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

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.

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.

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].

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 ddH 2 O, 1 µL of each primer, and 1 µL template DNA. The PCR thermal cycle program and primers are given in Table 1. The PCR products were sent for sequencing to SinoGenoMax, Beijing, China.

Phylogenetic Analyses
The sequences used in this study ( Table 2) 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 1000 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 1,000 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.
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.  (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. Therefore, we identified V. chiangmaiensis as a new species following the suggestions for species delineation [54].
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.

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 Tables 5 and 6.
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.