Two novel species and two new records of Distoseptispora from freshwater habitats in China and Thailand

Abstract During investigations into freshwater fungi from the Great Mekong Subregion, four Distoseptispora taxa were collected from China and Thailand. Based on morphological characteristics, and phylogenetic analyses of combined LSU, ITS, SSU, TEF1-α, and RPB2 sequence data, two new species Distoseptisporabangkokensis and D.lancangjiangensis are introduced, and two known species D.clematidis and D.thysanolaenae were first reported in freshwater habitat. Illustrations and descriptions of these taxa are provided, along with comparisons with extant taxa in the genus.


Introduction
Distoseptisporaceae was introduced by Su et al. (2016) based on morphological and phylogenetic analyses, with Distoseptispora as type genus. Distoseptisporaceae is placed in Distoseptisporales, which was introduced by Luo et al. (2019), and currently comprises two families, Aquapteridosporaceae and Distoseptisporaceae Hyde et al. 2021). Species of both families are commonly reported from freshwater habitats (Yang et al. 2015(Yang et al. , 2018u et al. 2016;Li et al. 2021;Hyde et al. 2016aHyde et al. , 2019Hyde et al. , 2020Luo et al. 2018Luo et al. , 2019Song et al. 2020;Dong et al. 2021).
During our ongoing study of freshwater fungi along the north-south gradient in the Asian/Australian region (Hyde et al. 2016b), we collected four species in the genus. Two new species, Distoseptispora bangkokensis and D. lancangjiangensis, are introduced in this study, D. clematidis and D. thysanolaenae are newly recorded from freshwater habitats for the first time in China. Morphological descriptions and illustrations of the species and an updated multi-gene phylogenetic tree are provided to reveal their taxonomic position among the species in the Distoseptisporales, and also provided the comparison of morphological characteristics, habitats and hosts information of species newly added to Distoseptispora after Monkai et al. (2020) (Table 2).

Isolation and morphology
Specimens of submerged decaying wood were collected from Dulongjiang, Nanpanjiang, Lancangjiang and Chao Phraya River in China and Thailand respectively. Multiple samples will be collected at each collection site at different times, allowing more strains to be obtained for each species. Methods of morphological observation and isolation follow Luo et al. (2018) and Senanayake et al. (2020). IFW (Tarosoft(R) Image Frame Work) was used for measurement of photomicrograph, and Adobe Photoshop CS5 software was used to process images for making photo-plates (Adobe Systems Inc., USA). Single spore isolation was performed according to the following steps: The conidia suspension from specimens, absorbed with a sterilized pipette, was placed on potato dextrose agar (PDA) and incubated at room temperature overnight. Germinated conidia were transferred to new PDA/MEA (Beijing land bridge technology CO., LTD., China) plates and incubated in an incubator at room temperature (25 °C). Specimens were deposited in the Kunming Institute of Botany, Academia Sinica herbarium (KUN-HKAS), and Mae Fah Luang University herbarium (MFLU). Cultures were deposited in the Dali University Culture Collection (DLUCC), China General Microbiological Culture Collection Center (CGMCC), and Mae Fah Luang University Culture Collection (MFLUCC). Facesoffungi number was obtained as described in Jayasiri et al. (2015) and Index Fungorum number was also registered (http://www. indexfungorum.org/Names/Names.asp). In this study, multiple samples were collected for each sample site and related environment, but unfortunately, there were still no more strains for the two new species in the paper.
DNA extraction, PCR amplification, and sequencing DNA extraction, PCR amplification, sequencing and phylogenetic analysis follow Dissayanake et al. (2020) with the following modifications. Fungal mycelia (200-500 mg) were scraped from grown on PDA/MEA plates using sterile scalpel, transferred to microcentrifuge tube with sterilized needles, and then grind with liquid nitrogen or quartz sand to break the cells. DNA was extracted using the Trelief TM Plant Genomic DNA Kit (TSP101) according to the manufacturer's instructions.

Phylogenetic analysis
Preliminary identification of genes obtained from fresh strains by GenBank database. The LSU, ITS, SSU, TEF1-α, and RPB2 used for phylogenetic analysis are selected based on the preliminary identification results and the related publications (Yang et al. 2018;Monkai et al. 2020). The sequences were aligned using MAFFT online service: Multiple alignment program for amino acid or nucleotide sequences MAFFT version 7 (Katoh and Standley 2013: http://mafft.cbrc.jp/ alignment/server/index.html), and edited manually in BioEdit v. 7.0 (Hall 1999 (Miller et al. 2010: http://www.phylo.org/portal2) and the estimated proportion of invariant sites is (GTRGAMMA+I) model.
Bayesian analyses were performed in MrBayes 3.2.6 (Ronquist et al. 2012) and the best-fit model (LSU, ITS, SSU, TEF1-α, and RPB2 are all GTR+I+G) of sequences evolution was estimated via MrModeltest 2.2 (Guindon and Gascuel 2003;Nylander 2004;Darriba et al. 2012). The Markov Chain Monte Carlo (MCMC) sampling approach was used to calculate posterior probabilities (PP) (Rannala and Yang 1996). Bayesian analyses of six simultaneous Markov chains were run for 10000000 generations with trees sampled every 1000 generations.
Phylogenetic trees were visualized using FigTree v1.4.0 (Rambaut 2012: http:// tree.bio.ed.ac.uk/software/figtree/), editing and typesetting using Adobe Illustrator (AI) (Adobe Systems Inc., the United States). The new sequences were submitted in GenBank and the strain information used in this paper is provided in Table 1. The alignments and phylogenetic trees were deposited in TreeBASE (http://www.treebase. org/, accession number: 28758).

Phylogenetic analysis
The  Etymology. Referring to the collecting location, Bangkok, Thailand.
Culture characteristics. Conidia cultivated on PDA within 12h and germ tubes produced at the ends. Colonies on PDA, reaching 6 cm in 1 month at room temperature (25 °C). Mycelium loose, flocculent, smooth edge, brown to dark brown, dark brown on the reverse.
Culture characteristics. Conidia cultivated on PDA within 12h and germ tubes produced at the apex. Colonies on PDA, reaching 4.5 cm in 1 month at room temperature (25 °C). Mycelium loose, flocculent, smooth edges, convex middle, pale brown to dark brown on the surface of PDA. Smooth, black on the reverse.

Discussion
Distoseptispora has been reported from both freshwater and terrestrial habitats. Of these, species have been collected from freshwater environments (Su et al. 2016;Hyde et al. 2016aHyde et al. , 2019Hyde et al. , 2020Luo et al. 2018;Xia et al. 2017Xia et al. , 2019Yang et al. 2018;Tibpromma et al. 2018;Crous et al. 2019;Monkai et al. 2020;Phukhamsakda et al. 2020;Song et al. 2020;Sun et al. 2020;Li et al. 2021). To date, 18 species of Distoseptispora have been reported from Thailand, 14 species from China. In this study, we collected four distoseptispora-like taxa from rivers and streams in China and Thailand. Phylogenetic analysis showed that all four species were wellplaced in Distoseptispora (Figure 1). Two new species and records are introduced based on morphological and phylogenetic analysis.
Based on the key morphological characteristics, viz. conidiophores, conidiogenous cells, and conidia, Subramanian (1992) redisposed seven genera, viz., Sporidesmium, Polydesmus, Sporidesmiella, Stanjehughesia, Repetophragma, Penzigomyces, and Ellisem-bia to accommodate several Sporidesmium-like taxa. Based on multi-gene phylogenetic analysis and morphology, Su et al. (2016) introduced a new Sporidesmium-like genus Distoseptispora. Some Sporidesmium-like taxa were introduced in different lineages and synonymized Ellisembia under Sporidesmium. Although Distoseptispora was only introduced from submerged wood in freshwater habitat in 2016 (Su et al. 2016), the genus has previously been reported from both freshwater and terrestrial habitats as species in other genera. For example, Cai et al. (2002), Ho et al. (2001Ho et al. ( , 2002 and Luo et al. (2004) reported Distoseptispora as other species (Ellisembia, Sporidesmiella, and Sporidesmium) from submerged wood in freshwater habitats, and Kodsueb et al. (2016), Mena-Portales et al. (2016) and Zhou et al (2001) reported from terrestrial habitats. However, none of these records had molecular data and it is impossible to consider the placement of these isolates. In these species distoseptispora/sporidesmium-like genera, it is therefore better to describe taxa based on molecular data.
Based on phylogenetic analysis, Xia et al. (2017) transferred Acrodictys martinii to Distoseptispora as Distoseptispora martinii. The species is characterized by solitary erect, unbranched conidiophores, monoblastic conidiogenous cells with percurrent extensions and subhyaline to pale brown and solitary, transversal ellipsoid, oblate or subglobose, muriform conidia, separated by septa, sometimes with pores in the septa and pale brown to brown. However, the current understanding of Distoseptisporaceae, D. martinii is significantly different from other Distoseptispora taxa; thus, needs to be verified in the future (Luo et al. 2018;Sun et al. 2020).