Morphology and Phylogeny Reveal Vamsapriyaceae fam. nov. (Xylariales, Sordariomycetes) with Two Novel Vamsapriya Species

Abstract

Phylogenetic analyses of combined LSU, rpb2, tub2 and ITS sequence data of representative Xylariales taxa indicated that Diabolocovidia, Didymobotryum and Vamsapriya cluster together and form a distinct clade in Xylariales. Morphological comparison also shows their distinctiveness from other families of Xylariales. Therefore, we introduce it as a novel family, Vamsapriyaceae. Based on morphological characteristics, Podosporium and Tretophragmia, which were previously classified in Ascomycota genera incertae sedis, are now included in the Vamsapriyaceae. In addition, three Vamsapriya species, V. chiangmaiensis sp. nov, V. uniseptata sp. nov, and V. indica are described and illustrated in this paper.

1. Introduction

Xylariales is a large order with both conspicuous and inconspicuous fruiting bodies, and unitunicate, perithecial ascomycetes [1,2]. Many species of Xylariales are saprobes and endophytes [3,4]. Some Xylariales species can produce secondary metabolites which are especially important for the pharmaceutical chemical industry [3,5,6].

Xylariales was established by Nannfeldt [7] to accommodate the type family Xylariaceae, along with Diatrypaceae, Hypocreaceae, Hyponectriaceae, Lasiosphaeriaceae and Polystigmataceae. The previous classification of Xylariales was mainly based on morphology [8,9,10,11,12,13]. With the development of molecular technology, the classification basis of Xylariales was gradually diversified [2,14,15,16]. Smith et al. [2] performed the first multigene analysis to find the familial relationships within Xylariales and treated the order with seven families. Lumbsch and Huhndorf [17] listed six families in Xylariales, while Senanayake et al. [18] revised Xylariales and accepted 11 families. Hyde et al. [19] redefined the families of Sordariomycetes and accepted 15 families in Xylariales based on morphology and multigene analysis, viz. Barrmaeliaceae, Cainiaceae, Clypeosphaeriaceae, Coniocessiaceae, Diatrypaceae, Graphostromataceae, Hansfordiaceae, Hypoxylaceae, Induratiaceae, Lopadostomataceae, Microdochiaceae, Polystigmataceae, Requienellaceae, Xylariaceae and Zygosporiaceae. Hyde et al. [20] introduced Fasciatisporaceae to accommodate Fasciatispora in Xylariales. However, the taxonomic position of many taxa in Xylariales are still uncertain, and they are treated as genera incertae sedis [20,21]. This may probably be due to monospecific genera with either sexual or asexual morph, with no additional collections and lack of molecular data, and sometimes due to the polyphyletic nature of some genera (such as Anthostomella and Xylaria) [22,23,24,25].

Vamsapriya was introduced by Gawas and Bhat [26] based on abundant asexual morphs of the genus, which is characterized by erect, cylindrical, dark brown, synnematous conidiophores, monotretic, clavate to cylindrical conidiogenous cells, and cylindrical or broadly fusiform or obclavate, brown to dark brown conidia [26,27,28,29,30,31,32,33]. The first sexual morph of Vamsapriya was described by Dai et al. [28], which has solitary, immersed ascomata visible as black dots, 8spored, unitunicate asci, and hyaline, fusiform apiospores. They linked the sexual morph of V. bambusicola (MFLUCC 11-0637) to the asexual morph of V. bambusicola (MFLUCC 11-0477) using ITS phylogenies [27,28]. The phylogenetic placement of Vamsapriya has been confusing. Dai et al. [27,28] and Jiang et al. [31] accepted Vamsapriya into the Xylariaceae. However, phylogenetic analyses using broader taxon sampling indicated that Vamsapriya was distant from Xylariaceae [19,34].

This study aims to resolve the phylogenetic position of Vamsapriya. Three Vamsapriya collections (V. chiangmaiensis sp. nov, V. uniseptata sp. nov, and V. indica) on bamboo from China and Thailand are described and illustrated herein. Vamsapriya, along with Diabolocovidia and Didymobotryum, formed a distinct monophyletic clade in the combined LSU, rpb2, tub2 and ITS phylogenetic analyses. A new family, Vamsapriyaceae, is thus established. Podosporium and Tretophragmia are also accepted in Vamsapriyaceae based on their morphology of hyphomycetous asexual morph.

2. Materials and Methods

2.1. Collection, Examination, Isolation and Conservation

Fresh specimens were collected from bamboo in terrestrial habitats in China and Thailand between August 2019 and September 2020. Sample collections and observations were followed by the method described in Senanayake et al. [35]. The samples were stored in envelopes and taken to the laboratory for examination. Morphological observations were done using a stereo microscope (LEICA M125 C, Wetzlar, Germany). The fungal structures were captured using a Nikon ECLIPSE Ni compound microscope (Nikon, Tokyo, Japan) fitted with a NikonDS-Ri2 digital camera (Nikon, Tokyo, Japan). The Tarosoft (R) Image Frame Work software was used to take the measurements. Adobe Photoshop CS6 software (Adobe Systems, San Jose, CA, USA) was used to do photo-plates.

Single spore isolation was carried out to obtain pure cultures following the method described in Senanayake et al. [35]. Germinated spores were transferred to pure potato dextrose agar (PDA) and cultivated under normal light at 26 °C for four weeks. Herbarium specimens were deposited in the Fungarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand, and the herbarium of the Guizhou Academy of Agriculture Sciences (GZAAS), Guiyang, China. Pure cultures were deposited in the Mae Fah Luang University Culture Collection (MFLUCC) and the Guizhou Culture Collection (GZCC). FacesofFungi (FoF) and Index Fungorum numbers were obtained as described in Jayasiri et al. [36] and Index Fungorum [37].

2.2. DNA Extraction, PCR Amplification and Sequencing

Genomic DNA was extracted from fresh fungal mycelia using the Genomic DNA Extraction Kit (GD2416 BIOMIGA, San Diego, CA, USA). Polymerase chain reactions (PCR) were carried out using a BIO-RAD T100 Thermal Cycler in a 20 μL reaction volume which contained 10 μL 2x PCR Master Mix, 7 μL ddH₂O, 1 μL of each primer, and 1 μL template DNA. The PCR thermal cycle program and primers are given in

Table 1. Primers and PCR protocol used in this study.

Locus	Primers	PCR Procedure	Reference
LSU	LR0R	94 °C 3 min; 35 cycles of 94 °C 30 s, 52 °C 30 s, 72 °C 1 min; 72 °C 8 min; 4 °C on hold	[38,39]
	LR5
ITS	ITS5
	ITS4

. The PCR products were sent for sequencing to SinoGenoMax, Beijing, China.

2.3. Phylogenetic Analyses

The sequences used in this study (

Table 2. Taxa names, strain numbers and corresponding sequences used for the molecular phylogenetic analyses.

Taxa	Strain Numbers	ITS	LSU	rpb2	tub2
Amphirosellinia fushanensis	HAST 91111209	GU339496	N/A	GQ848339	GQ495950
Amphirosellinia nigrospora	HAST 91092308	GU322457	N/A	GQ848340	GQ495951
Amphisphaeria sorbi	MFLUCC 13-0721	NR_153531	KP744475	N/A	N/A
Amphisphaeria thailandica	MFLU 18-0794	NR_168783	NG_068588	MK033640	MK033639
Anthostomella formosa	MFLUCC 14-0170	KP297403	KP340544	KP340531	KP406614
Anthostomella obesa	MFLUCC 14-0171	KP297405	KP340546	KP340533	KP406616
Anthostomelloides krabiensis	MFLUCC 15-0678	KX305927	KX305928	KX305929	N/A
Barrmaelia rhamnicola	CBS 142772	NR_153497	N/A	MF488999	MF489018
Barrmaelia rappazii	CBS 142771	NR_153496	N/A	MF488998	MF489017
Barrmaelia macrospora	CBS 142768	NR_167684	N/A	MF488995	MF489014
Biscogniauxia arima	WSP 122	NR_167683	N/A	GQ304736	AY951672
Biscogniauxia mangiferae	MFLU 18-0827	MN337232	MN336236	MN366247	MN509782
Biscogniauxia nummularia	MUCL 51395	NR_153649	NG_066378	KY624236	KX271241
Biscogniauxia repanda	ATCC 62606	KY610383	KY610428	N/A	KX271242
Brunneiperidium gracilentum	MFLUCC 14-0011	KP297400	KP340542	KP340528	KP406611
Brunneiperidium involucratum	MFLUCC 14-0009	KP297399	KP340541	KP340527	KP406610
Cainia anthoxanthis	MFLUCC 15-0539	NR_138407	NG_070382	N/A	N/A
Cainia graminis	MFLUCC 15-0540	KR092793	KR092781	N/A	N/A
Cainia desmazieri	CBS 137.62	MH858124	MH869702	N/A	N/A
Clypeosphaeria mamillana	CBS 140735	N/A	NG_067338	MF489001	MH704637
Collodiscula bambusae	GZUH 0102	KP054279	KP054280	KP276675	N/A
Collodiscula japonica	CBS 124266	JF440974	JF440974	KY624273	KY624316
Collodiscula fangjingshanensis	GZUH0109	KR002590	KR002591	KR002592	KR002589
Collodiscula leigongshanensis	GZUH0107	KP054281	KP054282	KR002588	KR002587
Coniocessia anandra	CBS 125766	MH863747	MH875215	N/A	N/A
Coniocessia maxima	CBS 593.74	NR_137751	NG_070051	N/A	N/A
Coniocessia cruciformis	CBS 126674	MH864206	MH875663	N/A	N/A
Coniolariella limonispora	CBS 283.64	KF719198	KF719210	N/A	N/A
Coniolariella gamsii	CBS 114379	GU553325	GU553329	N/A	N/A
Daldinia macaronesica	CBS 113040	JX658504	KY610477	KY624294	KX271266
Daldinia loculatoides	CBS 113279	MH862918	MH874491	KY624247	KX271246
Diabolocovidia claustri	CBS 146630	MT373367	MT373350	N/A	N/A
Diatrypella heveae	MFLUCC 17-0368	NR_154046	NG_069531	N/A	MG334557
Diatrypella tectonae	MFLUCC 12-0172	NR_154029	NG_069423	N/A	KY421043
Didymobotryum rigidum	JCM 8837	LC228650	LC228707	N/A	N/A
Entosordaria quercina	CBS 142774	NR_153499	N/A	MF489004	MF489022
Entosordaria perfidiosa	CBS 142773	NR_153498	N/A	MF489003	MF489021
Eutypa linearis	MFLUCC 11-0503	KU940150	KU863138	N/A	N/A
Fasciatispora arengae	MFLUCC 15-0326b	MK120301	MK120276	N/A	N/A
Fasciatispora arengae	MFLUCC 15-0326c	MK120302	MK120277	N/A	N/A
Graphostroma platystomum	CBS 270.87	JX658535	N/A	N/A	HG934108
Hansfordia pulvinata	CBS 254.59	KF893288	MH869394	N/A	N/A
Hansfordia pulvinata	CBS 144422	MK442587	MK442527	N/A	N/A
Hansfordia pruni	CBS 125767	MH863748	MH875216	N/A	N/A
Hansfordia pruni	CBS 276.51	MH856854	MH868374	N/A	N/A
Hypoxylon fragiforme	MUCL 51264	KC477229	NG_066364	N/A	KX271282
Hypoxylon neosublenormandii	MFLUCC 11-0618	NR_155174	NG_066168	N/A	N/A
Induratia sp.	SMH1255	MN250031	AY780069	N/A	AY780119
Induratia fengyangensis	CBS 126601	HM034852	HM034858	HM034847	HM034839
Induratia ziziphi	MFLUCC 17-2662	MK762705	MK762712	MK791281	MK776958
Induratia thailandica	MFLUCC 17-2669	MK762707	MK762714	MK791283	MK776960
Lopadostoma dryophilum	CBS 133213	NR_132028	N/A	KC774526	MF489023
Lopadostoma americanum	CBS 133211	NR_132027	N/A	N/A	N/A
Lopadostoma fagi	CBS 133206	NR_132029	N/A	KC774531	N/A
Lopadostoma turgidum	CBS 133207	NR_132036	N/A	KC774562	MF489024
Microdochium lycopodinum	CBS 125585	NR_145223	KP858952	KP859125	KP859080
Microdochium phragmitis	CBS 285.71	NR_132916	NG_058147	KP859122	KP859077
Nemania abortiva	WSP 71221	GU292816	N/A	GQ844768	GQ470219
Nemania beaumontii	HAST 405	GU292819	N/A	GQ844772	GQ470222
Nemania bipapillata	HAST 90080610	GU292818	N/A	GQ844771	GQ470221
Nemania primolutea	HAST 91102001	EF026121	N/A	GQ844767	EF025607
Podosordaria mexicana	WSP 176	GU324762	N/A	GQ853039	GQ844840
Podosordaria muli	WSP 167	GU324761	N/A	GQ853038	GQ844839
Poronia pileiformis	WSP 88113001	GU324760		GQ853037	GQ502720
Poronia punctata	CBS 656.78	KT281904	KY610496	KY624278	KX271281
Requienella fraxini	CBS 140475	NR_138415	N/A	N/A	N/A
Requienella seminuda	CBS 140502	NR_154630	MH878683	MK523300	N/A
Rosellinia buxi	JDR-99	GU300070	N/A	GQ844780	GQ470228
Rosellinia necatrix	HAST 89062904	EF026117	KF719204	GQ844779	EF025603
Vamsapriya aquatica	DLUCC:970	MZ420740	N/A	N/A	N/A
Vamsapriya bambusicola	MFLUCC 11-0477	KM462835	KM462836	KM462834	KM462833
Vamsapriya breviconidiophora	MFLUCC 14-0436	MF621584	MF621588	N/A	N/A
Vamsapriya chiangmaiensis	MFLUCC 21-0065	MZ613171	MZ613168	N/A	N/A
Vamsapriya indica	MFLUCC 12-0544	KM462839	KM462840	KM462841	KM462838
Vamsapriya indica	DLUCC: 2062	MZ420747	MZ420762	MZ442699	N/A
Vamsapriya indica	MFLUCC 21-0066	MZ613172	MZ613169	OK560921	N/A
Vamsapriya khunkonensis	MFLUCC 13-0497	KM462830	KM462831	KM462829	KM462828
Vamsapriya uniseptata	GZCC 21-0892	MZ613173	MZ613170	N/A	N/A
Vamsapriya yunnana	KUMCC 18-0008	MG833874	MG833873	MG833875	N/A
Xylaria arbuscula	CBS 126415	KY610394	KY610463	KY624287	KX271257
Xylaria bambusicola	MFLUCC 11-0606	KU940160	KU863148	KU940183	N/A
Xylaria hypoxylon	CBS122620	AM993141	KM186301	KM186302	KX271279
Zygosporium oscheoides	MFLUCC 14-0402	MF621585	MF621589	N/A	N/A
Zygosporium minus	HKAS99625	MF621586	MF621590	N/A	N/A

Abbreviations: ATCC: American Type Culture Collection, Virginia, USA; CBS: Centraalbureau voor Schimmelcultures, Utrecht, Netherlands; CPC: Culture collection of Pedro Crous, housed at CBS; GZCC: Guizhou Culture Collection, Guiyang, China; GZUH: The herbarium of Guizhou University, Guiyang, China; HAST: Herbarium, Research Center for Biodiversity, Academia Sinica, Taipei, China; HKAS: The Herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica, Kunming, China; JDR: Herbarium of Jack D. Rogers; KUMCC: The Kunming Institute of Botany Culture Collection, Kunming, China; MFLU: The Fungarium of Mae Fah Luang University, Chiang Rai, Thailand; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; MUCL: Mycothèque de l’Université Catholique de Louvian, Belgium; WSP: Washington State University, U.S.A. The newly generated sequences are indicated in red. Ex-type strains are in bold.

) were downloaded from GenBank according to the results of blast searches and previous studies [27,28,29,30,31,32,33]. Alignments for each locus were carried out in MAFFT v7.212 [40]. AliView [41] was used for checking the alignments and changing the format. Terminal ends and ambiguous regions of the alignment were deleted manually. Four single gene alignments were combined using the Sequence Matrix [42].

Single gene analyses were done to compare the topologies and clade stabilities, respectively. Single and combined phylogenies were subjected to Bayesian posterior probability (BYPP), maximum likelihood (ML) and maximum parsimony (MP) analyses. The BYPP analysis was performed in MrBayes v. 3.2.6 [43]. MrModeltest v. 2.3 [44] was used to estimate the best model. GTR+I+G model was chosen for LSU and rpb2; SYM+I+G (Xylariales analysis) and GTR+G (Vamsapriya analysis) models were chosen for ITS; HKY+I+G model was chosen for tub2. Six chains were run and trees were sampled every 1000th generation, the temperature value of the heated chain was set at 0.15. The first 25% sampled trees were discarded as “burn-in”, and the remaining trees were used for calculating BYPP in the majority rule consensus tree. The ML analyses were carried out using IQ-TREE [45] on the IQ-TREE web server (http://iqtree.cibiv.univie.ac.at, 6 September 2021) under partitioned models. The best-fit substitution models were determined by W-IQ-TREE [45]: TIM3e+I+G4 for LSU; TIM3+F+I+G4 for rpb2; TIM2+F+I+G4 for tub2; SYM+I+G4 for ITS. Ultrafast bootstrap analysis was implemented with 1,000 replicates. The MP analyses were carried out with a heuristic search in PAUP v. 4.0 b10 [46]. Bootstrap analysis was used to estimate clade stability, including 1000 replicates, each with 10 replicates of random stepwise addition of taxa [47].

Phylogenetic trees were viewed using FigTree v1.4.4 [48] and modified in Adobe Illustrator CS6 software (Adobe Systems, USA). The sequences generated from our collections were deposited in GenBank.

3. Results

3.1. Phylogenetic Analyses

In Xylariales phylogenetic analyses, the final combined dataset of Xylariales consists of 84 strains representing fifteen families along with the outgroup Amphisphaeria sorbi (MFLUCC 13-0721) and A. thailandica (MFLU 18-0794) in Amphisphaeriales. The aligned sequence matrix comprises LSU (1–829), rpb2 (830–1875), tub2 (1876–3579) and ITS (3580–4305), sequence data for a total of 4305 characters, including coded alignment gaps. Among them, 1894 characters were constant, 374 variable characters were parsimony-uninformative and 2037 characters were parsimony informative. The matrix had 2693 distinct alignment patterns. The BYPP, ML, and MP analyses based on combined sequence data provided similar tree topology. For BYPP, the standard deviation of split frequencies was reached at 0.0099 after 2,980,000 generations. The most likely tree (−ln = 66,531.894) is presented (Figure 1). The MP analysis resulted in two trees with TL = 15,668, CI = 0.302, RI = 0.524, RC = 0.158, HI = 0.698.

The single locus trees (Supplemental Figures S1–S4) and the multi-locus (LSU, rpb2, tub2 and ITS) tree (Figure 1) showed similar tree topology. In multigene analyses, Vamsapriya species clustered with Diabolocovidia claustri and Didymobotryum rigidum, and they formed an internal distinct clade with maximum support (ML-bs = 100%, MP-bs = 100%, BYPP = 1.00). Xylariaceae, Induratiaceae and Clypeosphaeriaceae clustered together, which is a sister to Vamsapriyaceae without significant support. Moreover, V. chiangmaiensis (MFLUCC 21-0065) formed a sister clade to V. yunnana; however, the support for this relationship in Figure 1 is extremely poor and does not exist in Figure 2, and V. uniseptata (GZCC 21-0892) is sister to V. indica. Our isolate MFLUCC 21-0066 grouped in V. indica clade with MFLUCC 12-0544 and DLUCC:2062, indicating they are phylogenetically the same species. Two Anthostomella (Xylariaceae) species, A. formosa (MFLUCC 14-0170) and A. obesa (MFLUCC 14-0171) formed a distinct clade and is sister to Cainiaceae.

The ITS based on Vamsapriya analyses contained 12 taxa and rooted with Diabolocovidia claustra (CBS 146630) and Didymobotryum rigidum (JCM-8837). The manually adjusted ITS alignment contained 563 characters. The best scoring RAxML tree with a final likelihood value of −1737.963458 is presented (Figure 2). Maximum parsimony analysis comprised 563 characters, of which 446 were constant, 54 were parsimony-informative, and 63 were parsimony-uninformative; the tree length is 184, CI = 0.739, RI = 0.597, RC = 0.441, HI = 0.261. The results showed our strain MFLUCC 21-0066 clustered together with V. indica (MFLUCC 12-0544 and DLUCC:2062) with good supports (ML-bs = 94%, MP-bs = 89%, BYPP = 1.00). Vamsapriya chiangmaiensis (MFLUCC 21-0065) formed a distinct clade, and Vamsapriya uniseptata (GZCC 21-0892) grouped with three V. indica (ML-bs = 79%, MP-bs = 61%).

3.2. Taxonomy

Vamsapriyaceae Y.R. Sun, Yong Wang bis & K.D. Hyde, fam. nov.

Index Fungorum number: IF558620; Facesoffungi number: FoF09926

Etymology: Name reflects the type genus

Type genus: Vamsapriya Gawas & Bhat

Saprobic on dead wood. Sexual morph: Ascomata solitary, scattered, immersed, subglobose, black, ostiolate. Peridium thin-walled, brown. Paraphyses hyaline, septate. Asci 8-spored, unitunicate, cylindrical, short pedicellate, with a J+ apical ring. Ascospores apiosporous, fusiform to broad fusiform, hyaline. Asexual morph: Hyphomycetous. Colonies on natural substrate effuse, black, velvety. Mycelium immersed, septate, branched. Synnemata present or absent; when present (Didymobotryum, Podosporium, Tretophragmia, Vamsapriya), synnemata erect, rigid, dark brown, composed of compact parallel conidiophores. Conidiophores erect, straight or curved, cylindrical, dark brown, septate. Conidiogenous cells mono- or polytretic, integrated, terminal, clavate to cylindrical, brown. Conidia catenate or solitary, acrogenous, simple, pigmented, multi-shaped, septate; when absent (Diabolocovidia, adapted from Crous et al. [49]), conidiophores micronematous, flexuous, mostly reduced to a terminal conidiogenous cell. Conidiogenous cells monoblastic, subcylindrical to clavate, pale brown, smooth. Conidia catenate, acrogenous, brown, ellipsoid to obovoid, thin-walled, aseptate.

Notes: A new family, Vamsapriyaceae, is introduced to accommodate Diabolocovidia, Didymobotryum, Podosporium, Tretophragmia, and Vamsapriya. Their phylogenetic position, which is distinct from other families, supports the establishment of the new family within Xylariales. Although the phylogeny of Podosporium and Tretophragmia could not be inferred due to the lack of molecular data, their morphological characters resemble Didymobotryum and Vamsapriya in having brown to dark, simple, straight synnemata, monotretic conidiogenous cells and solitary, obclavate, multi-septate, dark brown conidia [50,51,52,53]. We thus temporarily accept Podosporium and Tretophragmia in Vamsapriyaceae based on morphology. Sequence data are needed to resolve their phylogenetic affinities.

Vamsapriya Gawas & Bhat, Mycotaxon 94: 150 (2006) [2005]

Index Fungorum number: IF29041; Facesoffungi number: FoF00372

Type species: Vamsapriya indica Gawas & Bhat, Mycotaxon 94: 150 (2006) [2005]

Saprobic on dead wood. Sexual morph: Ascomata solitary, scattered, immersed, subglobose, black, ostiolate. Peridium thin-walled, brown. Paraphyses hyaline, septate. Asci 8-spored, unitunicate, cylindrical, straight, short pedicellate, with a J+ apical ring. Ascospores uniseriate or overlapping uniseriate, fusiform to broad fusiform, apiosporous, hyaline, pointed at both ends, surrounded by a mucilaginous sheath. Asexual morph: Hyphomycetous. Colonies on natural substrate effuse, black, velvety. Mycelium immersed, septate, branched. Conidiophores macronematous, synnematous, erect, straight or curved, dark brown, cylindrical, septate. Synnemata erect, rigid, dark brown, composed of compact parallel conidiophores. Conidiogenous cells monotretic, integrated, terminal, clavate to cylindrical. Conidia catenate or solitary, acrogenous, cylindrical, oblong, fusiform or obclavate, brown to dark brown, septate, verruculose.

Notes: Vamsapriya species are reported from tropical and subtropical regions, and most species are found in terrestrial as saprobes [26,27,28,29,30,31]. Nine species are accepted in the Vamsapriya, of which six have molecular data. Vamsapriya is the only holomorphic genus in Vamsapriyaceae, and V. bambusicola is the only species with a sexual-asexual connection in Vamsapriya. Bamboo seems to be the host preference for Vamsapriya species [26,27,28,29,30,31,32,33].

Vamsapriya indica Gawas & Bhat, Mycotaxon 94: 150 (2006) [2005]

Index Fungorum number: IF550801; Facesoffungi number: FoF00374, Figure 3

Saprobic on dead bamboo culms. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies effuse, dark brown, hairy. Conidiophores macronematous, synnematous, single, erect, cylindrical, straight or slightly flexuous, dark brown, smooth-walled. Synnemata erect, straight or slightly flexuous, dark brown, rigid, with cylindrical to clavate apical fertile part, composed of compactly arranged conidiophores, 1300–1900 um long, 80–150 μm wide at the base, 30–40 μm wide in the middle, 60–140 μm wide at the apical fertile region, with basal portion immersed. Conidiogenous cells monotretic, integrated, terminal, brown, cylindrical to clavate, apically rounded, smooth-walled, 4.5–8.5 × 3–4.5 μm ($\overline{x}$ = 6.5 × 4 μm, n = 30). Conidia catenate, acrogenous, cylindrical, rounded at the apex, taper and subtruncate at the base, olivaceous brown to brown, 2–8-septate, slightly constricted at the septa, smooth, 20–48 × 4.5–6.5 μm ($\overline{x}$ = 32 × 5.5 μm, n = 20).

Cultural characters: Conidia germinated on PDA within 12 h, germ tubes produced from both ends. Colonies reached 20 mm diam. within four weeks at 26 °C, cottony, flat, circular, edge entire, white from above, white to yellow from the below.

Material examined: Thailand, Chiang Mai Province, Mae Taeng District, Pa Pae, Mushroom Research Center, on bamboo culms, 10 September 2020, H.W. Shen, M38 (MFLU 21-0088; living culture, MFLUCC 21-0066).

Notes: Vamsapriya indica is the type species of Vamsapriya [26]. Dai et al. [27] recollected V. indica from Thailand and provided the culture characters and sequences data. Bao et al. [32] reported it from a bamboo plant in a freshwater habitat in China. Including our collection, all of these four isolates are recorded from bamboo. Morphological comparison is shown in

Table 3. Synopsis of characters of Vamsapriya indica collections.

Taxon	Host	Habitat/Location	Synnemata	Conidiogenous Cells	Conidia
V. indica (M 393674)	Bamboo	Terrestrial/India	700–1100 μm long, 60–160 μm wide at the base, 30–60 μm wide in the middle, 30–80 μm wide at the apical fertile region	Monotretic, clavate, dark brown, 4–9 × 2.5–4.5 μm	Catenate, acrogenous, brown, cylindrical, vermiform, 10–80 × 4–6 μm, 2–12-septate
V. indica (MFLU 13-0370)	Bamboo	Terrestrial/Thailand	700–1100 μm long, 60–160 μm wide at the base, 30–60 μm wide in the middle, 30–80 μm wide at the apical fertile region	Monotretic, ellipsoidal, brown to dark brown, 4–9 × 2.5–4.5 μm ($\overline{x}$ = 6.5 × 3.7 μm, n = 20)	Catenate, cylindrical, pale brown to dark brown 35–290 × 4–6.5 μm ($\overline{x}$ = 66.6 × 5.6 μm, n = 20), 1–3-septate when young, more than 20–septate at maturity
V. indica (HKAS 115803)	Bamboo	Freshwater/China	1145–1475 μm long, 105–235 μm wide at the base, 50–80 μm wide in the middle, 70–155 μm wide at the apical fertile region	Monotretic, clavate, dark brown 5–9 × 3–5 μm ($\overline{x}$ = 20 × 5 μm, n = 30)	Catenate, brown to dark brown, cylindrical to obclavate, 15–30 × 4–6.5 μm ($\overline{x}$ = 20 × 5 μm, n = 30), 1–4-septate
V. indica (MFLU 21-0088)	Bamboo	Terrestrial/Thailand	1300–1900 μm long, 80–150 μm wide at the base, 30–40 μm wide in the middle, 60–140 μm wide at the apical fertile region	Cylindrical to clavate, brown, 4.5–8.5 × 3–4.5 μm ($\overline{x}$ = 6.5 × 4 μm, n = 30)	Catenate, olivaceous brown to brown, cylindrical, 20–48 × 4.5–6.5 μm ($\overline{x}$ = 32 × 5.5 μm, n = 20), 2–8-septate

. Our collection has longer synnemata than those of the three isolates.

Vamsapriya chiangmaiensis Y.R. Sun, Yong Wang bis & K.D. Hyde, sp. nov.

Index Fungorum number: IF558618; Facesoffungi number: FoF09927, Figure 4

Etymology: Name reflects the collected site.

Holotype: MFLU 21-0087

Saprobic on dead bamboo culms. Sexual morph: Ascomata 650–1000 × 650–850 μm, solitary scattered, immersed within the host cortex, visible as black, circular dots, in cross section globose to subglobose. Ostiole raised, centric, periphysate ostiolar canal. Peridium composed of hyaline inner layer and dark brown to dark outer layer. Paraphyses long, hyaline, unbranched, septate, 1.5–4 μm wide ($\overline{x}$ = 2 μm, n = 15). Asci 8-spored, unitunicate, straight or slightly curved, cylindrical, short pedicellate, with apical ring, 140–190 × 6.5–12 μm ($\overline{x}$ = 160 × 9 μm, n = 15). Ascospores uniseriate, fusiform, 17–26 × 5.5–8 μm ($\overline{x}$ = 20.5 × 6.5 μm, n = 30), constricted apiosporous with a large cell 12.5–22 μm length, guttulate; basal cell 3.5–6.5 μm length, hyaline, smooth-walled, surround a gelatinous mucilaginous sheath. Asexual morph: Undetermined.

Culture characters: Ascospores germinated on PDA within 12 h, germ tubes produced from one end. Colonies reached 45 mm diam. within four weeks at 26 °C, flat, circular, cottony. White from above; brown to dark brown in the center, white to pale brown around from below.

Material examined: Thailand, Chiang Mai Province, Mae Taeng District, Mushroom Research Center, on bamboo culms, 15 July 2020, Y.R. Sun, M35 (MFLU 21-0087, holotype; ex-type living culture, MFLUCC 21-0065).

Notes: Vamsapriya chiangmaiensis is the second species that has a sexual morph in Vamsapriya. It is similar to V. bambusicola in having solitary, subglobose ascomata, 8-spored, unitunicate, cylindrical asci and fusiform hyaline ascospores. It can be distinguished by the longer asci (140–190 μm vs. 115–140 μm). In addition, polymorphic nucleotides from the ITS region showed 37 base differences, and the details are given in

Table 4. Nucleotide differences in the ITS regions of V. bambusicola and V. chiangmaiensis. Numbers are in reference to the nucleotide position of DNA sequences (V. bambusicola) submitted in GenBank.

Species	ITS
	42	52	73	74	82	106	127	167	171	173	194	196	203	206	207	208	209	213	214
V. bambusicola (MFLUCC 11-0477)	C	T	C	G	T	T	A	A	C	C	C	G	C	C	T	C	T	A	A
V. chiangmaiensis (MFLUCC 21-0065)	T	C	G	T	C	C	G	C	T	T	G	A	T	A	C	T	C	T	T
	216	221	229	232	233	235	239	421	432	442	446	447	448	451	461	465	557	558
V. bambusicola (MFLUCC 11-0477)	T	A	A	C	T	T	G	T	C	T	C	T	C	C	T	G	T	T
V. chiangmaiensis (MFLUCC 21-0065)	A	G	T	T	C	C	A	C	T	C	T	G	T	T	C	A	C	A

. Therefore, we identified V. chiangmaiensis as a new species following the suggestions for species delineation [54].

Vamsapriya uniseptata N.G. Liu & K.D. Hyde , sp. nov.

Index Fungorum number: IF558619; Facesoffungi number: FoF09928, Figure 5.

Etymology: Name reflects the 1-septate conidia.

Holotype: GZAAS 21-0378

Saprobic on submerged decaying wood in terrestrial habitat. Colonies on natural substrate effuse, black, velvety. Asexual morph: Hyphomycetous. Mycelium mostly immersed, composed of septate, branched, hyaline to brown hyphae. Conidiophores macronematous, synnematous, erect, straight or broadly curved, dark brown, cylindrical, septate. Synnemata erect, rigid, dark brown, composed of compact parallel conidiophores, up to 1300 µm long, 30–50 µm wide in the middle. Conidiogenous cells monotretic, integrated, terminal, clavate, brown to dark brown. Conidia catenate, acrogenous, olivaceous brown, smooth, oblong, rounded at the apex, taper and subtruncate at the base, 1-septate at the middle, septa thickened and darkened, slightly constricted at the septa, with a large globule in each cell, 14–19 × 3.5–4.5 μm ($\overline{x}$ = 16.5 × 5 µm, n = 30). Sexual morph: Unknown.

Cultural characters: Conidia germinated on PDA within 12 h and germ tubes produced from both ends. Colonies reached 30 mm within four weeks at 26 °C, flat, circular, cottony, white from above, from below brown to dark brown in the center, white to pale brown around.

Material examined: China, Guizhou Province, Xingyi City, Qingshuihe Town, 8 August 2019, N.G. Liu, Q1 (GZAAS 21-0378, holotype; ex-type living culture, GZCC 21-0892).

Notes: Vamsapriya uniseptata is distinguishable by having smaller, 1-septate conidia, while other Vamsapriya species have elongated phragmoconidia. In the BLASTn search, the closest match of the ITS sequence of V. uniseptata is V. khunkonensis (MFLUCC 13-0497, MFLUCC 11-0475 (93.4%)), followed by V. indica (MFLUCC 12-0544 (91.7%)). The LSU sequence of V. uniseptata is V. indica (DLUCC:2062 (99.8%)) and V. khunkonensis (MFLUCC 11-0475 (99.7%)). Vamsapriya uniseptata can be distinguished from V. khunkonensis in the multigene phylogenetic analyses. The ITS region of V. indica (MFLUCC 13-0497) differs by 23 base pairs (527 bp without gaps). Based on distinct morphology and phylogeny, V. uniseptata is introduced as a novel taxon.

3.3. Other Accepted Genera in Vamsapriyaceae

Diabolocovidia Crous, Persoonia 44: 331 (2020)

Index Fungorum number: IF835401; Facesoffungi number: FoF09929.

Parasitic on leaves in terrestrial habitats. Mycelium composed of septate, branched, hyaline to pale brown hyphae. Asexual morph: Hyphomycetous. Conidiophores solitary, erect, flexuous, mostly reduced to a terminal conidiogenous cell. Conidiogenous cells monoblastic, terminal, subcylindrical to clavate, pale brown, smooth. Conidia catenate, acrogenous, brown, ellipsoid to obovoid, thin-walled, un-septate, verruculose [49]. Sexual morph: Unknown.

Type species: Diabolocovidia claustri Crous

Notes: Diabolocovidia is a monotypic genus introduced by Crous et al. [49] to accommodate Diabolocovidia claustri, which was isolated from leaves of Serenoa repens in the U.S.A. Diabolocovidia claustri is characterized by mononematous, micronematous conidiophores in Xylariaceae. In their phylogenetic analyses, Diabolocovidia is basal to Vamsapriya [49]. Diabolocovidia is the only genus without synnemata in Vamsapriyaceae.

Didymobotryum Sacc., Syll. fung. (Abellini) 4: 626 (1886)

Index Fungorum number: IF8009; Facesoffungi number: FoF09930.

Saprobic on decaying plants materials in terrestrial habitats. Colonies on natural substrate effuse, olivaceous to dark brown, velvety. Mycelium mostly immersed, composed of septate, branched, thick-walled, subhyaline hyphae. Asexual morph: Hyphomycetous. Conidiophores macronematous, synnematous, erect, straight or broadly curved, dark brown, cylindrical, septate. Synnemata erect, rigid, dark brown, composed of compact parallel conidiophores. Conidiogenous cells monotretic, integrated, integrated or discrete, cylindrical to clavate, olivaceous brown to dark brown. Conidia catenate, dry, acrogenous, cylindrical, verrucose, 1-septate, slightly constricted at the septa, olivaceous brown to brown. Sexual morph: Unknown.

Type species: Didymobotryum rigidum (Berk. & Broome) Sacc.

Notes: Didymobotryum was introduced by Saccardo [55] typified by D. rigidum. Didymobotryum species have a worldwide distribution [56,57,58,59]. Six species are accepted in the Index Fungorum [37] but only D. rigidum has molecular data (ITS: LC228650, LSU: LC228707).

Podosporium Schwein., Trans. Am. phil. Soc., New Series 4(2): 278 (1832) [1834]

Index Fungorum number: IF9487; Facesoffungi number: FoF09931.

Saprobic on decaying plants materials in terrestrial habitats. Colonies on natural substrate effuse, brown, velvety. Mycelium mostly immersed, composed of septate, flexuous branched hyphae. Asexual morph: Hyphomycetous. Conidiophores arranged in synnemata, brown, septate, sometimes branched at the apex. Synnemata erect, rigid, brown to dark. Conidiogenous cells mono- or polytretic, integrated or discrete, subulate or cylindrical, darkly pigmented. Conidia solitary, obclavate or bacilliform, multi-septate, brown to dark brown. Sexual morph: Unknown.

Type species: Podosporium rigidum Schwein.

Notes: Podosporium was introduced by Schweinitz [60] with P. rigidum as the type species. Since then, many Podosporium species have been discovered worldwide [60,61,62,63]. Most of them are saprobes in terrestrial habitats [60,61,62,63]. There are 67 species listed in the Index Fungorum [37] but no sequence data are available.

Tretophragmia Subram. & Natarajan, Proc. Natl. Inst. Sci. India, B, Biol. Sci. 39: 550 (1974) [1973]

Index Fungorum number: IF10265; Facesoffungi number: FoF09932.

Saprobic on plants materials in terrestrial habitats. Colonies on natural substrate effuse, dark, velvety. Asexual morph: Hyphomycetous. Conidiophores macronematous, synnematous, brown, septate, erect, straight or broadly curved. Synnemata rigid, brown to dark, simple, erect, straight, consisting of a stalk and a capitate, broadened, fertile head. Conidiogenous cells monotretic, subulate or cylindrical, darkly pigmented. Conidia solitary, obclavate to fusiform or irregular in shape, straight, curved or bent, multi-septate, dark brown. Sexual morph: Unknown.

Type species: Tretophragmia nilgirensis (Subram.) Subram. & Natarajan

Notes: Tretophragmia was introduced in 1974. The asexual morph of Tretophragmia is similar to Didymobotryum, Podosporium and Vamsapriya, while no sexual morph is reported. Seifert et al. [53] treated Tretophragmia as a synonym of Podosporium. However, Tretophragmia is accepted in the Index Fungorum [37] and the MycoBank [64] as a separate genus. So far, only two species of Tretophragmia have been described [37] and no sequence data are available. Thus, based on morphology and until DNA sequences data are available, we regard this as a separate genus.

4. Discussion

In this study, three Vamsapriya species, V. chiangmaiensis, V. indica and V. uniseptata were collected from bamboo in terrestrial habitats. In our phylogenetic analyses of combined LSU, rpb2, tub2 and ITS sequence data, Diabolocovidia, Didymobotryum and Vamsapriya formed a distinct clade in Xylariales. Morphological comparison also shows their distinctiveness from other families in Xylariales. Therefore, we propose Vamsapriyaceae as a new family in Xylariales. The sexual morph of Vamsapriya differs from those of Xylariaceae in having hyaline apiospores [28,30]. It is noteworthy that the sexual morph of Vamsapriya is similar to Induratiaceae in having 8-spored asci with J+ apical ring and hyaline, apiospores, but Induratia (Induratiaceae) differs in having geniculosporium asexual morphs [34]. Apioclypea is morphologically similar to the sexual morph of Vamsapriya in having 8-spored, pedunculate, cylindrical asci and biseriate, fusiform, hyaline ascospores with a mucilaginous sheath, but its asexual morph is unknown [19,21].

Clypeosphaeriaceae and Induratiaceae are two other families that are phylogenetically related to Vamsapriyaceae, but they are distinct in morphology. Apioclypea, Aquasphaeria, Brunneiapiospora, Clypeosphaeria, Crassoascus, and Palmaria (Clypeosphaeriaceae) lack asexual morph descriptions and Diabolocovidia, Didymobotryum, Podosporium and Tretophragmia (Vamsapriyaceae) do not have sexual morph descriptions for the comparisons in

Table 5. Asexual morph comparison of the genera in Clypeosphaeriaceae, Induratiaceae and Vamsapriyaceae.

Family	Genus	Asexual Morph
		Synnemata	Conidiogenous Cells	Conidia
Vamsapriyaceae	Diabolocovidia	Absent	Monoblastic, subcylindrical to clavate, pale brown	Catenate, acrogenous, brown, ellipsoid to obovoid, unseptate
	Didymobotryum	Present	Monotretic, integrated, terminal, clavate to cylindrical, pale brown to brown	Catenate, olivaceous brown to brown, cylindrical
	Podosporium	Present	Mono- or polytretic, subulate or cylindrical, darkly pigmented	Solitary, obclavate or bacilliform, multi-septate, brown to dark brown
	Tretophragmia	Present	Monotretic, subulate or cylindrical, darkly pigmented	Solitary, obclavate to fusiform or irregular in shape, multi-septate, dark brown
	Vamsapriya	Present	Monotretic, clavate to cylindrical	Catenate or solitary, acrogenous, cylindrical, oblong, fusiform or obclavate, brown to dark brown, septate
Induratiaceae	Emarcea	Absent	Integrated, terminal, pale brown, forming a rachis with numerous small, pimple-like denticles	Hyaline, smooth, falcate, granular, apex subobtuse, base truncate
	Induratia	Absent	Terminal, solitary or sometimes two celled at the ends of branches, cylindrical, pale brown, bearing inconspicuous denticles	Narrowly ellipsoidal to subglobose, hyaline

and

Table 6. Sexual morph comparison of the genera in Clypeosphaeriaceae, Induratiaceae and Vamsapriyaceae.

Family	Genus	Sexual Morph
		Asci	Ascospores
Vamsapriyaceae	Vamsapriya	8-spored, unitunicate, cylindrical, short pedicellate, with J+ apical ring	Apiosporous, fusiform to broad fusiform, hyaline, with sheath
Induratiaceae	Emarcea	8-spored, unitunicate, cylindrical, pedicellate, with J+ ring	Overlapping uniseriate, long fusiform, hyaline, 2-celled
	Induratia	8-spored, unitunicate, cylindrical, short pedicellate, with a J+ apical ring	Uniseriate, naviculate to ellipsoidal, mostly hyaline, constricted apiosporous
Clypeosphaeriaceae	Aquasphaeria	8-spored, unitunicate, cylindrical, with J- apical ring	Biseriate, cylindrical and ovoid, hyaline
	Apioclypea	8-spored, pedunculate, cylindrical, fissitunicate	Biseriate, fusiform, hyaline, with sheath
	Brunneiapiospora	8-spored, unitunicate, cylindrical, pedicellate with J+ or J− ascal ring	Hyaline to light brown apiospores with a mucilaginous sheath
	Clypeosphaeria	8-spored, unitunicate, cylindrical to broadly cylindrical, pedicellate, with J+ or J− ascal ring	Ellipsoidal to fusiform, unicellular to septate, hyaline to dark brown ascospores, sometimes with sheaths or appendages
	Crassoascus	8-spored, unitunicate, cylindrical, short pedicellate, with J+ ring	Bright brown to dark brown, multiseptate, fusiform ascospores, with hyaline refractive cap-like appendages at each end
	Palmaria	Cylindric to clavate, with a J− subapical ring	Apiosporous, hyaline, 1-septate, obclavate, with a mucilaginous sheath

.

Diabolocovidia claustri was isolated on leaves of Serenoa repens by Crous et al. [49]. Although it has a close phylogenetic relationship with Vamsapriya, they are quite different in morphology. Diabolocovidia has micronematous rather than synnematous conidiophores, blastic rather than tretic conidiogenous cells, and ellipsoid to obovoid, aseptate conidia [49]. The phenomenon that Diabolocovidia mixes with synnematous and tretic genera like Didymobotryum and Vamsapriya reminds us of an example that Vanakripa with blastic conidiogenous resides in the phialidic genus Conioscypha [65]. These probably indicate the polyphyletic nature of some hyphomycetous groups. However, since D. claustri is the only species represented by one isolate in Diabolocovidia, we suggest using more collections to confirm its phylogenetic placement in the future.

When introducing Vamsapriya, Gawas and Bhat [26] pointed out Vamsapriya (conidia catenate, cylindrical to vermiform, phragmosporous) exhibits a combination of morphological characters of Didymobotryum (conidia catenate, ellipsoidal-cylindrical, didymosporous) [51,53,54] and Podosporium (conidia solitary, obclavate, phragmosporous) [56,61,63]. However, as more species are added to these three genera, such generic concepts based on conidial morphology have been dispelled. For example, V. uniseptata resembles species of Didymobotryum in having catenate, oblong, and 1-septate conidia, but it clusters with the type species of Vamsapriya, V. indica. Vamsapriya breviconidiophora and V. yunnana resemble Podosporium species in having obclavate, solitary, and multi-septate conidia, but they are grouped with V. aquatica, which has catenate, cylindrical to obclavate, multi-septate conidia in the phylogenetic tree. Either the authors did not follow the generic concepts strictly when introducing species, or these three genera are probably congeneric. We tend to infer the latter; however, the conclusion requires a detailed re-examination of herbarium specimens and molecular data.