflorus, Licuala sp.	Arecaceae	Australia, Brunei, China (Hong Kong)	[57,60,61]
O. asymmetrica	Calamus conirostris	Arecaceae	Brunei	[57]
O. atypica	Licuala longicalycata	Arecaceae	Thailand	[62]
O. atypica	Licuala longicalycata	Arecaceae	Thailand	[59]
O. australiensis	Archontophoenix sp.	Arecaceae	Australia	[31]
O. bambusicola	Indocalamus sp.	Poaceae	China (Hong Kong)	[18]
O. batuapoiensis	Daemonorops oxycarpa	Arecaceae	Brunei	[57]
O. belalongensis	Licuala sp.	Arecaceae	Brunei	[57]
O. bruneiensis	Calamus sp., Licuala sp.	Arecaceae	Brunei	[57]
O. calami	Calamus sp., Salacca wallichiana	Arecaceae	Australia, Burma, China (Hong Kong), Indonesia, Myanmar, Philippines	[31,57,60,61,63]
O. calamicola	Calamus sp.	Arecaceae	Thailand	[20]
O. caryotae	Caryota sp.	Arecaceae	China (Guangdong Province)	[29]
O. chinensis	Pandanus sp.	Arecaceae	China (Guangdong Province)	[27]
O. circularis	Myrsine sp.	Primulaceae	Brazil	[31]
O. coperniciae	Copernicia alba	Arecaceae	Thailand	[28]
O. cyrtospora	Licuala ramsayi	Arecaceae	Australia	[57]
O. cyrtostachicola	Cyrtostachys renda	Arecaceae	Thailand	[19]
O. daemonoropis	Daemonorops sp.	Arecaceae	Philippines	[31]
O. daemonoropsicola	Daemonorops margaritae	Arecaceae	Australia, China (Hong Kong), Malaysia, Thailand	[57,58,60,61]
O. dispariapicis	Daemonorops oxycarpa	Arecaceae	Brunei	[57]
O. elaeicola	Calamus sp., Elaeis sp., Livistona sp., Pandanus sp.	Arecaceae	Brazil, China (Hong Kong, Taiwan), Democratic Republic of the Congo, Honduras, Nigeria, Sierra Leone, Tanzania	[31,60,64−67]
O. elaeidis	Elaeis sp.	Arecaceae	China (Taiwan), Democratic Republic of the Congo, Tanzania, Zaire	[31,60,65,66]
O. extensa	Licuala ramsayi	Arecaceae	Australia	[57]
O. foliata	Licuala sp.	Arecaceae	Thailand	[29]
O. fortunei	Trachycarpus fortunei	Arecaceae	China (Guizhou Province)	[26]
O. froehlichii	Calamus radicalis	Arecaceae	Australia	[31]
O. froehlichiae	Calamus sp., Linospadix sp.	Arecaceae	Australia	[31]
O. frondicola	Licuala sp., Archontophoenix sp.	Arecaceae	Australia, Malaysia, Thailand	[31,58,59]
O. garethjonesii	Eleais sp.	Arecaceae	Thailand	[20]
O. gigantea	Palm	Arecaceae	Australia, Indonesia	[31,58]
O. grisea	Arenga sp., Calamus sp., Heliconia sp., Licuala sp., Ptychosperma sp.	Arecaceae	China (Taiwan), Malaysia, Indonesia, Venezuela	[31,59,66,68]
O. hoehnelii	Arenga sp., Calamus sp., Licuala sp.	Arecaceae	Philippines	[31,59−61]
O. hongkongensis	Daemonorops sp., Calamus sp.	Arecaceae	Australia, China (Hong Kong)	[57,60,61]
O. ianei	Trachycarpus sp.	Arecaceae	China (Hubei Province), UK	[58]
O. inaequalis	Wallichia siamensis	Arecaceae	Thailand	[19]
O. insignis	Eugenia sp.	Myrtaceae	Brazil	[31]
O. licualae	Archontophoenix sp., Calamus sp., Jessenia sp., Licuala sp.	Arecaceae	Australia, China (Hong Kong), Ecuador, Malaysia, Philippines, Thailand	[31,57−60]
O. licualicola	Licuala sp.	Arecaceae	Brunei, Myanmar	[57,63]
O. linospadicis	Linospadix microcarya	Arecaceae	Australia	[30,31]
O. livistonae	Licuala sp., Livistona sp.	Arecaceae	Brunei, Philippines, Thailand	[31,57,59,69]
O. livistonica	Calamus sp., Licuala sp., Livistona sp.	Arecaceae	China (Hong Kong), Japan, Thailand	[31,57,59−61]
O. livistonicola	Licuala sp.	Arecaceae	Australia	[57]
O. luteaspora	Calamus sp.	Arecaceae	Australia	[31]
O. maculosa	Palm	Arecaceae	Indonesia	[31,70]
O. magnicolla	Calamus sp., Licuala sp.	Arecaceae	Brunei	[56]
O. manokwariensis	Calamus sp., Daemonorops sp.	Arecaceae	China (Hong Kong), Indonesia	[31,57,60]
O. maquilingiana	Daemonorops sp.	Arecaceae	Philippines	[31]
O. mauritiae	Mauritia flexuosa	Arecaceae	Ecuador	[57]
O. megalospora	Calamus sp.	Arecaceae	Brunei	[57]
O. metroxylonis	Metroxylon sagu	Arecaceae	Thailand	[20]
O. miscanthicola	Miscanthus floridulus	Poaceae	China (Hong Kong)	[54]
O. narathiwatensis	Eleiodoxa conferta	Arecaceae	Thailand	[56]
O. nigra	Archontophoenix sp., Licuala sp., Livistona sp.	Arecaceae	Australia, China (Hong Kong), Malaysia	[31,58,60,61]
O. nigricans	Ptychosperma sp.	Arecaceae	Indonesia	[12]
O. nonamyloidea	Livistona sp.	Arecaceae	Indonesia	[31]
O. nonspecifica	Calamus sp., Licuala sp.	Arecaceae	Australia, Brunei	[57]
O. nontincta	Licuala sp.	Arecaceae	Brunei	[57]
O. nypae	Nypa fruticans	Arecaceae	Brunei	[31]
O. nypicola	Nypa fruticans	Arecaceae	Brunei	[31]
O. obducens	Calamus sp., Linospadix microcarya	Arecaceae	Australia, China (Hong Kong)	[31,57,60,61]
O. oedema	Mauritia flexuosa	Arecaceae	Brunei, China (Hong Kong), Guiana, Malaysia, Papua New Guinea, Seychelles	[31,57,58,60,61]
O. opaca	Rhopalostylis sp., Ripogonum sp.	Arecaceae	New Zealand	[31,73]
O. oraniopsidis	Calamus sp., Laccospadix sp., Licuala sp., Oraniopsis sp.	Arecaceae	Australia, India, Thailand	[31,57,59]
O. palmae	Licuala sp.	Arecaceae	China (Guangdong Province)	[29]
O. palmicola	Eleais guineensis	Arecaceae	Thailand	[20]
O. pandani	Pandanus sp.	Arecaceae	China (Hong Kong), French Polynesia, Tubuai, United States	[31,60,61]
O. pandanicola	Livistona chinensis, Pandanus sp., Pritchardia sp.	Arecaceae	Indonesia, Philippines, United States	[31,57,60,61]
O. parasitica	Licuala ramsayi	Arecaceae	Australia	[30,31,57]
O. parvula	Calamus sp., Orania sp., Phoenix sp.	Arecaceae	China (Hong Kong), Philippines	[31,57,60,61]
O. perangusta	Licuala sp.	Arecaceae	Brunei	[57]
O. phoenicis	Phoenix paludosa	Arecaceae	Thailand	[22]
O. poliothea	Palm	Arecaceae	Venezuela	[31]
O. pusillispora	Licuala sp.	Arecaceae	Brunei	[57]
O. pyriforme	Licuala sp.	Arecaceae	China (Guangdong Province)	[29]
O. ragae	Palm	Arecaceae	Indonesia	[31]
O. ragae	Arenga sp.	Arecaceae	China (Hong Kong), Irian Jaya	[31,60,61]
O. rattanica	Calamus sp., Daemonorops sp., Eleiodoxa sp.	Arecaceae	Brunei, China (Hong Kong), Thailand	[57,60]
O. rattanicola	Calamus sp., Daemonorops sp.	Arecaceae	Australia, China (Hong Kong)	[57]
O. rhapidicola	Rhapis excelsa	Arecaceae	Thailand	[20]
O. rhopalostylidis	Rhopalostylis sapida	Arecaceae	New Zealand	[23]
O. rimicolla	Calamus pogonacanthus	Arecaceae	Brunei	[57]
O. rubella	Calamus sp.	Arecaceae	Australia	[31,57,70]
O. sabalensis	Sabal sp., Serenoa sp.	Arecaceae	USA	[71,31]
O. saltuensis	Archontophoenix sp., Cocos nucifera, Licuala sp., Livistona sp.	Arecaceae	Australia, Brunei, Indonesia, Papua New Guinea, Seychelles, Sri Lanka	[31,57,58]
O. selenosporellae	Rhopalostylis sapida	Arecaceae	New Zealand	[23,31,72,73]
O. sinensis	Livistona chinensis	Arecaceae	China (Guangdong Province)	[29]
O. tayabensis	Calamus sp.	Arecaceae	Philippines	[31]
O. uniseriata	Calamus radicalis	Arecaceae	Australia	[57]
O. wallichianensis	Wallichia siamensis	Arecaceae	Thailand	[19]

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Despite the substantial diversity within this genus, only 23 species currently have sequence data available in GenBank. Some species lack informative genetic markers such as ITS for Oxydothis calamicola, O. hohnelli, and O. rhapidicola, or LSU and SSU sequences data for O. chinensis, O. cyrtostachicola, O. daemonoropsicola, O. fortunei, O. inaequalis, and O. yunnanensis. Previous studies on Oxydothis taxonomy relied on ITS, LSU, and SSU sequences^{[20,22,26,28,56]}. However, recent advancements in phylogenetic studies such as that by Zhang et al.^[29], introduced five new Oxydothis species using a multigene dataset including ITS, LSU, SSU, tef1-α, and rpb2. Only nine species incorporate tef1-α in their datasets, further supporting the phylogenetic placement of Oxydothis species in Oxydothidaceae. In the present study, we contribute to this approach by providing a combined ITS, LSU, SSU, and tef1-α phylogeny for introducing O. ailaoshanensis. The use of multiple gene markers in phylogenetic analyses has enhanced our ability to resolve species-level relationships within Oxydothis.

While ITS, LSU, and SSU have previously been used as the primary markers for differentiating species, the addition of protein-coding genes such as tef1-α and rpb2 has improved resolution by increasing phylogenetic signal and reducing ambiguities in species placement^[29]. For Oxydothis ailaoshanensis, the multigene analysis including tef1-α, coupled with morphological data such as the unique ascomata and spore characteristics (Fig. 2), has successfully differentiated it from close relatives such as O. metroxylonis. Nevertheless, some Oxydothis species still lack sequences for key informative gene regions, limiting comprehensive phylogenetic analyses. Future studies should focus on obtaining missing sequence data, especially for protein-coding genes, across more species in this genus. This could further clarify phylogenetic relationships and species boundaries within Oxydothis.

An ITS BLAST search of the sequences linked Oxydothis to some leaf-litter-based ascomycetes (i.e., AF502894, AF502896, AF502740) and various uncultured fungal strains (i.e., KT328718, GU174316, AM999626, KC222801). However, these strains lack associated morphological data, which limits deeper insights into their morpho-phylogenetic relationships. This absence of morphological connections emphasizes the need for comprehensive morphological and molecular characterization of these strains. The present findings suggest that Oxydothis diversity remains underexplored in this region, with potentially numerous species still awaiting discovery.

Acknowledgments

Rungtiwa Phookamsak sincerely acknowledges the Introducing Talents Start-up Fund of Kunming Institute of Botany, Chinese Academy of Sciences, Yunnan Revitalization Talent Support Program 'Young Talent' Project (Grant No. YNWR-QNBJ-2020-120), Yunnan Revitalization Talent Support Program "High-end Foreign Expert" Project and the Independent Research Department of Economic Plants and Biotechnology, Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences (Grant No. Y537731261). Jianchu Xu thanks the Yunnan Department of Sciences and Technology of China (Grant No. 202302AE090023, 202303AP140001). Dhanushka N. Wanasinghe is funded by the Distinguished Scientist Fellowship Program (DSFP), King Saud University, Kingdom of Saudi Arabia.