﻿Identification of two new species and a new host record of Distoseptispora (Distoseptisporaceae, Distoseptisporales, Sordariomycetes) from terrestrial and freshwater habitats in Southern China

﻿Abstract During our investigation of saprophytic fungi in Guizhou and Hainan provinces, China, three hyphomycetes were collected from terrestrial and freshwater habitats. Based on morphological characteristics and phylogenetic analyses of combined ITS, LSU, tef1-α, and rpb2 sequence data, two new species are introduced: Distoseptisporahainanensis and D.lanceolatispora. Additionally, one known species, D.tectonae, previously unreported from Edgeworthiachrysantha, is newly reported. Detailed descriptions, illustrations, and a phylogenetic tree to show the two new species and the new host record of Distoseptispora are provided. In addition, a checklist of Distoseptispora species with their locations, lifestyles, habitats, and hosts is provided.

In this study, three fresh hyphomycetous fungal collections were encountered during a microfungal investigation in Hainan and Guizhou provinces.Based on multi-gene phylogeny and morphological comparison, two new species, Distoseptispora hainanensis and D. lanceolatispora are introduced.In addition, a new host record of D. tectonae from Edgeworthia chrysantha is also reported.

Sample collection, isolation, and morphological study
Fresh specimens were collected from Hainan and Guizhou provinces in China.Fungal colonies were mounted on a slide with distilled water and were observed and examined using a stereomicroscope (SMZ 745, Nikon, Tokyo, Japan).Micro-morphological characteristics were captured with a Nikon EOS 90D digital camera combined with an ECLIPSE Ni-U compound microscope (Nikon, Tokyo, Japan).The sizes of the fungal structures were measured using the Tarosoft (R) Image Frame Work program (IFW 0.97 version), and the photo plates were processed with Adobe Photoshop CC 2019 (Adobe Systems, San Jose, CA, USA).
Single spore isolations were carried out following the methods described in Senanayake et al. (2020).Germinated conidia were transferred to fresh potato dextrose agar (PDA) plates and incubated at 25-27 °C for four weeks.Culture characteristics, including color, shape, and size, were recorded.Herbarium specimens were deposited in the herbarium of the Guizhou Academy of Agriculture Sciences (GZAAS), Guiyang, China, and the living cultures were deposited at the Guizhou Culture Collection, China (GZCC).Faces of Fungi and Index Fungorum numbers were obtained following the protocols outlined by Jayasiri et al. (2015) and Index Fungorum (2024), respectively.
Polymerase chain reaction (PCR) amplifications were carried out in a 50 µL reaction volume containing 44 μL of 1.1 × T3 Super PCR Mix (TsingKe Biotech, Chongqing, China), 2 µL of DNA template, and 2 µL of each forward and reverse primer.The amplification condition for LSU and ITS consisted of initial denaturation at 94 °C for 3 min, followed by 35 cycles of 45 s at 94 °C, 50 s at 56 °C, and 1 min at 72 °C, and a final extension period of 10 min at 72 °C.The amplification condition for the tef1-α gene consisted of initial denaturation at 94 °C for 3 min, followed by 30 cycles of 30 s at 94 °C, 50 s at 56 °C, and 1 min at 72 °C, a final extension period of 10 min at 72 °C.The amplification condition for the rpb2 gene consisted of initial denaturation at 95 °C for 5 min, followed by 35 cycles of 15 s at 95 °C, 50 s at 56 °C, and 1 min at 72 °C, a final extension period of 10 min at 72 °C.The quality of PCR amplification products was examined with 1% agarose electrophoresis gels stained with ethidium bromide, and the PCR products were sent to TsingKe Biotech, Chongqing, China for purification and sequencing.
The MP analysis employed 1,000 random taxa additions to infer trees.Branches of zero length were collapsed, and all multiple parsimonious trees were saved.The maxtrees value was set to 5,000.For trees generated using different optimal criteria, parsimony score values were determined for tree length (TL), consistency index (CI), retention index (RI), and homoplasy index (HI).To assess clade stability, the bootstrap (BT) method was used with 1,000 iterations, each consisting of 100 trials of random stepwise addition of taxa (Hillis and Bull 1993).
The posterior probabilities (PP) were determined based on Bayesian Markov chain Monte Carlo sampling (Huelsenbeck and Ronquist 2001).The best nucleotide substitution model for each data partition was determined using the program MrModeltest 2.2 (Nylander 2004).The GTR + I + G substitution model with gamma rates and Dirichlet base frequencies was selected for all LSU, ITS, tef1-α, and rpb2 sequences.To calculate the posterior probabilities, four simultaneous Markov chains were run for one million generations, with trees sampled every 100 th generation, resulting in a total of 10,000 trees.A burn-in parameter of 0.25 was set, indicating that 75% of the trees were remined during the burn-in phase, and the remaining trees were used for calculating the posterior probabilities in the majority rule consensus tree.
Culture characteristics.Colonies grown on PDA circular, dense, fluffy, with raised center and lobate edge, pale gray in the center, grayish brown in the outer ring from the front view, dark brown in the center, and blackish brown in the outer ring from the reverse view.
Culture characteristics.Colonies grown on PDA circular, dense, flat, dry, gray to dark gray, radially striated, and a ring in the middle of the colonies with an entire edge from the front view, dark brown to black with a circular, gray edge from reverse view, not pigmented.

Distoseptispora tectonae
Culture characteristics.Conidia germinating on PDA within 24 h, colonies circular, dense, umbonate, spreading, fluffy.The surface is slightly rough with reddish-gray mycelium, colonies somewhat raised in the middle, and with a filiform edge.The reverse side is dark gray with a circular, pale reddish-gray edge, not pigmented.

Discussion
Distoseptispora is one of the sporidesmium-like taxa and is well-known for its asexual morph, which has considerable morphological variations (Su et al. 2016;Yang et al. 2018Yang et al. , 2021)).However, the phylogenetic analyses suggest a lack of correlation between phylogenetic relationships and morphological analyses.1).In contrast, morphological analysis reveals significant differences, especially in the characteristics of conidiophores, conidiogenous cells, and conidia (Crous et al. 2019;Hyde et al. 2019;Luo et al. 2019;Dong et al. 2021;Yang et al. 2021;Zhang et al. 2022).This disparity is common within the genus.We recommend adopting a combination approach using molecular and morphological methods for more effective identification within this genus.
Worth noting, among the various species of Distoseptispora, D. martinii (J.L.Crane & Dumont) J.W. Xia & X.G.Zhang stands out due to its unique morphological characteristics, especially its oblate or subglobose conidia, distinguishing it from other species within Distoseptispora (Xia et al. 2017).The species was initially introduced as Acrodictys martinii J.L. Crane & Dumont by Crane and Dumont (1975) based on morphological characteristics.Then, it underwent several taxonomic revisions based solely on morphology (Baker et al. 2002;Delgado 2009).Later, Xia et al. (2017) reclassified Acrodictys martinii as D. martinii based on genetic analysis.However, the morphological traits of D. martinii greatly diverge from typical Distoseptispora features (Crane and Dumont 1975;Xia et al. 2017).Therefore, we suggest additional collections and analysis of D. martinii specimens to ensure the reliability of the provided DNA sequence data.
In recent years, Distoseptispora species have been reported worldwide, such as in China, Hungary, Hawaii, Malaysia, and Thailand (Shoemaker and White 1985;McKenzie 1995;Wu and Zhuang 2005;Zhang et al. 2022).Studies on Distoseptispora have been particularly extensive in China and Thailand (Hyde et al. 2016(Hyde et al. , 2019(Hyde et al. , 2020;;Su et al. 2016;Yang et al. 2018Yang et al. , 2021;;Luo et al. 2019;Sun et al. 2020;Hu et al. 2023).To date, 73 species of Distoseptispora have been documented, of which 55 have been recorded in China (including known species, see Table 2).Our collections further highlight the distribution of the genus in Distoseptispora K.D. Hyde, McKenzie & Maharachch.was introduced by Su et al. (2016) with D. fluminicola McKenzie, Hong Y. Su, Z.L. Luo & K.D. Hyde, as the type species.

Figure 1 .
Figure 1.Phylogenetic tree generated from ML analysis based on a combination of LSU, ITS, tef1-a, and rpb2 sequence data.Bootstrap support values of ML and MP equal to or greater than 75%, and PP value equal to or greater than 0.95 are given near the nodes as ML/PP/MP.The tree is rooted with Aquapteridospora aquatica (MFLUCC 17-2371).Ex-type strains are indicated by the superscript T. The new collections are in bold red text.
), D. lanceolatispora forms a unique clade adjacent to D. neorostrata D.F. Bao, Z.L. Luo & H.Y. Su with 100% ML, 1 PP, and 98% MP support.Based on a pairwise nucleotide comparison of ITS and LSU sequences, D. lanceolatispora deviates from

Table 1 .
Names, strain numbers, and corresponding GenBank accession numbers of taxa used in this study.
T " denotes ex-type strain.Newly generated sequences are indicated in black bold."N/A": no data available in GenBank.

Table 2 .
Distoseptispora species and their locations, lifestyles, habitats, hosts, and corresponding references.