﻿Diversity of Distoseptispora (Distoseptisporaceae) taxa on submerged decaying wood from the Red River in Yunnan, China

﻿Abstract The Red River Basin is located in the Indo-Burma biodiversity hotspot and is rich in lignicolous freshwater fungi, but no systematic research has been conducted. A systematic study on the species diversity of lignicolous freshwater fungi in the basin is ongoing. Seven distoseptispora-like specimens were collected from the Red River Basin in Yunnan. Phylogenetic analysis of ITS, LSU, tef1-α, and rpb2 genes and combined morphological data indicate that there are six distinct species of Distoseptispora, including two new species and four known species. Two new species were named D.suae and D.xinpingensis, and the four known species were D.bambusae, D.euseptata, D.obpyriformis and D.pachyconidia. This study provides detailed descriptions and illustrations of these six species and an updated phylogenetic backbone tree of Distoseptispora.

The Red River, one of the largest rivers in Southeast Asia, originates in Weishan County, Dali Bai Autonomous Prefecture, Yunnan Province, China, it has a total length of 1200 km with a catchment area of 169,000 km 2 (Haruyama 1995;Van den Bergh et al. 2007).Of this area, 50.3% is in Vietnam, 48.8% in China, and 0.9% in Laos (Van Maren 2007).The portion of the Red River Basin located in China is referred to as "Yuanjiang".This segment has a length of 677 km and is characterized by a plateau monsoon climate (Gu et al. 2018).Precipitation in the basin generally decreases from downstream to upstream and increases from the valleys to the mountains.The basin boasts a wealth of biological resources (Jiang 1980;Zhou and Cui 1997;Chen and Yu 2013;Gu et al. 2018).
Distoseptispora is a well-studied phylogenetic genus introduced by Su et al. (2016) to accommodate some Sporidesmium taxa with unbranched, olive green, cylindrical conidiophores, monoblastic, integrated, determinate, terminal, cylindrical conidiogenous cells, and acrogenous, distoseptate, cylindrical, smooth, darker conidia with slightly paler (but not hyaline), rounded apices of indeterminate length (Su et al. 2016;Yang et al. 2018Yang et al. , 2021;;Zhang et al. 2022).Based on morphological and phylogenetic analyses, 73 species have been accepted in Distoseptispora in recent years (http://www.indexfungorum.org/Names/Names.asp;accessed on 2 January 2024; Hu et al. 2023) (Yang et al. 2021;Konta et al. 2023).Members of Distoseptispora are primarily saprophytes found on woody substrates from freshwater habitats (45 species), predominantly in China and Thailand and some also have been found in terrestrial habitats (23 species); D. bambusae, D. clematidis, D. tectonae, D. thysanolaenae and D. xishuangbannaensis have been reported in both freshwater and terrestrial habitats (Hyde et al. 2016;Luo et al. 2018aLuo et al. , 2019;;Yang et al. 2018Yang et al. , 2021;;Monkai et al. 2020;Phukhamsakda et al. 2020;Sun et al. 2020;Dong et al. 2021;Li et al. 2021;Shen et al. 2021;Ma et al. 2022;Zhai et al. 2022;Zhang et al. 2022).The conidia of Distoseptispora vary significantly in their characteristics, especially in terms of shape and size.Zhang et al. (2022) reassessed both the generic and specific boundaries of Distoseptispora, and summarized the characteristics of species in this genus, encompassing various attributes such as the length of conidiophores, proliferation, and conidiogenesis in conidiogenous cells, and details about conidia, including their type (distoseptate or euseptate), number of septa, shape, length, color, proliferation, rostrate nature, and wall thickness.Even though Distoseptispora species formed three distinct clades in phylogenetic analysis results that received strong support, the morphological characters of these species (such as monoblastic/polyblastic, euseptate/distoseptate) only offer species-level differentiation and do not hold taxonomic significance for the broader categorization of Distoseptispora (Yang et al. 2021;Zhang et al. 2022).
A systematic investigation of lignicolous freshwater fungal diversity and distribution in the Red River Basin is ongoing.This study represents the first report of Distoseptispora species in the Red River Basin.A morphological examination combined with phylogenetic analysis combining internal transcribed spacer (ITS), large subunit nuclear ribosomal RNA (LSU), translation elongation factor 1-alpha (tef1-α) and second-largest subunit of RNA polymerase II (rpb2) sequence data, established that out of seven distoseptispora-like specimens collected in the Red River Basin, six species of Distoseptispora were identified, including two new species, named as D. suae and D. xinpingensis and four known species, viz.D. bambusae, D. euseptata, D. obpyriformis and D. pachyconidia.

Specimen collection, examination and isolation
Specimens of submerged decaying wood were collected from the Yuanjiang Basin (Red River) in Yunnan, China.The samples were incubated in a plastic box at room temperature for one week.Morphological observations were conducted following the methods of Luo et al. (2018a) and Senanayake et al. (2020) with a few modifications.Macromorphological characteristics of the samples were observed using an Optec SZ 760 compound stereomicroscope (Chongqing Optec Instrument Co., Ltd, Chongqing, China).Preliminary microscope slides were examined and photographed under a Nikon ECLIPSE Ni-U compound stereomicroscope (Nikon, Tokyo, Japan).Colonies' morphologies on native substrates were captured using a Nikon SMZ1000 stereo-zoom microscope (Nikon, Tokyo, Japan).The measurements of photomicrographs were obtained using Tarosoft (R) Image Frame Work version 0.9.7.Images were edited with Adobe Photoshop CS5 Extended version 12.0.0.0 software (Adobe Systems, San Jose, CA, USA).
Single spore isolations were carried out based on the method described by Luo et al. (2018a).The individually germinated conidia were transferred to fresh potato dextrose agar (PDA, from Beijing Bridge Technology Co., Ltd., Beijing, China) plates and incubated at room temperature in the dark.Some of the remaining germinated spores, along with their agar, were placed on water-mounted glass slides to photograph the origins of the germ tubes.
After observation and isolation, specimens were air-dried naturally, wrapped in absorbent paper, and stored in a ziplock bag with mothballs.These specimens were then deposited in the herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS), Kunming, China.The cultures were deposited with the China General Microbiological Culture Collection Center (CGMCC), and Kunming Institute of Botany Culture Collection (KUNCC).Fungal Names numbers are registered in the Fungal Names database (https://nmdc.cn/fungalnames/registe;accessed on 4 August 2023; Wang et al. 2023) and Facesoffungi numbers were obtained as described in Jayasiri et al. (2015).

DNA extraction, PCR amplification and sequencing
DNA extraction, PCR amplification, sequencing, and phylogenetic analysis were carried out following the methods described by Dissanayake et al. (2020).Mycelia used for DNA extraction were cultivated on PDA for 3-4 weeks at 24 °C.From each isolate, total genomic DNA was extracted from 100-150 mg of axenic mycelium, which was carefully scraped from the edges of the growing culture with a sterile scalpel.This material was transferred to a 1.5 mL microcentrifuge tube using sterilized inoculum needles.Mycelium was ground to a fine powder with liquid nitrogen or quartz sand to break the cells for DNA extraction.DNA was extracted with the TreliefTM Plant Genomic DNA Kit (TSP101) following manufacturer guidelines (Beijing Tsingke Biological Engineering Technology and Services Co., Ltd, Beijing, P.R. China).
Phylogenetic trees were visualized using FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/), editing and typesetting using Adobe Illustrator (AI) (Adobe Systems Inc., San Jose, CA, USA).The new sequences were submitted in GenBank and the strain information used in this paper is provided in Table 2.
Notes.Phylogenetic analysis showed that our new strain KUNCC 22-12668 clusters with the type strain of Distoseptispora bambusae (MFLUCC 14-0583) with 100% ML/1.00 PP support (Fig. 1).Furthermore, our new collection (Fig. 2) exhibits morphological characters identical to those of the type strain Distoseptispora bambusae (MFLUCC 14-0583).However, our collection has longer conidiophores and conidia.This observation aligns with Yang et al. (2018), suggesting that factors such as incubation time and habitat may influence the lengths of conidiophores and conidia.A comparison of the ITS, LSU, tef1-α and rpb2 sequences between our new strain KUNCC 22-12668 and the type strain (MFLUCC 14-0583) reveals only minimal base pair differences.Therefore, based on morphological evidence and phylogenetic affinity, our new strain KUNCC 22-12668 is identified as Distoseptispora bambusae and it is reported from freshwater habitat for the first time in Yunnan, China.
Culture characteristics.Conidia germinating on PDA within 12 hrs and germ tubes produced from apex of conidium.Colonies growing on PDA reaching 4-5 cm in 20 days at 26 °C in the dark, with dense, velvety, pale brown to dark brown mycelium from above, dark brown from below.

Distoseptispora obpyriformis
Culture characteristics.Conidia germinating on PDA within 12 hrs and germ tubes produced from apex and septa of conidium.Colonies growing on PDA reaching 4-5 cm in 20 days at 26 °C in the dark, with dense, velvety, middle papillae, pale to dark brown mycelium from above; dark brown from below.
Culture characteristics.Conidia germinating on PDA within 12 hrs and germ tubes produced from apex and septa of conidium.Colonies growing on PDA reach 2-3 cm in one month at 26 °C in the dark, with dense, velvety, pale brown to dark brown mycelium from above; dark brown from below.
Notes.Phylogenetically, our new strain KUNCC 23-13048 grouped with the strains of Distoseptispora pachyconidia (KUMCC 21-10724 and GZCC 22-0074) with 75% ML and 0.96% PP support (Fig. 1).Pairwise comparison of ITS, LSU, tef1-α and rpb2 sequences show negligible base pair differences.As previously reported, the conidia size and color of our new collection HKAS 125824 are significantly different from those originally described for D. pachyconidia (137-246 µm vs. 42-136 µm; pale brown to brown vs. pale-brown with a green tinge), as well as the number of conidial septa (14-45-distoseptate vs. 8-21-distoseptate) (Yang et al. 2018;Shen et al. 2021;Ma et al. 2022).Our new collection is also slightly different from the collection described by Ma et al. (2022), especially the number of conidial septa (14-45-distoseptate vs. up to 38-distoseptate) (Ma et al. 2022).However, based on slight differences in molecular data, this collection was not sufficient to qualify as a new species, and therefore, identify this collection as D. pachyconidia, which was first discovered in the Red River Basin of Yunnan.Etymology."suae" (Lat.) in memory of the Chinese mycologist Prof. Hong-Yan Su (4 April 1967-3 May 2022), who kindly helped the authors in many ways.
Culture characteristics.Conidia germinating on PDA within 24 hrs and swollen germ tubes produced from both ends and some septate.Colonies growing on PDA reaching 2-3 cm in two weeks at 26 °C in the dark, with dense, velvety, dark brown mycelium on the surface; in reverse brown to dark brown with entire margin.

Discussion
Systematic research on lignicolous freshwater fungi is ongoing in the Red River Basin.Seven distoseptispora-like species were discovered from submerged decaying wood.Based on multigene phylogenetic analysis and morphological studies, six Distoseptispora species were identified, D. suae and D. xinpingensis were introduced as new species with their unique morphology and phylogenetic placement.Previously introduced species, D. bambusae, D. euseptata, D. obpyriformis and D. pachyconidia were reported in the watershed for the first time.The Red River Basin may contain more interesting, particular, and undiscovered freshwater fungal species, as no studies have been reported on yet.
In the past seven years, more than 70 Distoseptispora species have been introduced based on morphological and molecular evidence.These species grow as saprophytes on a variety of decaying wood debris in tropical and subtropical freshwater and terrestrial habitats (Index Fungorum database; Hu et al. 2023).45 species have been reported on submerged bamboo stems and unknown wood debris in freshwater habitats, and 23 species have been reported on dead leaves, branches, and stems of various plants in terrestrial habitats, such as palms (Hyde et al. 2019), bamboo (Monkai et al. 2020), grasses (Hyde et al. 2023), and unknown broad-leaved trees (Hu et al. 2023), etc., and five species have been reported in both terrestrial and freshwater habitats (Hyde et al. 2016;Tibpromma et al. 2018;Phookamsak et al. 2019;Phukhamsakda et al. 2020;Sun et al. 2020;Shen et al. 2021;Ma et al. 2022;Zhang et al. 2022).China and Thailand are the countries that contribute the most Distoseptispora species, with 50 species reported in China and 25 species reported in Thailand.
Species of Distoseptispora are usually distinguished based on phylogenetic analysis combined with the morphological characteristics of conidiophores and conidia (Su et al. 2016;Monkai et al. 2020;Zhang et al. 2022).Important morphological characteristics are the color, shape, size, septation type (distoseptate/euseptate) and number of conidia, as well as the length of the conidiophores (Zhang et al. 2022).Phylogenetic studies of Distoseptispora are usually based on ITS, LSU, tef1-α, and rpb2 gene loci, and currently, all type species are well resolved on the phylogenetic tree (Hu et al. 2023;this study).Several previous studies have shown that new specimens of some species of Distoseptispora are significantly different in morphology from original descriptions, especially in the color and size of the conidia (Yang et al. 2018;Luo et al. 2019;Shen et al. 2021;Ma et al. 2022).These new specimens are usually collected from different habitats from the type specimens, and sometimes from the same habitat (Yang et al. 2018;Luo et al. 2019;Shen et al. 2021;Ma et al. 2022).However, the ITS, LSU, tef1-α and rpb2 sequences of these new specimens are not significantly different from the type specimens (Yang et al. 2018;Shen et al. 2021;Ma et al. 2022).Habitat and incubation time may affect the size of conidia, but this has not yet been determined and needs to be resolved in future studies (Yang et al. 2018;Shen et al. 2021;this study).Additionally, the brand and photography mode of the compound microscope may affect the color of the conidia.Of course, another possibility is that the four loci currently used to construct the phylogenetic analysis are not enough to provide more information to explain the morphological differences; combining more loci or a whole-gene phylogenetic study may explain these morphological differences.
The multigene phylogeny indicates that members of Distoseptispora are distributed in four distinct clades.However, there are no pronounced morphological differences sufficient to separate them (Zhang et al. 2022).Morphologically, Distoseptispora martinii stands apart from other species within the genus due to its ellipsoid, oblate or subglobose and muriform conidia.These characteristics align more closely with the general concept of Junewangia.Therefore, the phylogenetic placement of D. martinii requires further examination through nucleotide base sequence analysis.In subsequent studies, the culture of D. martinii (CGMCC 3.18651) could be encouraged to sporulate to determine if similar conidiophores and conidia are produced.Notably, in our study, the ex-type strain of D. suae (KUNCC 22-12476) produced conidiophores and conidia that mirrored those observed on the natural substrate, confirming this approach as promising.

Figure 1 .
Figure1.Maximum likelihood (ML) tree is based on combined LSU, ITS, tef1-α and rpb2 sequence data.Bootstrap support values with a ML greater than 65% and Bayesian posterior probabilities (PP) greater than 0.95 are given above the nodes, shown as "ML/PP".The tree is rooted to Aquapteridospora fusiformis (MFLUCC 18-1606) and A. lignicola (MFLUCC 15-0377).New species are indicated in blue and type strains are in bold.

Table 1 .
PCR thermocycling conditions for genes used in this paper.

Table 2 .
Taxa used in the phylogenetic analyses and their corresponding GenBank accession numbers.