Morphological Variety in Distoseptispora and Introduction of Six Novel Species

Distoseptispora is one of the sporidesmium-like taxa with great variation in asexual morphology and delineation of species. Phylogenetic analyses of four gene regions LSU, ITS, TEF1α, and RPB2 revealed the placement of several sporidesmium-like species in Distoseptispora (Distoseptisporaceae, Distoseptisporales, Sordariomycetes), collected on submerged decaying twigs from streams in China and Thailand. Based on morphological examination and molecular DNA data, six new species, Distoseptispora amniculi, D. atroviridis, D. effusa, D. fusiformis, D. hyalina, and D. verrucosa, are proposed. Among them, D. hyalina is the first sexual morph confirmed in the genus. A new geographical record is reported for D. lignicola in China. Conidial length proved to be of less taxonomic significance for some Distoseptispora species, whereas the type of conidial septa is informative at species level.

Distoseptispora is one of the sporidesmium-like genera introduced by Su et al. [9] with the type species D. fluminicola and the second species D. aquatica having long, cylindrical, distoseptate conidia. Yang et al. [10] emended the generic concept of Distoseptispora based on the morphological features of D. guttulata and D. suoluoensis, namely taxa with conspicuously longer conidiophores elongating percurrently and obclavate, rostrate, euseptate conidia. The generic circumscription was later expanded since more species with Germinated spores were grown on MEA/PDA medium at 25 • C for one month. Fungal mycelium was scraped off using a sterilized scalpel and transferred to a 1.5 mL microcentrifuge tube for genomic DNA extraction. A Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) was used to extract DNA following the manufacturer's instructions. DNA amplification was performed by polymerase chain reaction (PCR). LSU, SSU, ITS, TEF1α, and RPB2 gene regions were amplified using the primer pairs LR0R/LR5, NS1/NS4, ITS5/ITS4, 983F/2218R, and fRPB2-5F/fRPB2-7cR [31][32][33][34]. The amplifications were performed in a 25 µL reaction volume containing 9.5 µL ddH 2 O, 12.5 µL 2 × Taq PCR Master Mix with blue dye (Sangon Biotech, China), 1 µL of DNA template, and 1 µL of each primer (10 µM). The amplification condition for LSU, SSU, ITS, and TEF1α consisted of initial denaturation at 94 • C for 3 min, followed by 40 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 RPB2 gene consisted of initial denaturation at 95 • C for 5 min, followed by 37 cycles of 15 s at 95 • C, 50 s at 56 • C, and 2 min at 72 • C, final extension period of 10 min at 72 • C. Purification and sequencing of PCR products were carried out by Shanghai Sangon Biological Engineering Technology and Services Co., Shanghai, China.

Phylogenetic Analyses
The ex-type and additional strains of Distoseptisporaceae species and related families (Acrodictyaceae, Aquapteridosporaceae, Bullimycetaceae, Cancellidiaceae, Papulosaceae, and Pseudostanjehughesiaceae) were selected in the phylogenetic analyses (Table 1). Four gene regions LSU, ITS, TEF1α, and RPB2 were used for the multi-gene analyses. Sequences were optimized manually to allow maximum alignment and maximum sequence similarity. The sequences were aligned using the online multiple alignment program MAFFT v.7 (Available online: http://mafft.cbrc.jp/alignment/server/ (accessed on 3 August 2021)) [35]. The alignments were checked visually and improved manually where necessary. Maximum likelihood (ML), Bayesian inference (BI), and maximum parsimony (MP) analyses were employed to assess phylogenetic relationships. ML and BI analyses were performed through the CIPRES science Gateway V. 3.3 [36]. ML analyses were conducted with RAxML-HPC v. 8.2.12 [37] using a GTRGAMMA approximation with rapid bootstrap analysis followed by 1000 bootstrap replicates. For the BI approach, MrModeltest2 v. 2.3 [38] was used to infer the appropriate substitution model that would best fit the model of DNA evolution for the combined dataset. GTR + G + I substitution model was selected for LSU, ITS, TEF1α, and RPB2 partitions. BI analyses were performed in a likelihood framework as implemented in MrBayes 3.2.6 [39]. Six simultaneous Markov chains were run until the average standard deviation of split frequencies was below 0.01, with trees saved every 1000 generations. The first 25% of saved trees, representing the burn-in phase of the analysis, were discarded. The remaining trees were used for calculating posterior probabilities of recovered branches [40]. MP analyses were conducted with PAUP v. 4.0a167 [41]. A heuristic search was performed with the stepwise-addition option with 1000 random taxon addition replicates and tree bisection and reconnection branch swapping. All characters were unordered and of equal weight and gaps were treated as missing data. Maxtrees were unlimited, branches of zero length were collapsed, and all multiple, equally parsimonious trees were saved. Clade stability was assessed using a bootstrap analysis with 1000 replicates, each with 10 replicates of random stepwise addition of taxa [42].
The resulting trees were printed with FigTree v. 1.4.4 and the layout was created in Adobe Illustrator 2019 (Adobe Systems, San Jose, CA, USA). Sequences generated in this study were deposited in GenBank (Table 1).
Type species-Distoseptispora fluminicola McKenzie, H.Y. Su, Z.L. Luo, and K.D. Hyde Notes: Hyde et al. [11] provided the family description for the monotypic Distoseptisporaceae. The diagnosis of the sexual morph in the family was based on Miyoshiella triseptata, which was associated with "Distospetispora adscendens" (as Ellisembia adscendens) in the same collection [7,43]. However, neither the cultural study nor molecular data has proved their connection. We prefer to treat Miyoshiella triseptata as a possible sexual morph of sporidesmium-like taxa. The sexual description here is based on Distoseptispora hyalina. Distoseptispora martinii is unique in the genus by transverse ellipsoid or subglobose, muriform conidia [15]. Additional collections and further molecular evidence are needed to confirm its taxonomy.
Culture characteristics: conidia germinating on PDA within 24 h and swollen germ tubes produced from both ends. Colonies growing on PDA reaching 5-10 mm in two weeks at 25 • C in the dark, with dense, velvety, dark green mycelium on the surface; in reverse dark green with entire margin.
Distoseptispora hyalina were colonized close to D. amniculi on the same twig in sexual and asexual stages, respectively. However, they are separate taxa based on molecular evidence.
Distoseptispora Notes: Our collection GZAAS 20-0424 matches the original diagnosis of the holotype of Distoseptispora lignicola (MFLU 18-1458) well [13]. Comparison of their LSU and ITS sequences showed 100% and 99.43% (526/529bp) similarity, respectively. We therefore identify our two collections as D. lignicola and report a new geographical record of this species in China.

Discussion
The establishment of Distoseptispora [9] was based on morphology and molecular DNA data. More than 30 species in the genus are supported by sequence data. The genus forms monophyletic clade (Figure 1) distinct from other sporidesmium-like taxa. Members in the genus mainly occur in the asexual morph, forming effuse, hairy colonies on decaying wood, bamboo culms, plant stems, rachis, and fallen leaves from terrestrial and

Discussion
The establishment of Distoseptispora [9] was based on morphology and molecular DNA data. More than 30 species in the genus are supported by sequence data. The genus forms monophyletic clade (Figure 1) distinct from other sporidesmium-like taxa. Members in the genus mainly occur in the asexual morph, forming effuse, hairy colonies on decaying wood, bamboo culms, plant stems, rachis, and fallen leaves from terrestrial and freshwater habitats. The morphological concept of Distoseptispora is macronematous, mononematous, solitary or fasciculate conidiophores, sometimes elongating percurrently and rarely reduced to conidiogenous cells; monoblastic or polyblastic, cylindrical or clavate conidiogenous cells; conidia are cylindrical, obclavate, rostrate, ellipsoidal, obovoid or fusiform, subhyaline, olivaceous, dark green, brown or yellowish-brown to reddish-brown, euseptate or distoseptate, rarely muriform, sometimes born a secondary conidium, with or without septal pore and mucilaginous sheath. The characters delineating the genus Distoseptispora also cover the criteria of Ellisembia and Sporidesmium. Ellisembia was segregated from the widely circumscribed Sporidesmium by Subramanian [1] based on the septal type. However, given the non-taxonomic value of euseptate and distoseptate conidia among sporidesmium-like species, Su et al. [9] recognized the distoseptate Ellisembia as a synonym of the redefined Sporidesmium sensu stricto which forms a robust monoclade and accommodates species with distoseptate/euseptate, obclavate or subcylindrical conidia, and conidiophores with or without percurrent extensions. In this study, we follow Hyde et al. [12] in treating them as separate taxa because of their unclear relationship due to the absence of molecular DNA data from their type species.
Several sexual morphs have been linked to Ellisembia and Sporidesmium through cultural and/or molecular studies. Ellisembia folliculata (sexual morph: Lecythothecium duriligni) [50] and E. aurea [12] differ from the sexual morph D. hyalina by versicolorous ascospores and position within Chaetosphaeriaceae (Chaetosphaeriales, Sordariomycetes). Sporidesmium thailandense [10,48] and S. lignicola [13] can be distinguished by brown ascomata with a hyaline neck and compact, elongated cells of the ascomatal wall, outer layer undifferentiated from host tissue, and their systematic placement in Sporidesmiaceae (Sporidesmiales, Sordariomycetes). The morphology of sexual morphs of sporidesmium-like genera along with molecular DNA data characterizes their identification although they have similar asexual morphs.
It is challenging to identify some Distoseptispora species with highly similar morphology, such as those of D. multiseptata, D. phangngaensis, and D. xishuangbannaensis. Still, they can be well separated by molecular DNA data [10,16,18]. Some Distoseptispora species have a wide range of conidial length, for example, conidia of D. multiseptata are 95-290 µm long of the holotype but 300-700 µm long in the additional collection; those in D. phangngaensis 165-350 µm long and D. tectonigena 83-360 µm long [9,10,16]. The indeterminate conidial length may depend on the incubation period. Thus, conidial length is less taxonomically informative in separating some Distoseptispora species. The type of septa, however, is proven to have no taxonomic significance for generic delimitation of sporidesmium-like taxa, but it is informative at the species level [10,14].
Su et al. [9] accepted two former Ellisembia species E. adscendens and E. leonensis in Distoseptispora that were not validly published, based on the non-type strains "Distoseptispora adscendens" HKUCC 10820 and "D. leonensis" HKUCC 10822 lacking associated morphology. Ellisembia adscendens was initially introduced as Sporidesmium adscendens forming elongated black patches on the pileus of Polyporus versicolor Fr. (No. 1345) which was collected on the underside of timber in the Falkland Islands [51]. Ellisembia adscendens is a widespread species. It is similar to E. vaga [52] but differs by wider conidia. Ellisembia adscendens and E. vaga highly resemble a group of morphologically indistinguishable species in Distoseptispora, e.g., D. multiseptata and D. phangngaensis. They are probably members of Distoseptispora because of the typical Distoseptispora morphology in having short conidiophores and subcylindrical long conidia. On the other hand, the large number of specimens of Ellisembia adscendens may be a complex comprising several separate taxa.
Ellisembia leonensis [47] is characterized by relatively long conidiophores with percurrent extensions and fusiform to rostrate, distoseptate conidia. It matches the morphological concept of both Distoseptispora and Sporidesmium sensu stricto. At present, we avoid reassigning E. adscendens and E. leonensis in Distoseptispora until their systematic placement is confirmed by molecular data from type materials or resolved by epitypifications.