Multi-locus phylogenetic analyses reveal eight novel species of Distoseptispora from southern China

ABSTRACT Plant debris are habitat favoring the growth of various microbial species. Over the course of our mycological surveys in Jiangxi and Yunnan Provinces, China, eight new Distoseptispora species, viz. D. gasaensis, D. guanshanensis, D. jinghongensis, D. longnanensis, D. menghaiensis, D. menglunensis, D. nanchangensis, and D. yichunensis collected on dead branches of unidentified plants, were introduced by morphological and molecular phylogenetic analyses. Multi-locus (LSU, ITS, TEF1, and RPB2) phylogenetic analyses were performed using maximum-likelihood and Bayesian inference to infer their taxonomic positions within Distoseptispora. Both molecular phylogenetic analyses and morphological characters supported them as eight independent taxa within Distoseptispora. This work improves our understanding of the diversity of Distoseptispora in southern China. IMPORTANCE Distoseptispora as a single genus in Distoseptisporaceae was introduced by morphological and phylogenetic analyses. Members of this genus occur mainly as asexual morphs, forming effuse, hairy colonies on decaying wood, plant stems, bamboo culms, and fallen leaves and shafts in terrestrial and freshwater habitats. In the present study, saprobic hyphomycetes from plant debris were investigated, and eight new Distoseptispora species were introduced based on morphology and phylogenetic analyses of LSU, ITS, TEF1, and RPB2 sequence data. This study provides important data on the species diversity, ecological environment, and geographical area of Distoseptispora, greatly updates the classification of Distoseptispora, and improves our understanding of the taxonomy of Distoseptispora.

Distoseptispora is known as a saprobic fungal genus from woody hosts in aquatic and terrestrial habitats (11).Distoseptispora currently has 65 valid species, 44 of which are freshwater species and 21 are terrestrial species (14-17).Most Distoseptispora species are distributed in Asia, mainly in China (42 species) and Thailand (23 species) (4-7, 10, 14-32), while only a little published information is recorded in other regions (14, 15,18,33,34).Only two species, viz.D. hyalina and D. licualae are reported as sexual morphs based on multi-locus analysis (5,32).Distoseptispora species are not restricted to any particular host and are recorded in a variety of different plants, being the only genus in the Distoseptisporaceae, Distoseptisporales, Diaporthomycetidae, and Sordariomycetes (18).
With its complex geography, warm and humid climate, abundant light and rainfall, and numerous forested nature reserves, the southern regions of China have accumulated a rich resource of plant debris and countless taxa of dark sporulating fungi over the years.In a taxonomy and diversity survey of terrestrial fungi in Jiangxi and Yunnan Provinces, we collected 57 sporidesmium-like taxa.Based on multi-locus phylogenetic analyses and morphological evidence, eight Distoseptispora species were introduced as new to science in the present study.

Research Article
Microbiology Spectrum obpyriform, straight or slightly curved, smooth, 5-9-distoseptate, brown to pale brown, rostrate, tapering and paler toward the rounded apex, and truncate at the base.Culture characteristics: Colony on potato dextrose agar (PDA) reaching 81-86 mm diameter after 4 weeks in an incubator under dark conditions at 25°C, circular, with dense, gray mycelium in the middle, darker part of the inner ring, with sparser, white mycelium of the outer ring on the surface; reverse dark brown to black.
MycoBank number: MB849131 Etymology: In reference to the locality, Longnan County, where the fungus was collected.
Culture characteristics: Colony on PDA reaching 80-85 mm diameter after 4 weeks in an incubator under dark conditions at 25°C, irregularly circular, surface velvety, with gray-white and denser mycelium at the center, becoming black-brown and sparser toward the edge; reverse pale brown to black.
MycoBank number: MB849132 Etymology: In reference to the locality, Menghai County, where the fungus was collected.
Culture characteristics: Colony on PDA reaching 63-69 mm diameter after 4 weeks in an incubator under dark conditions at 25°C, circular, with dense, gray mycelium in the center, darker in the outer ring, with sparser, gray-white mycelium on the surface; reverse dark brown to black.
Culture characteristics: Colony on PDA reaching 76-85 mm diameter after 4 weeks in an incubator under dark conditions at 25°C, circular, surface velvety, dense, gray-brown mycelium with dark brown margin, reverse dark brown to black.

DISCUSSION
In recent years, the number of new taxa in the Distoseptispora has steadily increased, which have been discovered mostly in freshwater and some in terrestrial habitats.However, as terrestrial habitats are a vast habitat for saprobic fungi, there may be more species in this genus to be discovered and further research are needed to elucidate whether specific species in Distoseptispora are specific to their habitats.The establishment of Distoseptispora was based on morphology and phylogenetic stud ies.In all, 66 epithets for Distoseptispora have been listed in Index Fungorum (14), and of which 65 are accepted (15)(16)(17).Members of this genus occur mainly as asex ual morphs, forming effuse, hairy colonies on decaying wood, plant stems, bamboo poles, fallen leaves, and shafts from both terrestrial and freshwater habitats (5).The genus Distoseptispora (Distoseptisporaceae, Distoseptisporales) is mainly characterized by macronematous, mononematous, septate, unbranched, cylindrical, olivaceous to brown conidiophores; mono-or polyblastic, integrated, terminal, cylindrical, determinate or percurrently extending conidiogenous cells; and acrogenous, solitary, smooth or verruculose, euseptate or distoseptate conidia.However, Distoseptispora species show a high morphological similarity to Sporidesmium and Ellisembia with euseptate and distoseptate conidia, but it is hard to identify some Distoseptispora species by morpho logical features alone.Thus, with the availability of sequence data for Distoseptispora species, the introduction of molecular phylogenetic analyses led to a better understand ing of the species diversity of Distoseptispora (5,15,35,36).Studies conducted to date on Distoseptispora have no universally accepted standards in selecting loci for phylogenetic analyses (15).However, recent studies indicated that the use of LSU, ITS, TEF1, and RPB2 shows good phylogenetic resolution in resolving the phylogeny of Distoseptisporaceae (18,30).Other loci in the mitochondrial genome are a potential model for molecular evolutionary and phylogenetic studies and has been important for taxonomy, phyloge netics, and population genetics of fungi (37)(38)(39), but not a single mitogenome to date has been characterized in Distoseptispora.The characteristics of mitogenomes belonging to different representatives of Distoseptispora are needed to facilitate further investiga tions into the taxonomy, phylogenetics, conservation genetics, and evolutionary biology of this genus and other closely related species, to clarify the understanding of the evolution and potential technological uses of specific isolates of the genus.
Distoseptispora as a single genus in Distoseptisporaceae was introduced by Su et al. (6) with D. fluminicola as the type species based on morphological and phylogenetic analyses.The genus is known for its asexual morph, and only two sexual species, viz.D. hyalina and D. licualae are reported, but their related anamorph is still unknown (5,32).Morphology and especially the conidial shape in Distoseptispora species are highly diverse.Distoseptispora has shown convergent evolution as a member of sporidesmiumlike genera but has not been well represented in phylogenetic analyses to support morphological classification.A comparison of morphological characters and phyloge netic analysis of the various species in Distoseptispora reveals that the conidia of most species are obclavate to cylindrical or rostrate (e.g., D. aquatica, D. leonensis, and D. longispora), and a few are ellipsoidal to subglobose (e.g., D. martinii and D. atroviridis), and lanceolate (e.g., D. guttulata and D. multiseptata).However, these characteristics are not significantly correlated with the phylogenetic relationship.Combining the morphological characteristics and the phylogenetic evidence of all species in the genus Distoseptispora, we found that some Distoseptispora species are highly similar morpho logically, but can nevertheless be well separated by molecular DNA data.For example, conidia of D. multiseptata, D. phangngaensis, and D. xishuangbannaensis are all character ized as acrogenous, solitary, obclavate, multi-distoseptate (6,9,35); D. tropica and D. verrucosa also have an identical morphology of conidiophores and conidia (5,30), but there are have a distant phylogenetic relationship.In addition, there are a number of species that are sister clades in the phylogenetic tree but exhibit different morphological characters.For example, D. nabanheensis has obclavate, slightly constricted at the septa, brown to dark brown conidia, while D. clematidis has oblong, obclavate, cylindrical or rostrate, brown with green tinge conidia (15,40); D. longispora and D. longnanensis have a close phylogenetic relationship, but D. longispora has obclavate, rounded at the apex, truncate at the base, 31-56-distoseptate, brown to yellowish brown, slightly paler toward the apex, while D. longnanensis has obclavate, tapering toward the rounded apex, 4-8-septate, olivaceous to yellowish-brown or brown, becoming paler toward the apex, with a darkened scar at the base.Therefore, due to the unique phenomenon of Distoseptispora, it needs to be verified and extended in the future by morphological characterization and phylogenetic analyses.
Among the prevalent woody litter saprobes in terrestrial and freshwater ecosys tems, dematiaceous sporidesmium-like hyphomycetes are dominant (5).This genus is considered to be a saprobic lignicolous fungal genus that can decompose lignocellulose in wood and to participate in the decomposition and nutrient cycling of dead plant material in terrestrial and aquatic ecosystems (35,41).The diversity in a particular area or ecosystem is usually expressed as the number of species in the system, highlighting the fundamental role of taxonomy in biodiversity assessment and biology (42,43).However, knowledge of the role of the genus in decomposition and nutrient cycling, their geographical distribution, environment, host information, substrate specificity, and teleomorph relationships are relatively limited.Therefore, it is not possible to quantify their role in ecosystem functioning.The reports of Distoseptispora are mainly concentra ted in Thailand (Chiang Rai, Phitsanulok, Phang Nga) and China (Yunnan and Guizhou Province) (7,9,12), but three Distoseptispora species, D. meilingensis, D. yongxiuensis, and D. yunjushanensis, were found in Jiangxi, China (20), and D. septata, D. tropica, and D. wuzhishanensis were found in Hainan, China (30).In this paper, eight taxa were identified from terrestrial habitats in Jiangxi and Yunnan Provinces, China.After morphological comparison and phylogenetic analyses, it can be shown that these eight collections are new species of Distoseptispora, widening the diversity of the genus.Therefore, surveys of different geographical areas, ecological environments, and vegetation types are needed to help reveal the genus diversity, increase the fungal species number curve, and further clarify their taxonomic status through phylogenetic analyses.

Sample collection, isolation, and morphological observation
Samples of dead branches were collected randomly from humid environments and river banks in the forest ecosystems of Yunnan and Jiangxi Provinces, China, and returned to the laboratory in Ziploc plastic bags.Samples were processed and examined following the methods described in Ma et al. (44).Colonies on the surface of dead branches were examined and visually observed using a stereomicroscope (Motic SMZ-168, Xiamen, China) from low magnification (0.75 times) to high magnification (five times).Fresh colonies were picked with a sterile needle at 5× magnification under a stereomicroscope, placed on a slide with a drop of lactic acid-phenol solution (lactic acid, phenol, glycerol, sterile water; 1:1:2:2:1, respectively), and then placed under an Olympus BX 53 light microscope equipped with an Olympus DP 27 digital camera (Olympus Optical Co., Tokyo, Japan) for microscopic morphological characterization.The conidia of the target colony were captured directly from the specimen using the tip of a sterile toothpick dipped in sterile water.Conidia were then placed on the surface of PDA (20% potato + 2% dextrose + 2% agar, wt/vol) and incubated overnight in an incubator at 25°C.The single germinated conidia were transferred to fresh PDA plates according to the method of Goh (45) and incubated in an incubator at 25°C.Culture characteristics were examined and recorded after 3 days, followed by 3 days at regular intervals.Colony colors were assessed according to the charts of Rayner (46).All fungal strains were stored in 10% sterilized glycerin at 4°C for further studies.The studied specimens and cultures were preserved in the Herbarium of Jiangxi Agricultural University, Plant Pathology, Nanchang, China (HJAUP).The names of the new taxa names were registered in Index Fungorum (14).

DNA extraction, PCR amplification, and sequencing
Fungal hyphae were scraped from the surface of colonies grown on PDA plates, and genomic DNA was extracted using the Solarbio Fungal Genomic DNA Extraction Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer's protocol.DNA amplification was performed by polymerase chain reaction (PCR) using the respective loci (ITS, LSU, TEF1, and RPB2).Primer sets used for these genes were as follows: ITS: ITS5/ITS4 (47), LSU: 28S1-F/28S3-R (21), TEF1: EF1-983F/EF1-2218R (48,49), and RPB2: RPB2-5F2 (50)/fRPB2-7cR (51).The final volume of the PCR was 25 µL, containing 12.5 µL of 2 × Power Taq PCR MasterMix, 1 µL of each forward and reverse primer, 1 µL of DNA template, and 9.5 µL of ddH 2 O.The PCR thermal cycling conditions of ITS, LSU, and TEF1 were initialized at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 15 s, annealing at 54°C for 15 s, elongation at 72°C for 30 s, a final extension at 72°C for 10 min, and finally kept at 4°C.Regions of RPB2 were amplified with annealing at 59°C for 15 s, elongation at 72°C for 120 s, and others consistent with the above procedure.The PCR products were checked on 1% agarose gel electrophoresis stained with ethidium bromide.Purification and DNA sequencing were carried out at Beijing Tsingke Biotechnology Co., Ltd., Beijing, China.New sequences generated in this study were deposited in the NCBI GenBank (www.ncbi.nlm.nih.gov,Table 1).

Phylogenetic analyses
The newly generated sequences were aligned with other sequences obtained from GenBank (Table 1) using MAFFTv.7 (52) on the online server (https://maffth.cbrc.jp/alignment/server/, accessed on 19 April 2023) and manually optimized where required.Phylogenetic analyses were first performed for each locus separately, and then for a combined data set of four gene loci (ITS, LSU, TEF1, and RPB2).The tandem sequences for ITS, LSU, TEF1, and RPB2 were obtained by Phylosuite software v1.2.1 (53) under "Concatenate Sequence, " and missing sequence data in the comparisons were used as missing data with a question mark.Phylosuite software v1.2.1 (53) was used to construct the phylogenetic tree based on ITS, LSU, TEF1, and RPB2 tandem sequence  data.The concatenated aligned data set was analyzed separately using maximumlikelihood (ML) and Bayesian inference (BI).The best-fitting nucleotide substitution models for each alignment data set were selected using ModelFinder (54).Maximumlikelihood phylogenies were inferred using IQ-TREE ( 55) under an Edge-linked partition model for 10,000 ultrafast bootstraps (56).The final tree was selected among suboptimal trees from each run by comparing the likelihood scores using the TIM2e + I + G4 for ITS, TIM2 + F + R4 for LSU and TEF1, and TIM3e + I + G4 for RPB2 substitution model.Bayesian inference phylogenies were inferred using MrBayes 3.2.6 (57) under a partition model (two parallel runs, 2,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in.The best-fit model was GTR + F + I + G4 for ITS, LSU, and TEF1, and SYM + I + G4 for RRPB2.ModelFinder (54) was used to select the best fitting partition model (Edge-linked) using the BIC criterion to construct an IQ-TREE and using the AlCc criterion to construct a MrBayes.These trees were visualized using FigTree v.

FIG 1 FIG 2
FIG 1 Maximum-likelihood majority rule consensus tree for Distoseptisporaceae and related families using LSU, ITS, TEF1, and RPB2 sequence data.Bootstrap support values for maximum-likelihood greater than 70% and Bayesian posterior probabilities greater than 0.90 are shown near the nodes.The tree is rooted with Myrmecridium schulzeri (CBS 100.54) and M. banksiae (CBS 132536).The ex-type strains are in bold and the new isolates of this study are in red.Some branches were shortened according to the indicated multipliers.

FIG 3
FIG 3 Morphology of Distoseptispora guanshanensis (from holotype HJAUP M1063).(a) The surface of the colony after 4 weeks on PDA; (b) reverse of the colony after 4 weeks on PDA; (c) conidia; and (d and e) conidiophores, conidiogenous cells, and conidia.Scale bars (c to e), 40 µm.

FIG 7
FIG 7 Morphology of Distoseptispora menglunensis (from holotype HJAUP M2170).(a) The surface of the colony after 4 weeks on PDA; (b) reverse of the colony after 4 weeks on PDA; (c) conidia; and (d to g) conidiophores, conidiogenous cells, and conidia.Scale bars (c to g), 20 µm.

FIG 8
FIG 8 Morphology of Distoseptispora nanchangensis (from holotype HJAUP M1074).(a) The surface of the colony after 4 weeks on PDA; (b) reverse of the colony after 4 weeks on PDA; (c and d) conidia; and (e and f) conidiophores, conidiogenous cells, and conidia.Scale bars (c to f ), 20 µm.

FIG 9
FIG 9 Morphology of Distoseptispora yichunensis (from holotype HJAUP M1065).(a) The surface of the colony after 4 weeks on PDA; (b) reverse of the colony after 4 weeks on PDA; (c) conidiophores, conidiogenous cells, and conidia; and (d and e) conidia.Scale bars (c to e), 40 µm.

TABLE 1
Taxa used in the phylogenetic analyses and their GenBank accession numbers a,b,c November/December 2023 Volume 11 Issue 6 10.1128/spectrum.02468-2318

TABLE 1
Taxa used in the phylogenetic analyses and their GenBank accession numbers a,b,c (Continued) (Continued on next page) 1.4.4 (http:// tree.bio.ed.ac.uk/software/figtree, accessed on 19 April 2023), with editing and typeset ting using Adobe Illustrator CS v. 5.

TABLE 1
Taxa used in the phylogenetic analyses and their GenBank accession numbers a,b,c (Continued) The ex-type cultures are indicated using T after strain numbers; -stands for no sequence data in GenBank.b AFTOL: Assembling the Fungal Tree of Life; CBS: Central Bureau voor Schimmel cultures, Utrecht, The Netherlands; CGMCC: China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; CPC: Collection of P.W. Crous; DAR: Plant Pathology Herbarium, Orange Agriculture Institute, NSW, Australia; DLUCC: Dali University Culture Collection, Yunnan; GZCC: Guizhou Culture Collection China; HFJAU: Herbarium of Fungi, Jiangxi Agricultural University; HKUCC: The University of Hong Kong Culture Collection, Hong Kong, China; JAUCC: Jiangxi Agricultural University Culture Collection; KUMCC: Kunming Institute of Botany Culture Collection; KUN HKAS: Kunming Institute of Botany Academia Sinica, Yunnan, China; MFLU: the herbarium of Mae Fah Luang University, Chiang Rai, Thailand; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand.
a c Newly generated sequences are in bold.