Morphological and phylogenetic appraisal of Ophioceras (Ophioceraceae, Magnaporthales)

Abstract

Ophioceras is accommodated in the monotypic family Ophioceraceae (Magnaporthales, Sordariomycetes), and the genus is delimited based on molecular data. During an ongoing survey of bambusicolous fungi in southwest China, we collected a submerged decaying branch of bamboo from Sichuan Province, China and an Ophioceras species occurring on this substrate was observed and isolated. An Ophioceras taxon was delimited based on morphological characteristics and combined LSU, RPB1 and ITS sequence analyses and is described as Ophioceras sichuanense sp. nov. The species formed a well-supported clade basal to Ophioceras (100% ML, 1.00 PP). Based on the updated phylogenetic tree of Magnaporthales, Ceratosphaerella castillensis (generic type) and C. rhizomorpha formed a clade within Ophioceras and morphologically resemble Ophioceras. Therefore, Ceratosphaerella is synonymized under Ophioceras. The phylogenetic relationships of Ophioceras are discussed in relation to morphological similarities of genera in Magnaporthales. The generic circumscription of Ophioceras is emended.

Introduction

Klaubauf et al. [1] introduced the family Ophioceraceae to accommodate Ophioceras Sacc. which is typified by O. dolichostomum (Berk. & M.A. Curtis) Sacc. The family is recognized as black, immersed to superficial, globose to subglobose, perithecial ascomata with long, periphysate necks, 8-spored, unitunicate, subcylindrical to narrowly fusoid asci with a J-, apical ring, and filiform, hyaline to olivaceous, septate ascospores without sheaths [1–3]. Species of Ophioceraceae are saprobes on wood and herbaceous plants in aquatic or terrestrial habitats [1–6].

Saccardo [7] introduced Ophioceras to accommodate taxa with immersed, sub carbonaceous, globose perithecia, with conical-cylindrical, filiform ostioles, elongate asci and filiform, septate ascospores. The genus initially accommodated O. bacillatum (Cooke) Sacc., O. dolichostomum, O. friesii (Mont.) Sacc., O. hystrix (Ces.) Sacc., O. macrocarpum (Sacc.) Sacc., O. longisporum Sacc. and O. therryanum (Sacc. & Roum.) Sacc [7]. Ophioceras was accommodated in Magnaporthaceae based on limited molecular data [8–10]. However, the genus was excluded from Magnaporthaceae and accommodated in the separate family Ophioceraceae in Magnaporthales based on combined LSU and RPB1 phylogenetic analyses [1]. To date, the asexual morph of Ophioceras has not yet been reported [1,2,6], and only 40 epithets are listed under this genus in Index Fungorum [11]. However, only eleven Ophioceras species have molecular data in GenBank, and only LSU and ITS sequence data are most available for these species [12].

Ophioceras species are commonly discovered from wood in freshwater and are generally clumped under the name Ophioceras sp. [13,14], O. commune [15–17] or O. dolichostomum [18–21]. Twenty-three Ophioceras species are listed in Species Fungorum [22] and accepted in Hyde et al. [3]. Ophioceras species occur in disparate streams in different islands and continents and are therefore likely to have separated for millions of years ago, potentially explaining their evolution into distinct species. The number of existing Ophioceras species is therefore likely to exceed more than presently known. Hyde et al. [23] has shown that numerous new taxa await description across most under-collected and -studied.

Ceratosphaerella comprises C. castillensis (C.L. Sm.) Huhndorf, Greif, Mugambi & A.N. Mill. and C. rhizomorpha Huhndorf & Mugambi, introduced by Huhndorf et al. [8]. Phylogenetic analyses of the LSU sequence dataset showed that Ceratosphaerella grouped with Ophioceras in Magnaporthaceae [8]. Klaubauf et al. [1] only accommodated Ophioceras into Ophioceraceae, but they did not incorporate Ceratosphaerella in their analyses. Thus, Ceratosphaerella remains in Magnaporthaceae. To date, there are only two species included in Ceratosphaerella [11], and these taxa only have LSU sequence data in GenBank [12]. Ceratosphaerella castillensis has been reported only as a sexual morph, while C. rhizomorpha is holomorphic and has a Didymobotryum-like asexual morph [8]. In Luo et al. [2], Ceratosphaerella did not group within Magnaporthaceae but clustered with Ophioceras in Ophioceraceae. However, Luo et al. [2] did not verify the phylogenetic status of Ceratosphaerella in Ophioceraceae, leading to the uncertain placement of the genus.

This study aims to introduce the novel Ophioceras taxon on a submerged bamboo branch in Sichuan Province, China and resolve the congeneric status of Ceratosphaerella and Ophioceras in Ophioceraceae based on a morpho-molecular approach.

Materials and methods

Sample collection, observation and isolation

Decaying branches of bamboo submerged in freshwater were collected in the stream in the Shunan Artificial Bamboo Forest, Sichuan Province, P.R. China in July 2019. Samples were kept in a paper bag for further morphological examination in the laboratory. Pseudostromata visualized on decaying branches of bamboo were observed and examined under a stereo microscope (Motic series SMZ 140) and captured via digital phone camera (iPhone 7, Apple Inc., USA). Microscopic features (e.g., asci, ascospores and paraphyses) were prepared using the squashing mount technique in sterilized distilled water on clean slides for morphological study. Sections of pseudostromatic ascomata, ostiolar necks and peridial structures were by free-hand sectioning using Gillette razor blades. Melzer’s reagent and Indian ink were used to detect the J-/J+ apical ring of the asci and mucilaginous sheath surrounding the ascospores, respectively. Morphological features visualized under a Nikon ECLIPSE Ni compound microscope were photographed using a Canon EOS 600D digital camera. Measurements (n = 10–20) of pseudostromata, locules, peridium, paraphyses, asci and ascospores were carried out using Tarosoft (R) Image Frame Work version 0.9.7. Photographic plate and line drawings of fungal morphologies were edited and combined using Adobe Photoshop CS6 (Adobe Systems Inc., USA).

Single spore isolation based on the spore suspension technique [24] was carried out to obtain a pure fungal culture. Germinated ascospores were transferred to the new potato dextrose agar plates (PDA; Qingdao Daily Water Biotechnology co. LTD. Shandong, P.R. China) under aseptic conditions and grown under normal day/nightlight at room temperature. Culture characteristics (e.g., growth, shape, colour, margin, elevation, consistency) were checked and recorded after one week and four weeks.

The holotypic specimen is conserved in the herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), Yunnan, P.R. China. The isotype is stored in the herbarium of Mae Fah Luang University, Chiang Rai, Thailand (MFLU). Ex-type living cultures are preserved in the Kunming Institute of Botany Culture Collection (KUMCC) and Mae Fah Luang University Culture Collection (MFLUCC). Facesoffungi and Index Fungorum numbers were registered for the new taxon [11,25].

DNA extraction, amplification and sequencing

Fungal genomic DNA was extracted from fresh mycelia using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux^®, P.R. China) following manufacturer’s instructions (Hangzhou, P.R. China) and also extracted from fruiting bodies (= pseudostromata) directly using the Forensic DNA Kit (Omega^®, USA) for a duplicated strain. DNA amplification was performed by polymerase chain reaction (PCR). Two gene regions including the internal transcribed spacer (ITS) and 28S large subunit rDNA (LSU), were used to amplify PCR fragments using forward and reward primer pairs: ITS5/ITS4 [26] and LR0R/LR5 [27], respectively. PCR reactions were conducted in a 25 μL total volume, consisted of 2 μl of DNA template, 1 μl of each forward and reverse primer, 12.5 μl of 2× Power Taq PCR Master Mix (Beijing BioTeke Corporation, P.R. China) and 8.5 μl double-distilled water (ddH₂O). The PCR thermal cycle program for ITS and LSU was set up following Jiang et al. [28]. PCR fragments were purified and sequenced at TsingKe Biological Technology (Beijing) Co., Ltd, P.R. China.

Molecular phylogeny

The newly generated sequences (ITS and LSU) of fungal strains were initially subjected to the basic local alignment search tool (BLASTn) via the National Center for Biotechnology Information web portal (NCBI; https://blast.ncbi.nlm.nih.gov) for discovering closely related fungal taxa. In order to clarify the phylogenetic placement of the new isolate, the representative taxa in Magnaporthales were incorporated with the new taxon to generate the sequence data matrix for further analysis. These representative taxa of Magnaporthales were downloaded from the GenBank database (Table 1) based on recent publications [2,29].

Table 1. Detailed information of fungal taxa used in the phylogenetic analyses.

The newly generated sequences are indicated in blue, and the ex-type strains are in bold.

Species name	Culture collection/ Voucher no.	GenBank accession numbers
		LSU	RPB1	ITS
Aquafiliformis lignicola	MFLUCC 16–1341	MK835815	/	MK828615
Aquafiliformis lignicola	MFLUCC 18–1338	MK835814	/	MK828614
Bambusicularia brunnea	INA-B-92-45	NG_058671	KM485043	NR_145387
Barretomyces calatheae	CBS 129274	MH876639	KM485045	MH865202
Bifusisporella sorghi	URM 7442	NG_067852	MK060159	NR_164042
Budhanggurabania cynodonticola	BRIP 59305	NG_058678	KP162143	NR_137952
Buergenerula spartinae	ATCC 22848	DQ341492	JX134720	JX134666
Bussabanomyces longisporus	CBS 125232	NG_058668	KM485046	NR_145385
Ceratosphaeria aquatica	MFLU 18–2323	MK835812	/	MK828612
Ceratosphaeria lampadophora	SMH 4822	AY346270	/	/
Ceratosphaeria lignicola	MFLU 18–1457	MK835813	/	MK828613
Deightoniella roumeguerei	CBS 128780	MH876533	KM485047	MH865092
Falciphora oryzae	CBS 125863	NG_064356	KJ026706	NR_153972
Falciphoriella solaniterrestris	CBS 117.83	NG_058108	KM485058	NR_153995
Gaeumannomycella caricis	CBS 388.81	NG_058109	KM485059	NR_146245
Gaeumannomyces amomi	CMUZE002	DQ341493	/	AY265318
Gaeumannomyces radicicola	CBS 296.53	NG_058089	KM009194	NR_146246
Gaeumannomyces tritici	CBS 541.86	DQ341497	/	/
Kohlmeyeriopsis medullaris	CBS 117849	NG_058110	KM485068	NR_154068
Macgarvieomyces borealis	CBS 461.65	NG_058088	KM485070	NR_145384
Macgarvieomyces juncicola	CBS 610.82	KM484970	KM485071	KM484855
Magnaporthiopsis incrustans	M35	JF414892	JF710437	JF414843
Magnaporthiopsis maydis	CBS 133165	KX306614	/	KX306544
Magnaporthiopsis maydis	CBS 662.82A	NG_058111	KM485072	NR_154175
Magnaporthiopsis poae	M47	JF414885	JF710433	JF414836
Muraeriata africana	GKM 1084	EU527995	/	/
Muraeriata collapsa	SMH 4553	EU527996	/	/
Myrmecridium schulzeri	CBS 100.54	EU041826	/	EU041769
Myrmecridium sorbicola	CBS 143433	NG_063957	/	NR_158871
Nakataea oryzae	CBS 252.34	MH867001	KM485078	KM484862
Nakataea oryzae	CBS 288.52	MH868571	KM485080	MH857040
Neocordana malayensis	CBS 144604	NG_066327	/	NR_163364
Neocordana musae	CPC 18127	LN713290	/	NR_154265
Neogaeumannomyces bambusicola	MFLUCC 11–0390	NG_059556	/	NR_146247
Neopyricularia commelinicola	CBS 128308	NG_058112	KM485087	NR_154226
Omnidemptus affinis	ATCC 200212	NG_059478	JX134728	NR_154292
Omnidemptus graminis	CBS 138107	MK487734	/	NR_164058
Ophioceras aquaticus	IFRDCC 3091	JQ797433	/	JQ797440
Ophioceras aquaticus	MFLUCC 16–0906	MK835810	/	MK828611
Ophioceras castillensis (as Ceratosphaeria castillensis)	SMH 1865	EU527997	/	/
Ophioceras chiangdaoense	CMU 26633	NG_066356	/	/
Ophioceras commune	KUN-HKAS 92569	MH795820	/	MH795815
Ophioceras commune	KUN-HKAS 92587	MH795819	/	MH795814
Ophioceras commune	KUN-HKAS 92590	MK835809	/	MK828610
Ophioceras commune	KUN-HKAS 92640	MH795818	/	MH795813
Ophioceras dolichostomum	CMURp50	DQ341504	/	/
Ophioceras hongkongense	HKUCC 3624	DQ341509	/	/
Ophioceras leptosporum	CBS 894.70	NG_057959	JX134732	NR_111768
Ophioceras rhizomorpha (as Ceratosphaerella rhizomorpha)	GKM 1262	EU527998	/	/
Ophioceras sichuanense	KUN-HKAS 107677	MW057782	/	MW057779
Ophioceras sichuanense	KUMCC 20–0213	MT995046	/	MT995045
Ophioceras submersum	MFLUCC 18–0211	MK835811	/	/
Proxipyricularia zingiberis	CBS 133594	NG_063934	KM485091	AB274434
Pseudohalonectria fagicola	MFLUCC 15–1117	KX426219	/	/
Pseudohalonectria hampshirensis	MFLUCC 15–0774	KX426218	/	/
Pseudohalonectria lignicola	SMH 2440	AY346299	/	/
Pseudohalonectria lutea	CBS 126574	MH875622	/	MH864160
Pseudophialophora eragrostis	CM12m9	KF689638	KF689618	NR_146240
Pseudopyricularia cyperi	CBS 133595	NG_058113	/	NR_137920
Pseudopyricularia kyllingae	CBS 133597	NG_058114	KM485096	NR_155645
Pyricularia ctenantheicola	GR0001	KM484994	KM485098	KM484878
Pyricularia grisea	BR0029	KM484995	KM485100	KM484880
Pyriculariopsis parasitica	HKUCC 5562	DQ341514	/	/
Slopeiomyces cylindrosporus	CBS 609.75	KM485040	KM485158	KM484944
Slopeiomyces cylindrosporus	CBS 610.75	NG_057751	JX134721	NR_120170
Xenopyricularia zizaniicola	CBS 132356	KM485042	KM485160	KM484946

Abbreviations: ATCC: American Type Culture Collection, Virginia, USA; CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; CMU: Chiang Mai University, Chiang Mai, Thailand; CPC: Culture Collection of Pedro Crous, Netherlands; KUN-HKAS: the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica, Yunnan, P.R. China; HKUCC: Hong Kong University Culture Collection, Hong Kong, P.R. China; IFRDCC: Fungal Research & Development Centre Culture Collection, P.R. China; KUMCC: Kunming Culture Collection, Yunnan, P.R. China; MFLU: the Herbarium of Mae Fah Luang University, Chiang Rai, Thailand; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand.

Preliminary single-gene data matrixes were aligned via MAFFT v. 7.452 [30] and improved manually in BioEdit v. 5.0.6 [31]. The single-gene alignments of LSU and ITS data matrixes were prior analyzed by maximum-likelihood (ML) criterion using RAxML v. 7.0.3 [32,33] for checking if there are any conflicts between the tree topologies. The concatenated LSU-ITS and LSU-RPB1-ITS sequence datasets were further analyzed based on maximum-likelihood (ML) and Bayesian inference (BI) criteria and the tree topologies of these combined gene datasets were compared for checking the congruence of the tree topologies. The concatenated LSU-ITS and LSU-RPB1-ITS sequence datasets comprise 64 strains of ingroup taxa in Magnaporthales. Myrmecridium schulzeri (CBS 100.54) and M. sorbicola (CBS 143433) were selected as the outgroup taxa.

Maximum-likelihood (ML) criterion was analyzed by the online tool RAxML-HPC v.8 on XSEDE (8.2.12) via CIPRES Science Gateway v. 3.3 web portal [34]. The ML + thorough bootstrap parameters were set at default values but modified as 1000 replications of bootstraps (-N 1000) and using the GTRGAMMAI model.

The best-fit evolutionary models of nucleotide substitution for LSU, RPB1 and ITS loci were evaluated by MrModeltest 2.3 [35], and the GTR+I+G substitution model under the Akaike Information Criterion (AIC) was the best-fit evolutionary model for each locus. Bayesian inference (BI) analysis was performed by MrBayes v. 3.1.2 [36]. The Markov Chain Monte Carlo sampling (MCMC) sampling method was used to determine posterior probabilities (PP) [37,38]. One million generations of six simultaneous Markov chains were run and sampled every 100th generation. MCMC heated chain was set up with a “temperature” value at 0.15. The burn-in was set to 20% of all sampled trees, meaning that sampled trees beneath the asymptote (20%) were discarded. Posterior probabilities values were then calculated from the remaining 8000 trees in the majority rule consensus tree.

The final phylogram presented in this study was visualized in FigTree v. 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/). The phylogenetic tree was edited in Microsoft Office PowerPoint 2016 (Microsoft Inc., USA) and converted to jpeg file using Adobe Photoshop CS6 (Adobe Systems Inc., USA). New sequences generated from the present study were registered for GenBank accession numbers (Table 1). The final alignment and phylogram are submitted in TreeBASE submission ID: 28293 (http://purl.org/phylo/treebase/phylows/study/TB2:S28293?x-access-code=66338d666c9ae6b7c0a0aa779b50078d&format=html).

Nomenclature

The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

In addition, new names contained in this work have been submitted to Index Fungorum from where they will be made available to the Global Names Index. The unique Index Fungorum number can be resolved, and the associated information viewed through any standard web browser by appending the Index Fungorum number contained in this publication to the prefix www.indexfungorum.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central and LOCKSS.

Compliance with ethical standards

There is no conflict of interest (financial or non-financial) and all authors have agreed to submission of paper. The authors also declare that they have no conflict of interest and confirm that the field studies did not involve endangered or protected species.

Results

Molecular phylogeny

Based on the results from the nucleotide BLAST search tool of LSU sequence, our new strains (KUMCC 20–0213 and KUN-HKAS 107677) are closely related to species of Ophioceras, whereas ITS sequence revealed that our new strains are similar to the unidentified fungal endophyte isolate 4583 (86.79% similarity) and other taxa in Magnaporthales. The concatenated LSU-RPB1-ITS dataset included 2594 total characters with gaps (LSU: 1–905 bp, RPB1: 906–1877 bp, ITS: 1878–2594 bp). The best scoring ML tree with the final ML optimization likelihood value of -23570.931644 (ln) was selected to represent the phylogenetic relationships of taxa in Magnaporthales (Fig 1). All free model parameters were estimated using the GTRGAMMAI model, with 1354 distinct alignment patterns and 40.89% of undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.247576, C = 0.255381, G = 0.292293, T = 0.204750, with substitution rates AC = 1.508478, AG = 2.781197, AT = 1.835341, CG = 0.983246, CT = 6.427316, GT = 1.000000. The Tree-Length = 7.381586 and the gamma distribution shape parameter α = 0.605644. The evaluation of Bayesian posterior probabilities (BYPP) from MCMC was carried out with the final average standard deviation of split frequencies reached 0.009301.

Fig 1. Maximum likelihood tree based on a combined LSU, RPB1 and ITS sequence matrix for taxa in Magnaporthales.

Bootstrap support values for ML equal to or higher than 70% and the Bayesian posterior probabilities equal to or higher than 0.90 PP are defined above the nodes as ML/PP. Ex-type strains are in black bold, and new species and new combinations are indicated in red bold.

The tree topology from ML analysis showed similar results with the BI analysis and comparing LSU-ITS, and LSU-RPB1-ITS phylograms also revealed similarities in overall topologies (Fig 1 and S2 Fig). Thus, we will use the LSU-RPB1-ITS topology for further discussion. Five families of Magnaporthales were included in the presented phylogenetic analyses viz. Ceratosphaeriaceae, Magnaporthaceae, Ophioceraceae, Pseudohalonectriaceae and Pyriculariaceae. These five families formed well-resolved monophyletic clades within Magnaporthales with significant support (greater than 70% ML and 0.95 PP) in our combined gene analyses (Fig 1 and S2 Fig). Ophioceraceae has a close relationship with Ceratosphaeriaceae and Pseudohalonectriaceae. However, the phylogenetic relationships of these three families are not well resolved and pending further clarification.

Phylogenetic analyses of the LSU-RPB1-ITS sequence matrix revealed that the investigated specimen (KUN-HKAS 107677) and its pure culture (KUMCC 20–0213) are grouped together and form an independent lineage basal to Ophioceras in Ophioceraceae with high statistical support (100% ML, 1.00 PP; Fig 1). Considering the phylogenetic results and morphology, we propose a novel species, Ophioceras sichuanense, occurring on submerged bamboo in Sichuan Province, China.

Ceratosphaeria castillensis (SMH 1865) formed a robust clade with C. rhizomorpha (GKM 1262) (100% ML, 1.00 PP; Fig 1) within Ophioceras (84% ML, 0.90 PP; Fig 1). Ceratosphaeria castillensis (SMH 1865) and C. rhizomorpha (GKM 1262) clustered with Ophioceras aquaticus (IFRDCC 3091, MFLUCC 16–0906), O. dolichostomum (CMURp50), O. hongkongense (HKUCC 3624), O. submersum (MFLUCC 18–0211) and O. commune (KUN-HKAS 92569, KUN-HKAS 92587, KUN-HKAS 92590, KUN-HKAS 92640) in our all analyses (Fig 1, S1 and S2 Figs) and separated distantly from taxa in Magnaporthaceae. Thus, Ceratosphaerella is treated as a synonym of Ophioceras, the prior introduced genus, in Ophioceraceae.

Taxonomy

Ophioceraceae Klaubauf, Lebrun & Crous, Studies in mycology 79: 103 [1].

Type genus: Ophioceras Sacc.

Notes–To date, Ophioceraceae includes a single genus, Ophioceras. In the present phylogenetic study, Ophioceraceae formed a stable clade within Magnaporthales and distinguished from other families of Magnaporthales.

Ophioceras Sacc., Sylloge Fungorum 2: 358 (1883), emend. H.B. Jiang, Phookamsak & K.D. Hyde.

Facesoffungi number: FoF01255.

Synonym: Ceratosphaerella Huhndorf, Greif, Mugambi & A.N. Mill., Mycologia 100(6): 941 [8].

Type species: Ophioceras dolichostomum (Berk. & M.A. Curtis) Sacc.

Saprobic on bamboo, palm, bark or wood, and other herbaceous plants from aquatic or terrestrial environments. Sexual morph: Ascomata black, perithecial, immersed to superficial, scattered or gregarious, globose to subglobose, or ampulliform, glabrous with ostiolar necks, somewhat forming uni- to multi-loculate pseudostromata. Pseudostromata if present: locules immersed in pseudostroma, dark brown to black, subglobose to ampulliform, or irregular in shape, with a long, cylindrical, black, brittle, curved or straight, periphysate neck. Peridium composed of several layers, of pseudoparenchymatous cells, arranged in textura angularis, inner layers composed of hyaline, elongate cells, with compressed, dark brown to black cells towards the outer layers. Paraphyses filiform, hyaline, unbranched, septate, broad at the base, tapering at the tip. Asci unitunicate, 8-spored, subcylindrical to acerose or clavate, pedicellate or sessile, with a refractive J-, apical ring. Ascospores filiform or narrow fusiform, with rounded ends, slightly curved or sigmoidal, hyaline, pale brown or olivaceous, aseptate or septate, with or without guttulate, lacking a sheath. Asexual morph: Hyphomycetous, Didymobotryum-like. Colony on substrates dark brown to black, rhizomorphic-like threads, radiated from central of clustered ascomata on patched subiculum. Synnemata formed on rhizomorphic strands, dichotomously branched hyphae, straight or flexuous, branched, lighter brown head, black in mass, with conidiophores at the apical region. Conidiophores elongate, septate, with dark brown bands at the septa, verrucose. Conidiogenous cells pale brown, cylindrical, tretic, integrated, terminal, verrucose. Conidia pale brown, with darker brown at the septa, ellipsoid to cylindrical, 1–3 transverse septa, verrucose [8].

Notes–Ophioceras occurs on a wide range of hosts mainly distributed in America, Asia, Africa and Oceania [2,5,8,9,13–17,39–53]. To date, only O. bambusae and O. guttulatum have been reported from bamboo [51,53,54]. Eleven Ophioceras species have been reported from freshwater [2,10], of which nine species were found in China [10,17,41,53]. In this study, O. sichuanense is introduced as the second species occurring on submerged decaying branches of bamboo in China.

The genus Ophioceras is emended herein to accommodate the genus Ceratosphaerella that clustered with other Ophioceras species in Ophioceraceae. Ceratosphaerella is morphologically different from Ophioceras in having clavate asci and hyaline to pale brown, narrow fusiform ascospores, whereas Ophioceras has subcylindrical to acerose asci and hyaline to olivaceous, filiform ascospores [1,8]. However, Ophioceras resembles Ceratosphaerella in the ascomatal morphology and is also supported by phylogenetic analyses. Through ML and BI phylogenetic analyses based on a concatenated LSU-RPB1-ITS sequence matrix (Fig 1), C. castillensis (SMH 1865), which was previously treated in Magnaporthaceae, is phylogenetically closely related to C. rhizomorpha and clustered within Ophioceras in Ophioceraceae. Therefore, we treat Ceratosphaerella as a synonym of Ophioceras instead of a genus in Ophioceraceae. Using a morpho-phylogenetic approach, Ophioceras castillensis comb. nov. and O. rhizomorpha comb. nov. are hereby introduced.

Ophioceras sichuanense H.B. Jiang, Phookamsak & K.D. Hyde, sp. nov. Fig 2.

Fig 2. Ophioceras sichuanense (KUN-HKAS 107677, holotype).

a, b Pseudostromata with long ostiolar necks on host. c–e Section through pseudostromata. f Ascoma. g Ostiole. h Apical part of neck. i Peridium. j–l Asci. m Ascospores. n Paraphyses. Scale bars: d, e = 200 μm, f–h = 50 μm, i = 30 μm, j–n = 20 μm.

[urn:lsid:indexfungorum.org:names:557956].

Facesoffungi number: FoF09404.

Etymology–The specific epithet “sichuanense” refers to Sichuan Province, P.R. China, where the species was collected.

Holotype–KUN-HKAS 107677.

Saprobic on decaying branches of bamboo submerged in freshwater. Sexual morph: Pseudostromata 300–750 μm diam., 230–350 μm high (excluding necks), black, scattered, solitary, semi-immersed to superficial, 1–5-loculate, glabrous, ostiolate, papillate, carbonaceous. Locules 150–300 μm diam., 150–250 μm high (excluding necks), immersed within pseudostroma, clustered, subglobose to ampulliform, blackened, with a long black, periphysate neck, up to 1 cm. Peridium 20–35 μm wide, thick-walled, composed of several layers, of flattened to broad, pseudoparenchymatous cells, arranged in textura angularis to textura prismatica, inner layers composed of hyaline cells, outer layers composed of dark brown to black pseudoparenchymatous cells. Paraphyses 4–8 μm wide, filiform, hyaline, indistinct septate, unbranched, slightly rough with small guttules, broad at the base, tapering toward the tip. Asci 90–115 × 5–6.5 μm (x̅ = 103 × 5.8 μm, n = 20), 8-spored, unitunicate, cylindrical, sessile to subsessile, with short broad bulb-like at the base, apically rounded with a J-, apical ring. Ascospores 80–90 × 1–1.5 μm (x̅ = 85 × 1.3 μm, n = 15), overlapping, or in parallel, hyaline, filiform, slightly curved to sigmoidal, thin-walled, aseptate, smooth-walled, multi-guttulate. Asexual morph: not observed.

Culture characteristics: Ascospores germinated on PDA within 24 hours at room temperature under normal condition. Mycelium superficial to immersed in agar medium, branched, septate, smooth hyphae. Colonies growing slowly on PDA, reaching 20 mm in two weeks, cottony, circular, with entire edge, raised, white from above and below.

Material examined: P.R. China, Sichuan Province, Yibin City, Shunan Artificial Bamboo Forest, on submerged decaying branches of bamboo, 25 July 2019, H.B. Jiang & R. Phookamsak, SC011 (KUN-HKAS 107677, holotype), ex-type living culture, KUMCC 20–0213.

GenBank accession numbers: ITS = MT995045, LSU = MT995046, SSU = MT995047 (KUMCC 20–0213; from pure culture); ITS = MW057779, LSU = MW057782, SSU = MW057847, TEF1-α = MW082017 (KUN-HKAS 107677; from fruiting bodies).

Notes–Ophioceras sichuanense can be distinguished from other Ophioceras species because it forms multi-loculate pseudostromata. Ophioceras sichuanense resembles O. guttulatum, O. leptosporum and O. tenuisporum. However, O. sichuanense differs from O. guttulatum in having smaller asci and ascospores (asci: 90–115 × 5–6.5 μm vs. 130–160 × 14–17 μm; ascospores: 80–90 × 1–1.5 μm vs. 100–128 × 4–5 μm) [17]. Ophioceras sichuanense resembles O. leptosporum and O. tenuisporum due to the size ranges of asci and ascospores and extremely long ostiolar necks. However, the species differs from the latter two species in their ascospore septation (Table 2). Although O. bambusae and O. sichuanense were collected from bamboos, O. sichuanense has longer asci and ascospores (asci: 90–115 × 5–6.5 μm vs. 90–95 × 5.5–6.5 μm; ascospores: 80–90 × 1–1.5 μm vs. 75–80 × 1.5 μm) [54]. Present phylogenetic analyses show O. sichuanense formed a clade basal to Ophioceras and close to O. chiangdaoense and O. leptosporum. Ophioceras sichuanense and O. chiangdaoense are different in the dimensions of the ostiolar neck, asci and ascospores (Table 2).

Table 2. Synopsis of Ophioceras species.

Species name	Ascomata	Asci	Ascospores	Habitat	Origin	Host/substrate	References
Ophioceras aquaticus	310–620 μm diam., 1-loculate, superficial to submerged, with a 500–800 μm long neck	85–100 × 9–10 μm, cylindrical	42–68 × 3–4 μm, filiform, slightly acute at each end, falcate, sigmoid, hyaline, 3–5-septate	Submerged	China: Yunnan	Wood	[2, 10]
O. arcuatisporum	313–324 × 252–340 μm, 1-loculate, superficial to immersed, with a long neck, up to 800 μm	276–307 × 15–20 μm, fusoid to narrowly cylindrical	170–239 × 4–7 μm, narrowly fusoid to cylindrical, falcate, hyaline to pale orange, 5–12-septate	Submerged	USA: Minnesota	Typha sp., herbaceous debris, grass	[49]
O. bambusae	1 mm long, 2/3 mm diam., 1-loculate, immersed, with a 2–2.5 mm long neck	90–95 × 5.5–6.5 μm, cylindrical	75–80 × 1.5 μm, filiform, with both blunt ends, curved, hyaline, indistinctly septate	Terrestrial	Indonesia: Java	Bamboo	[54]
O. castillensis	525–650 μm diam., 1-loculate, superficial, with a 250–400 μm long neck	70–90 × 10–14 μm, clavate	29–40 × 4–5.5 μm, narrowly fusiform, slightly curved, hyaline to pale brown, 3-septate	Terrestrial	Nicaragua	Bark and wood	[8]
O. cecropiae	200–250 μm diam., 1-loculate, immersed, with a long neck, up to 2 mm	75–90 × 6.5–7.5 μm, cylindrical to subfusoid	60–70 × 2 μm, filiform, straight to slightly curved, hyaline, septate	Terrestrial	Venezuela	Cecropia sp.	[55]
O. chiangdaoense	200–310 × 170–310 μm, 1-loculate, immersed, with a 93–273 μm long neck	85–125 × 11–17 μm, cylindrical	54–75.5 × 4–5.5 μm, filiform, falcate, fusoid at both ends, hyaline, 3-septate	Terrestrial	Thailand: Chiang Mai	Decaying leaves of Dracaena loureiroi	[9]
O. commune	150–350 × 260–400 μm, 1-loculate, immersed to superficial, with a 375–1660 μm long neck	64–118 × 4–12 μm, cylindrical	50–110 × 2 μm, filiform, arcuate or sigmoidal, hyaline, 3–7-septate	Submerged	Panama: Barro Colorado Island	Wood, herbaceous debris	[2, 49]
O. dolichostomum	500 μm diam., 1-loculate, immersed, with a 1–5 mm long neck	100–130 × 8–12 μm, cylindrical	94–110 × 2–3 μm, filiform, falcate, sigmoid, hyaline, 3–7-septate	Submerged	USA: Florida	Dead wood	[2, 46]
O. filiforme	3–5 mm long, 150–180 μm diam., 1-loculate, immersed, erumpent to superficial, with a long neck	100–120 × 10–13 μm, clavate or fusoid	80–100 × 3–3.5 μm, filiform, hyaline to yellowish, multi-septate	Terrestrial	Indonesia: Java	Rotten leaf sheaths of Amomum sp.	[56]
O. fusiforme	360–500 × 330–450 μm, 1-loculate, immersed to erumpent, with a 250–800 μm long neck	70–112 × 6–12 μm, cylindrical	64–104 × 1.5–3 μm, filiform, fusoid, tapering at both ends, straight to falcate, 3–5-septate	Submerged	USA: Indiana	Decorticated woody debris	[49]
O. guttulatum	400–600 × 1200–1800 μm, 1-loculate, superficial to immersed, with a 500–1500 μm long neck	130–160 × 14–17 μm, broadly cylindrical	100–128 × 4–5 μm, cylindrical, falcate, pale yellow to hyaline, 3–5-septate	Submerged	China: Hong Kong	Wood	[17]
O. hongkongense	500–640 × 700–800 μm, 1-loculate, superficial to immersed, with a more than 600 μm long neck	100–125 × 12–14 μm, elongated fusoid to broadly cylindrical	72–101 × 3.5–4.5 μm, cylindrical, falcate, tapered at both ends, hyaline, 3–5-septate	Submerged	China: Hong Kong	Wood	[17]
O. indicus	400–650 μm diam., 1-loculate, immersed, with a long neck, up to 1.5 mm	65–90 × 8.5–11.5 μm, cylindrical to subfusoid	60–85 × 2.5–3.5 μm, filiform, tapering towards base, slightly curved, hyaline to subhyaline, 7–10-septate	Terrestrial	India: New Delhi	Dried twigs of Ficus infectoria	[47]
O. leptosporum	250–300 μm diam., 1-loculate, immersed or superficial, with a 1–2 mm long neck	70–95 × 5–6 μm, cylindrical	70–80 × 1–1.5 μm, filiform, apex rounded, base acute, hyaline to faintly tinted, straight to slightly curved or sigmoid, 3–7-septate	Submerged	UK: Exeter	Rotten stems of Umbelliferae sp.	[4]
O. miyazakiense	Data unavailable	Data unavailable	Data unavailable	Terrestrial	Japan: Kyushu	Decaying litter	[57]
O. palmae	164–320 × 244–288 μm, 1-loculate, partly immersed, with a 180–376 μm long neck	76–96 × 10–14 μm, broadly cylindrical	79–90 × 1.2–2 μm, filiform, tapering at both ends, sigmoid, hyaline, 5-septate	Terrestrial	Philippines: Mt. Makiling	Calamus ornatus	[17]
O. parasiticum	600–800 μm diam.	100–140 × 9–11 μm	48–70 × 2.5–3.3 μm, 3–9-septate	Terrestrial (parasite)	China	Data unavailable	[9]
O. petrakii	600–750 × 555–675 μm, 1-loculate, immersed, with a neck	171–182 × 12–15 μm, cylindrical	152–171 × 3–4 μm, filiform, apex rounded, base acute, slightly curved or sigmoid, hyaline, multi-septate	Terrestrial	India: Karnataka	Dead stems of Vitex negundo	[45]
O. rhizomorpha	500–900 × 500–750 μm, 1-loculate, superficial, with a 300–600 μm long neck	115–145 × 13–16 μm, clavate	39–49 × 3.5–4.5 μm, narrowly fusiform, slightly curved, hyaline to pale brown, 3-septate	Terrestrial	Kenya	Bark or wood	[8]
O. sichuanense	230–350 × 300–750 μm, pseudostromatic, 1–5-loculate, semi-immersed to superficial, with long necks, up to 1 cm	90–115 × 5–6.5 μm, cylindrical	80–90 × 1–1.5 μm, filiform, slightly curved to sigmoidal, hyaline, aseptate	Submerged	China: Sichuan	Decaying branches of bamboo	This study
O. sorghi	300–400 μm diam., 1-loculate, immersed, with a 350–700 μm long neck	85–110 × 12–14 μm, cylindrical to clavate	75–95 × 3–4 μm, filiform, cylindrical, with rounded apex and slightly thinner rounded base, slightly curved, hyaline, 3–12-septate	Terrestrial	Central African Republic: M’ Baiki	Sorghum vulgare	[4, 43]
O. submersum	300–400 × 500–600 μm, 1-loculate, immersed, with a long neck	115–137 × 10–11 μm, cylindrical	87–109 × 3–4 μm, filiform, rounded at both ends, slightly curved or sigmoid, hyaline, multi-septate	Submerged	Thailand	Wood	[2]
O. tambopataense	Data unavailable	Data unavailable	Data unavailable	Terrestrial	Peru	Decaying leaf of palm	[57]
O. tenuisporum	240–625 × 260–775 μm, 1-loculate, superficial to partially immersed, with a long neck, up to 20 mm	82–114 × 4–6 μm, cylindrical to narrowly fusoid	66–94 × 1–1.5 μm, filiform, more broadly rounded at one end than the other, curved to sigmoid, hyaline, 3-septate	Submerged	Panama: Barro Colorado Island	Twig	[49]
O. venezuelense	730–890 × 745–868 μm, 1-loculate, partially immersed to superficial, with a 250–800 μm long neck	148–180 × 11–18 μm, cylindrical to narrowly fusoid	130–158 × 2–4 μm, filiform, falcate, more broadly rounded at one end than the other, straight to slightly curved, hyaline, (4)–5–(6)-septate	Submerged	Venezuela: Portuguesa	Wood, herbaceous debris	[49]
O. zeae	450–650 μm diam., 1-loculate, superficial or partially immersed, with a 350–900 μm long neck	55–65 × 7–8 μm, cylindrical to narrowly fusoid	39–50 × 2.3–2.5 μm, cylindrical to fusoid, with rounded ends, straight or slightly curved, hyaline, 3-septate	Terrestrial	Central African Republic: Boukoko	Dead Zea mays	[4, 42]

Ophioceras castillensis (C.L. Sm.) H.B. Jiang, Phookamsak & K.D. Hyde, comb. nov. Fig 3.

Fig 3. Ophioceras castillensis (redrawn from Huhndorf et al. [8], NY isotype).

a, b Ascomata. c Ascus. d Apical ring. e Ascospores. f Peridium. g Paraphyses. Scale bars: a = 1 mm, b = 200 μm, f = 20 μm, c, e, g = 10 μm, d = 5 μm.

[urn:lsid:indexfungorum.org:names:557957].

Facesoffungi number: FoF09405.

Basionym: Ceratosphaeria castillensis C.L. Sm., Bull. Lab. Nat. Hist. Iowa State Univ. 2: 403 (1893).

Synonym: Ceratosphaerella castillensis (C.L. Sm.) Huhndorf, Greif, Mugambi & A.N. Mill., Mycologia 100(6): 944 [8].

Type information: Nicaragua, Castillo Viejo, on bark, Feb–Mar 1893, C.L. Smith, Central American Fungi 13, (isotype, NY).

Detailed description and illustration: see Huhndorf et al. [8].

Known hosts/ habitat and distribution: Saprobic on bark or wood in terrestrial habitat. To date, Ophioceras castillensis is only reported from Costa Rica, Nicaragua, and Puerto Rico [8].

Notes–In this study, Ceratosphaerella castillensis is transferred to Ophioceras as O. castillensis based on a concatenated LSU-RPB1-ITS analyses coupled with morphological similarity of the ascomata and ascomatal wall which is typical Ophioceras. Ophioceras castillensis can be separated from other Ophioceras species in forming ascomata on large clusters, superficial on sparse, subicular hyphae and having clavate asci and hyaline to pale brown, fusiform, 3-septate ascospores [8]. Detailed morphological comparison and taxa habitats in Ophioceras are described in Table 2.

Ophioceras rhizomorpha (Huhndorf & Mugambi) H.B. Jiang, Phookamsak & K.D. Hyde, comb. nov. Fig 4.

Fig 4. Ophioceras rhizomorpha (redrawn from Huhndorf et al. [8], all from Mugambi 1262).

a Ascomata and synnemata on the substrate. b Ascoma. c Peridium. d Ascus. e Apical ring. f Hyaline to pale brown ascospores. g Paraphyses. h, i Conidiophores. j Conidiogenous cell bearing conidium. k Conidium. Scale bars: a = 1 mm, b = 300 μm, c = 50 μm, g = 30 μm, d = 20 μm, i, k = 15 μm, f, h, j = 10 μm, e = 5 μm.

[urn:lsid:indexfungorum.org:names:557958].

Facesoffungi number: FoF09406.

Basionym: Ceratosphaerella rhizomorpha Huhndorf & Mugambi, Mycologia 100(6): 944 [8].

Type information: Kenya, Kagamega National Park, on decaying wood on the ground, 17 January 2007, G.K. Mugambi, GKM1262 (holotype EA, isotype F).

Detailed description and illustration: see Huhndorf et al. [8].

Known hosts/ habitat and distribution: Saprobic on decaying wood of terrestrial habitat. To date, Ophioceras rhizomorpha is only reported from Kenya.

Notes–Ophioceras rhizomorpha was reported with a Didymobotryum-like asexual morph on a natural substrate [8]. The species resembles O. castillensis in forming ascomata on the large subicular, and having clavate asci and hyaline to pale brown, fusiform, 3-septate ascospores [8]. Ophiocera rhizomorpha, however, differs from O. castillensis in having larger ascomata, necks, peridia, paraphyses, asci and ascospores [8] (see Table 2).

Discussion

Ophioceraceae currently accommodates only Ophioceras. Maharachchikumbura et al. [58] accommodated the family in Magnaporthales (Diaporthomycetidae, Sordariomycetes) based on literature review and phylogenetic analysis. Ophioceraceae has limited taxon sampling, and most taxa in this family lack reliable protein coding genes to clarify phylogenetic affinities. For example, Ophioceras arcuatisporum (strains A9-1, A167-1B) has only SSU sequence data available in GenBank. In preliminary phylogenetic analysis of SSU sequence matrix, the species formed a stable clade within Magnaporthaceae rather than Ophioceraceae. Ophioceras arcuatisporum needs to be re-visited and re-illustrated, incorporating details from molecular data.

Luo et al. [2] performed combined LSU and TEF1-α phylogenetic analyses to investigate the relationships of taxa in Magnaporthales. In their phylogeny, Ceratosphaeria grouped with Pseudohalonectria (Pseudohalonectriaceae) and separated from Ophioceras (Ophioceraceae). Based on the fact that Ceratosphaeria differs from Pseudohalonectria in having narrow cylindric-fusiform to filiform and longer ascospores, Ceratosphaeriaceae was thus introduced as a new family within Magnaporthales to accommodate Ceratosphaeria [2]. In the present study, we performed an updated phylogenetic tree based on a concatenated LSU-RPB1-ITS sequences and showed that Ceratosphaeria (Ceratosphaeriaceae) clustered with Ophioceras (Ophioceraceae) with low statistical support, suggesting that gene selection in the data matrix affects the tree topology at the familial levels in Magnaporthaceae. Ceratosphaeria is morphologically similar to Ceratosphaerella [8]. Although Ceratosphaeria clustered with Ophioceras with low statistical support, Ceratosphaeria possibly belongs to Ophioceraceae. However, the phylogenetic status of Ceratosphaeria needs to be clarified with more evidence in the future studies.

In the present study, we synonymize Ceratosphaerella under Ophioceras based on molecular phylogeny coupled with similar ascomatal morphology. Phylogenetic analyses based on the LSU sequence dataset (S1 Fig) and the concatenated LSU-ITS (S2 Fig) and the LSU-RPB1-ITS (Fig 1) sequence datasets have always shown that O. castillensis (≡ C. castillensis) and O. rhizomorpha (≡ C. rhizomorpha) formed a stable clade within Ophioceras. Ophioceras castillensis, O. rhizomorpha and most Ophioceras species lack protein coding genes and other reliable genes to clarify phylogenetic placement as well as limited taxon sampling. The ex-type strain of O. rhizomorpha and the reference strain of O. castillensis were sequenced only for LSU locus. Hence, more reliable gene loci (e.g., SSU, ITS, RPB1 and TEF1-α) from the ex-type strain of O. rhizomorpha should be obtained and the epitype of O. castillensis should be designated and incorporated with morpho-molecular based taxonomic treatment. Furthermore, the new collections and sequence data of taxa in Ophioceras are required to provide a better taxonomic resolution for robust species delineations in this genus.

Many genera in Magnaporthales have similar morphological characteristics with Ophioceras (Table 3). However, these genera are considered distinct genera based on phylogenetic investigations [1,2,8,59–61]. Pseudohalonectria (Pseudohalonectriaceae) is also similar to Ophioceras in its ascomata and asci. However, Pseudohalonectria varied in shape of ascospores, such as ellipsoidal, fusiform or filiform [62]. In the present study, Pseudohalonectria (Pseudohalonectriaceae) formed an independent lineage separate from other families in Magnaporthales; however, Pseudohalonectria could not be resolved at the species level such as in Perera et al. [62]. It may be because molecular data of most taxa in this genus are unavailable in GenBank database. Moreover, some sequences of P. lignicola deposited in GenBank are likely to be misidentified [62]. Therefore, sequences of Pseudohalonectria species used for phylogenetic analyses are limited.

Table 3. Morphological comparisons of similar genera to Ophioceras.

Generic name	Ascomata	Asci	Ascospores	References
Aquafiliformis (Magnaporthaceae)	Globose to subglobose, with long beak	Cylindrical to clavate, with an inconspicuous apical ring	Aseptate, filiform, hyaline	[2]
Ceratosphaeria (Ceratosphaeriaceae)	Globose to pyriform, with a long black or yellow neck	Cylindrical, with a conspicuous, non-amyloid, apical ring	Multi-septate, filiform, hyaline	[2]
Gaeumannomycella (Magnaporthaceae)	Subglobose to elliptical, with a lateral, central cylindrical neck	Cylindrical to elongated clavate, apical ring not observed	0–3-septate, narrowly fusiform, hyaline	[61]
Gaeumannomyces (Magnaporthaceae)	Subglobose to elliptical, with a cylindrical neck	Cylindrical to elongated clavate, with an apical refringent ring	Indistinctly septate, filiform, hyaline to pale brown	[60]
Kohlmeyeriopsis (Magnaporthaceae)	Ellipsoid, with a long cylindrical periphysate neck	Fusoid to cylindrical, with a large apical ring	Indistinctly septate, filamentous, tapering towards the base, hyaline	[1, 63]
Muraeriata (Magnaporthaceae)	Lageniform to globose, with long beak	Cylindrical to ventricose, with a tall, narrow, apical ring	3-septate, narrowly fusiform, ends slightly curved, hyaline	[8]
Neogaeumannomyces (Magnaporthaceae)	Globose to subglobose, with a long, periphysate neck	Cylindrical, with an apical ring	2–3-septate, filiform to long fusiform, hyaline	[59]
Ophioceras (Ophioceraceae)	Subglobose to ampulliform, with a long cylindrical periphysate neck	Subcylindrical to narrowly fusoid or clavate asci with a small, refractive, non-amyloid apical ring	Aseptate to multi-septate, filiform, narrowly fusiform, hyaline pale brown or olivaceous	[2, 8, this study]
Slopeiomyces (Magnaporthaceae)	Globose, with periphysate neck bearing hyphae	Clavate, with a non-amyloid apical ring	3–4-septate cylindrical to fusoid, tapering somewhat to base, hyaline	[1]

Supporting information

S1 Fig. RAxML tree generated from an analysis of the LSU matrix of taxa in Magnaporthales.

(TIF)

S2 Fig. RAxML tree generated from an analysis of the LSU-ITS matrix of taxa in Magnaporthales.

(TIF)

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

The authors are grateful for the support of the Mushroom Research Foundation, Chiang Rai, Thailand (to H-BJ) and Yunnan Provincial Science and Technology Department grant no. 202003AD150004 (to RP and H-BJ under Jianchu Xu). KDH thanks the Foreign Experts Bureau of Yunnan Province, Foreign Talents Program (2018; grant no. YNZ2018002), Impact of Climate Change on Fungal Diversity and Biogeography in the Greater Mekong Subregion (grant no: RDG6130001). RP sincerely acknowledges the CAS President’s International Fellowship Initiative (PIFI) for young staff (grant no. Y9215811Q1), the “High-level Talent Support Plan” Young Top Talent Special Project of Yunnan Province and Chiang Mai University for financial support. SCK thanks the CAS President’s International Fellowship Initiative (PIFI) young staff under the grant number: 2020FYC0002 and the National Science Foundation of China (NSFC) under the project code 31851110759 for funding. AHB and AME thank the International Scientific Partnership Program ISPP at King Saud University through ISPP#0089. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.