Multi-Gene Phylogenetic Analyses Revealed Five New Species and Two New Records of Distoseptisporales from China

Eight hyphomycetes were collected as part of an investigation into the diversity of hyphomycetous fungi in China. Based on morphology and multi-loci (LSU, ITS, tef1α, and rpb2) phylogenetic analyses, five new taxa, including a new Aquapteridospora species A. hyalina and four novel Distoseptispora species, viz D. aquisubtropica, D. septata, D. tropica, and D. wuzhishanensis were introduced in Distoseptisporales (Sordariomycetes). Two new habitat records, viz Distoseptispora pachyconidia and D. xishuangbannaensis were firstly reported. Also provided in this study are detailed descriptions of eight new collections and a revised phylogenetic tree for the Distoseptisporales.

During a survey of hyphomycetes in China, eight hyphomycetous taxa were collected from Hainan and Guizhou Provinces. Based on morphological evidence and phylogenetic analyses of sequence combinations of LSU, ITS, tef1α, and rpb2, five new species in genera Aquapteridospora and Distoseptispora, namely A. hyalina, D. aquisubtropica, D. septata, D. tropica, and D. wuzhishanensis, and two new habitat records of D. pachyconidia and D. xishuangbannaensis, were identified, and their full descriptions and illustrations are provided in the present report.

Sample Collection, Specimen Examination, and Isolation
Fresh specimens of decaying wood were randomly collected from freshwater and terrestrial habitats in Hainan and Guizhou Province, China ( Figure 1). Samples were brought back to the laboratory in plastic bags with the collection details including localities and dates. Samples were incubated at room temperature in ziplock bags or sterile moist plastic boxes for about two weeks. Colonies on decaying wood surface were examined, observed, and photographed for their appearance with stereomicroscopes (SMZ 745 and SMZ 800N, Nikon, Tokyo, Japan) from low (0.75 times) to high (5 times) magnification. Fresh colonies were picked with sterile needles at a stereomicroscope magnification of 5 times and placed on a slide with a small amount of distilled water, and then placed under a Nikon EOS 90D digital camera attached to ECLIPSE Ni compound microscope (Nikon, Tokyo, Japan) for microscopic morphological characteristics. The dimensions of conidiophores, conidiogenous cells, and conidia were measured using Tarosoft (R) Image Frame Workprogram. In the species descriptions, arithmetic means as " ̅ ", and "n" stand for the number of measured elements. Photoplates were processed with Adobe Pho-toShop CC 2019 (Adobe Systems, San Jose, CA, USA).  Single spore isolations were performed on water agar (WA) and germinated conidia were aseptically transferred to fresh potato dextrose agar (PDA) following the method of Senanayake et al. [30]. Cultures were grown on PDA and incubated in an incubator at 25 • C for 5 weeks and morphological characters, including color, shape, and size were recorded.
The dried specimens were deposited at the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences (HKAS), Kunming, China, and the Herbarium of Guizhou Academy of Agriculture Sciences (GZAAS), Guiyang, China. Cultures were deposited in Guizhou Culture Collection, China (GZCC). Index Fungorum and Faces of Fungi numbers were acquired by the guideline in Jayasiri et al. [31] and Index Fungorum (2022) [32].

DNA Extraction, PCR Amplification, and Sequencing
Fresh fungal mycelia were scraped with sterilized toothpicks and transferred to 1.5 mL microcentrifuge tubes. Genomic DNA was extracted using the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, Shanghai, China), following the manufacturer's protocol. The primer pairs of LR0R/LR5, ITS5/ITS4, 983F/2218R, and frpb2-5f/frpb2-7cr were used to amplify the large subunit ribosomal DNA (LSU) [33], the internal transcribed spacer (ITS) [34], the translation elongation factor 1 alpha (tef1α) [35] and the RNA polymerase II second largest subunit (rpb2) gene regions [36], respectively. The amplification reactions were completed in a 50 µL reaction volume, including 2 µL DNA template, 2 µL of each forward and reverse primers, and 44 µL of 1.1 × T3 Supper PCR Mix (Qingke Biotech, Chongqing, China). Amplification reactions were carried out as follows (Table 1). The quality of PCR amplification products was verified on 1% agarose electrophoresis gels stained with ethidium bromide. Purification and sequencing of PCR products were completed at Beijing Tsingke Biological Engineering Technology and Services Co., Ltd. (Beijing, China).
The "ALTER" (http://www.sing-group.org/ALTER/, accessed on 29 October 2022) website was used to convert the aligned fasta file to the phylip format for ML analyses [40]. ML analyses were performed through the CIPRES science Gateway V. 3.3 (https://www. phylo.org/portal2/home.action, accessed on 29 October 2022) [41]. The ML analysis was carried out using the RAxML-HPC v.8 on XSEDE (8.2.12) tool using a GTRGAMMA approximation with rapid bootstrap analysis followed by 1000 bootstrap replicates [42]. Maximum Parsimony (MP) analysis was carried out by using PAUP on XSEDE (4.a168) online website [43]. The 1000 random taxa were added for a heuristic search to infer MP trees. The value of MaxTrees, which collapsed branches of zero length and saved all multiple parsimonious trees, was set to 5000. Parsimony score values of tree length (TL), consistency index (CI), retention index (RI), and homoplasy index (HI) were calculated for trees generated under different optimum criteria. Clade stability was estimated using a bootstrap analysis with 1000 replicates, and the taxa was added for the random stepwise of each with 10 replicates [44].
The aligned fasta file was converted to the nexus format file for BI analysis by using AliView v. 1.27 [40]. Bayesian Inference (BI) analyses were performed by using MrBayes on XSEDE (3.2.7a) via CIPRES [42]. The best-fit evolutionary model for the individual and combined datasets was determined using MrModeltest v. 2.3. 10 [45]. GTR + G + I substitution model was selected for LSU, ITS, tef1α, and rpb2. The posterior probabilities (PP) were determined based on Bayesian Markov chain Monte Carlo (BMCMC) sampling [46]. Four simultaneous Markov chains were run for 10,000,000 generations, and trees were sampled every 1000th generation (resulting in 10,000 trees). The first 2500 trees, which represented the burn-in phase of the analysis, were discarded. The posterior probabilities (PP) in the majority rule consensus tree were calculated by the remaining 7500 trees.
Phylogenetic     Note: T denote ex-type strain. Newly generated sequences are indicated in bold. "-"means no data available in GenBank.
Culture characteristics: Conidia were germinated on water agar and produced germ tubes within 10 h. Colonies grown on PDA, circular, flat, dense, fluffy, reaching 35 mm diam. after 25 days of incubation at 25 • C, olivaceous-green, paler brown and dark brown, reverse-side dark brown.

Discussion
In recent years, studies on the number of saprotrophic fungi have received extensive attention [55,56]. For example, freshwater fungi occur in streams and other aquatic bodies [2,57], Calabon et al. [51] listed 3870 species, and numerous taxa are still being discovered [1]. Calabon et al. [51] listed 22 Distoseptispora species from freshwater and this study brings the total to 40 species. Analyses of morphological characteristics and molecular data indicate that eight collections, including five new species, namely A. hyalina, D. aquisubtropica, D. septata, D. tropica, and D. wuzhishanensis are introduced. Five Aquapteridospora epithets (A. lignicola, A. fusiformis, A. bambusinum, A. jiangxiensis, and A. aquatica) are listed in Index Fungorum [32,58]. Morphologically, A. hyalina corresponds well with the generic concept of Aquapteridospora [4,6,58]. However, the new isolate differs from other new species of genus Aquapteridospora by having conidia that have a truncate obtuse at their septum. Multi-gene analyses indicated that A. hyalina, a species phylogenetically distinct in the genus Aquapteridospora, was most closely related to A. fusiformis with weak support.
Morphologically, the asexual morph of Distoseptispora resembles Sporidesmium taxa [9]. Yang et al. [7] reported the existence of the sexual morph of Distoseptispora. Combining the morphological characteristics and the phylogenetic evidence of all species in genus Distoseptispora, we found that some Distoseptispora species form sister clades in phylogeny but have different morphologies. For example, D. martini and D. aquisubtropica have a close phylogenetic relationship. However, D. martini has distinctive oblate or subglobose conidia, while D. aquisubtropica has obclavate or lanceolate conidia. In addition, there are some species with similar morphologies but are genetically unrelated. For example, D. tropica and D. verrocosa have an identical morphology of conidiophores and conidia, but the two species have a distant phylogenetic relationship. Considering this phenomenon, additional molecular data and morphological characteristics are required for verification and expansion.
Previous studies of Distoseptispora have primarily been conducted in China (Guizhou, Yunnan, Sichuan, and Jiangxi Provinces) and in Thailand, which are subtropical and tropical regions. In this paper, eight collections were discovered in freshwater and terrestrial habitats in the Provinces of Hainan and Guizhou, China. Thus, it is unclear whether it has a close relationship with the climate. It may be a result of the limited geographical regions sampled.  Data Availability Statement: All sequences generated in this study were submitted to GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 16 May 2022).