Polyphasic Identification of Distoseptispora with Six New Species from Fresh Water

Twelve new specimens of sporidesmium-like taxa were collected from freshwater habitats in China and Thailand. Phylogenetic analysis of nuc 28S rDNA (LSU), internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1-α) and second-largest subunit of RNA polymerase II (RPB2) sequence data, combined with morphological data, revealed that they are Distoseptispora species. Among them, six new species, including D. aqualignicola, D. aquamyces, D. crassispora, D. curvularia, D. nonrostrata and D. pachyconidia, are introduced. Two new combinations, D. adscendens and D. leonensis, are transferred from Ellisembia. A new habitat and geographical record are reported for D. clematidis, collected from a freshwater habitat in China. New RPB2 sequence data for D. dehongensis are provided.

With increasing molecular data, multi-locus phylogenetic relationships between Sporidesmium and Ellisembia species have been investigated to gain a better understanding of their taxonomy, and it turned out that these species were distributed in different subclasses in Sordariomycetes, including Diaporthomycetidae, Hypocreomycetidae, Sordariomycetidae and Xylariomycetidae [5,7]. Therefore, some previously described species were revised and transferred to newly established genera, such as Pseudosporidesmium. The newly collected sporidesmium-like taxa were placed in new genera, such as Pseudostanjehughesia and Distoseptispora, and Ellisembia was synonymized with Sporidesmium [7].
Recently, Yang et al. [24] used multi-locus analysis and reported, for the first time, a sexual morph in Distoseptispora, namely, D. hyalina, which was characterized by an immersed to semi-immersed, subglobose to ellipsoidal, dark-brown perithecial with a short neck; a relatively thick peridium; sparse, persistent, hyaline paraphyses; cylindrical asci with non-amyloid apical annuli; and hyaline, filiform, 0-3-septate ascospores with mucilaginous sheaths. To date, it is the only known sexual morph in Distoseptispora. The hosts of Distoseptispora species are diverse, including Carex sp., Pandanus sp. and Tectona grandis, as well as bamboo [3,18,19]. Distoseptispora is the only genus in Distoseptisporaceae, Distoseptisporales, Diaporthomycetidae and Sordariomycetes.
In this study, 12 fresh specimens of sporidesmium-like taxa were collected from freshwater habitats in China and Thailand and were preliminarily identified as Distoseptispora species. In order to clarify the classification of these specimens, an updated phylogeny of Distoseptisporales and their relatives is provided based on a concatenated nuc 28S rRNA (LSU)-internal transcribed spacer (ITS)-translation elongation factor 1-alpha (TEF1-α)second-largest subunit of RNA polymerase II (RPB2) dataset. Six new species and two new combinations are introduced in this study based on phylogenetic analyses combined with morphological data. The characters of species are discussed.

Sample Collection, Morphological Studies and Isolation
In this study, decaying wood samples were collected from various regions of Thailand (Chiang Rai Province) and China (Sichuan and Yunnan Provinces). Decaying wood samples were place in zip-lock plastic bags with sterile wet cotton. The locations, collectors, dates of collection and countries were recorded for all collections, and they were taken to the laboratory for detailed observation and the isolation of pure cultures.
Fungal structures were observed and examined using a Nikon SMZ-171 dissecting microscope. Micro-morphological characters were examined using a Nikon ECLIPSE Ni compound microscope and photographed with a Canon 600D digital camera fitted to the microscope. The Tarosoft (R) Image Frame Work program and the Oplenic program were used to measure the fungal structures. Adobe Photoshop CS6 extended v. 13.0 software (Adobe Systems, San Jose, CA, USA) was used to process images. Single-spore isolation was obtained following the method described in Chomnunti et al. [26]. Germinated spores were aseptically transferred to potato dextrose agar (PDA) and incubated at 20-25 • C for 2-4 weeks. Colony characteristics were recorded, including color and shape. Dry specimens were deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand, and the Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China. Living cultures were deposited in the Mae Fah Luang University Culture Collection (MFLUCC) and the Kunming Institute of Botany Culture Collection (KUMCC). Facesoffungi and Index Fungorum numbers were acquired according to Jayasiri et al. [27] and Index Fungorum (http://www.indexfungorum.org/names/names.asp, accessed on 15 March 2021), respectively.

DNA Extraction, PCR Amplification and Sequencing
Fresh fungal mycelia were scraped off with a needle and transferred into a 1.5 mL centrifuge tube. A Plant genomic DNA extraction kit (generic) (TreliefJM, Kunming, China) was used to extract DNA from fresh mycelia, according to the manufacturer's instructions.
DNA amplification for LSU, ITS, TEF1-α and RPB2 genes was performed by polymerase chain reaction (PCR). The primers and PCR thermal cycle programs are shown in Table 1. PCR products were confirmed on 1% agarose electrophoresis gels stained with ethidium bromide. PCR products were sent to TsingKe Biological Technology (Kunming, China) for purification and sequencing.

Phylogenetic Analyses
The raw sequences were initially checked with Finch TV v. 1.4.0, and each gene was subjected to a BlastN search in NCBI's GenBank to assess the confidence level. Sequences of Distoseptisporaceae species, along with the newly generated sequences and related taxa, following Dong et al. [9] and Phukhamsakda et al. [25], were downloaded from GenBank. The gene sequences were aligned using the online multiple alignment program MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html, accessed on 7 April 2022) [32] and manually improved, where necessary, in BioEdit v. 7.2 [33], then concatenated with Mesquite v. 3.11.
Maximum likelihood (ML) and Bayesian inference (BI) methods were used for the phylogenetic analyses. Maximum likelihood analysis was performed with RAxML-HPC v. 8 on XSEDE in the CIPRES Science Gateway (https://www.phylo.org/portal2/home.action/, accessed on 8 April 2022) [34,35], with the following changes made to the default settings: maximum hours to run: 10; model for bootstrapping phase: GTRGAMMA; analysis type: rapid bootstrap analysis/search for best-scoring ML tree (-f a); bootstrapping type: rapid bootstrapping (-x); bootstrap iterations: 1000 (the maximum value allowed).
For BI analysis, the best model for each gene was selected using MrModelTest 2.3 [36], and GTR+I+G was selected as the best-fitting model for LSU, ITS TEF1-α and RPB2. BI analysis was conducted using MrBayes 3.2.6 for posterior probabilities (PPs) by Markov chain Monte Carlo sampling (BMCMC) [37,38]. Six simultaneous Markov chains were run for one million generations, and trees were sampled every 100 generations. The first 2500 trees, representing the burn-in phase of the analyses, were discarded and the remaining trees were used for the calculation of PPs in the majority rule consensus tree (the critical value for the topological convergence diagnostic was 0.01). The phylogenetic tree was visualized in FigTree v. 1.4.2 [39] and edited in Adobe Illustrator CS6 (Adobe Systems, USA). Newly generated sequences were submitted to GenBank. All of the sequences used in this study are listed in Table 2.   The ex-type cultures are indicated using " T " after strain numbers; newly generated sequences are indicated in bold. "-" stands for no sequence data in GenBank.

Phylogenetic Analyses
The combined LSU, ITS, TEF1-α and RPB2 dataset was analyzed for species of Distoseptisporales and their relatives, using Myrmecridium banksiae (CPC 19852) and Myrmecridium schulzeri (CBS 100.54) as outgroup taxa. The dataset consists of 94 strains with an alignment length of 3743 total characters. The RAxML analysis resulted in a best-scoring likelihood tree selected with a final ML optimization likelihood value of −36376.671888. The matrix had 1984 distinct alignment patterns, with 35.27% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.241400, C = 0.263989, G = 0.281489, T = 0.213122; substitution rates AC = 1.230237, AG = 3.213547, AT = 1.297497, CG = 0.852333, CT = 6.671646, GT = 1.000000; gamma distribution shape parameter α = 0.254430. The phylogenetic trees generated from BI analyses were topologically similar to the one generated via the ML analyses, and the latter is shown in Figure 1.

Taxonomy in Distoseptispora
Distoseptispora adscendens (Berk.) R. Zhu & H. Zhang, comb. nov. (Figure 2).  Notes: Sporidesmium adscendens was synonymized as Ellisembia adscendens on the basis of pseudoseptate conidia and conidiophores with irregular percurrent proliferation [13]. An ascomycetes, Miyoshiella triseptata (≡Lasiosphaeria triseptata), collected in Hungary, was found to be associated with S. adscendens in the host of Platanus orientalis and therefore reported as its sexual morph [40,41]. Shenoy et al. [42] reported a strain, HKUCC 10820, of E. adscendens with LSU and RPB2 sequence data. The strain was later proved to phylogenetically cluster in the genus Distoseptispora by Su et al. [7] and was therefore named Distoseptispora adscendens in their phylogenetic tree but not validly introduced as a new combination. Many subsequent authors followed them in using the name D. adscendens [5,8,17,23].
Culture characteristics: Conidia germinating on PDA within 24 h. Germ tubes produced from the conidial apex. Colonies on PDA reaching 34 mm diameter after 22 days at 20-25 • C, circular, dry, dark-brown to black on surface and reverse, raised, with entire margin.
Culture characteristics: Conidia germinating on PDA within 24 h. Germ tubes produced at end of conidia. Colonies on PDA reaching 40 mm diameter after 45 days at 20-25 • C, circular, flat, surface rough, grey from above, dark-brown from below, edge entire.
Culture characteristics: Conidia germinating on PDA within 24 h. Germ tubes produced from the two sides of the conidia. Colonies on PDA reaching 40 mm diameter after 40 days at 20-25 • C, circular, flat, rough surface, grey from above, dark-brown from below, edge entire.
Notes: Distoseptispora bambusae was initially collected from culms of bamboo by Sun et al. [22] in Guizhou, China, and Chiangrai, Thailand. The morphological characters and size of our new collection are consistent with the holotype, except for the wider range of conidial length ((29-)43-94(-105) µm vs. 45-74 µm). Phylogenetically, the new collection clusters with D. bambusae, with strong support (100% MLBS/1.00 BIPP; Figure 1). Our isolate was collected from dead, submerged, decaying wood, which constitutes a new habitat record. Culture characteristics: Conidia germinating on PDA within 24 h. Germ tubes produced from the conidial base. Colonies on PDA reaching 34 mm diameter after 3 weeks at 20-25 • C, brown with sparse mycelium from above, dark-brown from below, rough surface, dry, flat, entire at edge.
Culture characteristics: Conidia germinating on PDA within 24 h. Germ tubes produced from the two sides of the conidia. Colonies on PDA reaching 20 mm diameter after 20 days at 20-25 • C, circular, raised, velvety, aerial, medium-sparse, dark-brown from both above and below.
Culture characteristics: Conidia germinating on PDA within 24 h. Germ tubes produced from the conidial base. Colonies on PDA reaching 30 mm in diameter after 20 days at 20-25 • C, circular, raised, convex or dome-shaped with dark-brown papillate surface, brown at the margins, dark-brown in reverse.
A comparison of sequence data for these two species showed a difference of 15 (2.81%, no gaps) noticeable nucleotides in ITS gene regions. Therefore, we introduce D. curvularia as a new species.  (Figures 9 and 10).
Material examined: Thailand, Mukdahan Province, small river of Nong Bo Na Kae, found on dead, submerged, decaying wood of unidentified plants, 13   , cluster with ex-type strain KUMCC 18-0090 of D. dehongensis, with strong support (100% MLBS/1.00 BIPP; Figure 1). Morphologically, the two new isolates fit well with the characters of D. dehongensis in having monoblastic or polyblastic conidiogenous cells and obpyriform to obclavate, straight or curved conidia with fewer than ten distosepta [45], except that the conidia of MFLU 19-0544 and MFLU 17-1671 show some percurrent proliferation, which is not observed in the holotype [45]. Additionally, the conidia of MFLU 19-0544 are pale-brown, while those of the holotype and MFLU 17-1671 are olivaceous. Comparisons of sequence data between the three strains showed no differences in LSU genes and three to six nucleotide differences in ITS genes. The holotype was collected from Yunan Provice, China [45], while our isolates were collected from Thailand, their locations constituting new geographic records. We provide new RPB2 sequence data for D. dehongensis in this study. Notes: The case of Distoseptispora leonensis is the same as that of D. adscendens, that is, it is the only strain of D. leonensis (HKUCC 10822) reported by Shenoy et al. [42], with LSU and RPB2 sequence data, which has been proven to phylogenetically cluster in the genus Distoseptispora [7] and has been named as D. adscendens in the phylogenetic tree, though it has not been validly introduced as a new combination. The morphology of strain HKUCC 10822 was also referred to Wu and Zhuang [14] (P137), who described it as having macronematous, mononematous, cylindrical, straight or slightly flexuous, smooth, 5-7-septate, mid-brown to brown conidiophores 110-130 µm long, 6-8 µm wide; integrated, terminal, cylindrical, smooth, pale-brown to brown conidiogenous cells 5-15 × 4.5-5 µm in size; and acrogenous, obclavate, fusiform or ellipsoidal, rostrate, 8-10-distoseptate conidia, 50-75 µm long, 15-18 µm wide at the broadest part, with conical-truncate basal cells 3-4.5 µm wide at the base. This description is consistent with the description and illustration of the holotype of S. leonensis given in Ellis [11], even regarding the sizes of conidiophores and conidia, though the conidiophore and conidial proliferations are points of difference. The above characters fit the generic features of Distoseptispora well.  , acrogenous, solitary, oblong, obclavate or narrowly obpyriform, mostly non-rostrate, rarely rostrate, straight or curved, 4-10-distoseptate, truncate at the base, smooth, thick-walled, pale-olivaceous or pale-brown.
Culture characteristics: Conidia germinating on PDA within 24 h. Germ tubes produced from conidial bases. Colonies on PDA reaching 30-35 mm diameter after 5 weeks at 20-25 • C, circular, raised, fluffy, dense, convex or dome-shaped, with dark-brown papillate surface, brown in the center, lighter on the outside, dark-brown at the margins, consistently dark-brown in reverse.
Culture characteristics: Conidia germinating on PDA within 24 h. Germ tubes produced from both ends. Colonies on PDA reaching 35-38 mm diameter after 5 weeks at 20-25 • C, dark-brown at the margins, greenish-glaucous at the center, olivaceous-brown in reverse, with smooth margin.
Notes: Distoseptispora rayongensis was introduced by Hyde et al. [47] and collected from a freshwater habitat in Rayong Province, Thailand. In our phylogenetic analysis, our new isolate was grouped with D. rayongensis, with strong support (99% MLBS/1.00 BIPP; Figure 1). Morphologically, our isolate was the same as D. rayongensi, except that it had shorter conidiophores (62-76 µm vs. 75-125 µm). Additionally, in the holotype, conidia showed percurrent proliferation and conidia forming from the conidial apices, which was not observed in our isolate. Comparisons of nucleotides between the holotype and our isolate showed no differences in ITS and TEF1-α regions and 1 and 35 (3.5%, including 21 gaps) nucleotide differences in LSU and RPB2 regions, respectively.
Distoseptispora tectonae Doilom & K.D. Hyde, Fungal Diversity 80: 222 (2016) (Figure 15). thicker region in the middle of septa, while the distoseptate condition is characterized by sac-like septa, usually narrower in the middle of septa or exhibiting cracks, as in D. clematidis, D. crassispora and D. nonrostrata (indicated by the arrows in Figures 6f, 7d and 12g in this paper).
According to Figure 1, most species in Clades 1 and 2 have distoseptate conidia, just like the generic type D. fluminicola. The species with euseptate conidia are all grouped in Clade 3, except for D. guttulata and D. martinii. Distoseptispora martinii was first described as Acrodictys martinii, then was transferred to Junewangia and Rhexoacrodictys [48][49][50]. Recently, it was proved to be a new combination in Distoseptispora on the basis of phylogenetic analysis [16]. Morphologically, it has ellipsoid, oblate to subglobose, muriform conidia instead of obclavate to obpyriform, transversely distoseptate conidia. Distoseptispora uttulate is another exception in Distoseptispora, as it has 11-14-euseptate conidia. Distoseptispora lignicola was described as euseptate, but it can be seen from the illustrations that it is euseptate only at the rostra. At the main part of the conidia, it should be distoseptate, due to the obvious narrowing in the middle of the septa [8] (Figure 5g,h). Distoseptispora rayongensis has the same characteristics, such that its septa should be recognized as distoseptate ( [47], Figure 99k; this study, Figure 10g). Distoseptispora meilingensis is the only species in Clade 3 that has distoseptate conidia.
(3) Type of conidial wall-thick or thin, smooth or verrucose All species in Clades 1 and 2 have thick-walled conidia, except for D. guttulata and D. martini, which have thin-walled conidia. The species with euseptate and thin-walled conidia are all grouped in Clade 3, except for D. meilingensis, which has thick-walled conidia. Distoseptispora bambusae and D. euseptata were described as thick-walled, with wall thicknesses less than 1 µm-thicker than those in D. Suoluoensis and D. Lancangjiangensis but not as thick as the ones in Clades 1 and 2 (ca. 2-4 µm). Thus, rather than thickwalled, it is more proper to call them medium-thick-walled. All Distoseptispora species have smooth conidial walls, except for D. aquamyces, D. suoluoensis and D. verrucose, which are verrucose-walled.
(4) Conidial shapes and color Conidial shapes in Clades 1 and 2 are mostly obclavate, sometimes obpyriform, oblong, subcylindrical/cylindrical, rarely fusiform or ellipsoid/oblate/subglobose, while those in Clade 3 are obclavate. D. euseptata also has obpyriform conidia. Conidial color in most species is brown with a green tinge, and a few have a single color. For example, in Clade 1, conidia are brown in D. tectonigena and D. longispora, and olivaceous green in D. multiseptata. Conidial color may vary within a species. For example, in D. tectonae, conidia are brown in the holotype (MFLU 15-3417) and specimen MFLU 20-0262 and olivaceous in specimen MFLU 15-2693. Another example is D. dehongensis: conidial color is pale brown in specimen MFLU 19-0544 and olivaceous in the holotype and specimen MFLU 17-1671.
(5) The length of conidiophores and conidia, and numbers of septa The lengths of conidiophores in the three clades are irregular, including 0 (maximum < 100 µm), 1 (minimum < 100 µm and maximum > 100 µm) and 2 (minimum > 100 µm). The conidial lengths of species in Clade 1 are relatively long, mostly longer than 100 µm, even up to 700 µm. Therefore, they have more conidial septa-many more than 20 and even up to 80. The conidial lengths of species in Clade 3 are relatively short, all mostly shorter than 100 µm. Conidial length may vary within a species, as in D. multiseptata. The conidial lengths of collection MFLU 15-1144 (mostly 300-600 µm long, up to 700 µm long) are significantly longer than those of the holotype (up to 380 µm long) [5]. Yang et al. [5] suspected that the length of conidia may depend on the length of incubation.
(6) Conidial rostrateness Conidia are described as rostrate when they have a beak-like extension or appearance, according to Dictionary of the Fungi (9th edition), where "beak" means a pointed part on the tip (https://dictionary.cambridge.org, accessed on 8 May 2022). Given this definition, we re-checked all species from the published illustrations and listed those with rostrate conidia in Figure 1. Species with rostrate conidia that were not reported as such in their original descriptions are labeled in red. From Figure 1, we can see that most species possess rostrate conidia. Species with only non-rostrate conidia are D. atroviridis, D. dehongensis, D. fusiformis, D. hydei, D. martinii and D. saprophytica.
(7) Conidial proliferation Conidial proliferation has been described in Clade 1 (D. amniculi, D. bangkokensis, D. tectonigena, D. rayongensis, D. yunjushanensis) and Clade 3 (D. suoluoensis). Distoseptispora saprophytica was thought to produce catenate conidia occasionally [9]. However, it is more proper to use the term conidial proliferation, because the upper conidia were obviously immature [9] (Figure 14g). Although no conidial proliferation was reported in D. xishuangbannaensis and D. thailandica, we can see obvious proliferation in their illustrations ( [18], Figure 65c,d and Figure 64c). Conidial proliferation is not a species-delimiting factor because an individual species may exhibit variation in this character, as in D. clematidis, D. dehongensis and D. multiseptata. These species were not reported as being characterized by conidial proliferation with respect to their holotype, though proliferation was reported in the new collections ( [18], Figure 3; this study, Figures 3 and 7). To sum up, no significant morphological differences were found among Clades 1, 2 and 3. Therefore, there is not enough evidence to segregate Clades 2 and 3 from Clade 1, in which the type species D. fluminicola is included, although Clades 1, 2 and 3 all received strong support (100% MLBS/1.00 BIPP; Figure 1). Conidial differentiation on the basis of euseptate and distoseptate does not receive molecular support, as such differences are found in both Clades 1 and 3. This is the same as the situation for euseptate/distoseptate conidia in Sporidesmium. Subramanian [13] segregated the species with distoseptate conidia from Sporidesmium and introduced the genus Ellisembia to accommodate them. However, Su et al. [7] proved that the euseptate/distoseptate difference between Sporidesmium and Ellisembia does not have molecular support and proposed Ellisembia as a synonym of Sporidesmium. Yang et al. [24] also inferred that euseptate/distoseptate differences have no taxonomic significance for generic delimitation of sporidesmium-like taxa, though they are informative at the species level.