Morphological and Phylogenetic Characterisations Reveal Four New Species in Leptosphaeriaceae (Pleosporales, Dothideomycetes)

Leptosphaeriaceae is a widely distributed fungal family with diverse lifestyles. The family includes several genera that can be distinguished by morphology and molecular phylogenetic analysis. During our investigation of saprobic fungi on grasslands in Yunnan Province, China, four fungal taxa belonging to Leptosphaeriaceae associated with grasses were collected. Morphological observations and phylogenetic analyses of the combined SSU, LSU, ITS, tub2, and rpb2 loci based on maximum likelihood and Bayesian inference were used to reveal the taxonomic placement of these fungal taxa. This study introduces four new taxa, viz. Leptosphaeria yunnanensis, L. zhaotongensis, Paraleptosphaeria kunmingensis, and Plenodomus zhaotongensis. Colour photo plates, full descriptions, and a phylogenetic tree to show the placement of the new taxa are provided.

Grasslands comprise a biome subjected to alternating drought, where grass and grasslike species dominate, and arboreous trees are uncommon [27]. In the grassland biome, several living organisms, such as herbivorous mammals, insects, and fungi (pathogenic, saprobic, and symbiotic), play essential roles in maintaining biomass and biodiversity [28]. Regarding fungi, a checklist of Ascomycetes on grasses was provided by Karunarathna et al. [29], which lists 3,165 fungal species. Studies of fungi on grasses include those of Thambugala et al. [30], Goonasekara et al. [31], and Brahmanage et al. [32].
This study describes four new Leptosphaeriaceae species that were collected from herbaceous plants in Kunming and Zhaotong, Yunnan Province, China. Phylogenetic analyses results based on SSU, LSU, ITS, tub2, and rpb2 loci, colour photo plates, complete descriptions of the four new species, and a summary of the morphological characteristics of Leptosphaeria, Paraleptosphaeria, and Plenodomus are provided.

Sample Collection, Isolation, and Identification
Herbaceous plants with fungal fruiting bodies were collected from Kunming and Zhaotong in Yunnan Province, China, stored in plastic bags, and returned to the mycology laboratory at the Kunming Institute of Botany. The samples were examined under an Olympus SZ-61 dissecting microscope (Tokyo, Japan). Fungal fruiting bodies were manually sectioned and mounted in double distilled water (ddH 2 O). Micro-morphological characteristics were captured using an OLYMPUS SZ2-ILST compound microscope connected to an Industrial Digital Camera 16NP USB3.0 (Panasonic, Osaka, Japan) microscope imaging system. Photo plates were processed using Adobe Photoshop CS6 Extended version 13.0.1 (Adobe Systems, San Jose, CA, USA). As described by Senanayake et al. [33], cultures were obtained via single-spore isolation and incubated under normal light at room temperature (25 • C). Germinating ascospores or conidia were observed under a stereo microscope and transferred to new potato dextrose agar (PDA) plates. Herbarium specimens were deposited in the herbarium at the Kunming Institute of Botany, Chinese Academy of Sciences (HKAS), and the Herbarium Mycologicum Academiae Sinicae, Beijing, China (HMAS), and living cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC). The Index Fungorum [24] and Faces of Fungi (FoF) [34] numbers were registered for the new species.

DNA Extraction, PCR Amplification, and DNA Sequencing
Genomic DNA was extracted from fresh mycelia grown on PDA at 28 • C for two weeks using the Biospin Fungus Genomic DNA Extraction Ki-BSC14S1 (BioFlux ® , Hangzhou, China) according to the manufacturer's protocol. The E.Z.N.A. Forensic DNA Kit-D3591 (Omega Biotek, Inc., Norcross, Georgia) was used to extract DNA directly from fruiting bodies. Polymerase chain reactions (PCRs) were carried out for five genetic markers: internal transcribed spacer region (ITS) [35], partial 28S large subunit nuclear ribosomal DNA (LSU) [36], partial small subunit ribosomal RNA (SSU) [35], β-tubulin (tub2) [37], and partial RNA polymerase second largest subunit (rpb2) [38]. The primers and amplification conditions used are listed in Table 1. The total volume of PCR mixtures for amplification was 25 µL, containing 8.5 µL ddH 2 O, 12.5 µL 2xF8FastLong PCR MasterMix (Beijing Aidlab Biotechnologies Co. Ltd., Beijing, China), 2 µL of the DNA template, and 1 µL each of reverse and forward primer (10 pM). The PCR products were sequenced by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd., Shanghai, China.
Maximum likelihood trees were inferred using RAxML-HPC2 in the XSEDE v. 8.2.12 [45] in CIPRES Science Gateway v. 3.3 online platform [46] under the GTRGAMMA nucleotide substitution model with 1000 bootstrap replicates. Bayesian inference analysis was conducted using MrBayes on XSEDE v. 3.2.7a [47], under the substitution model GTR + I + G for all loci, estimated by MrModeltest v. 2.3 [48] using PAUP v. 4.0b10 [49]. Six simultaneous Markov chains were run for 10,000,000 generations, with trees sampled every 1000th generation. The run was configured to stop when the standard deviation of split frequencies dropped below 0.01, and the first 25% of the trees were discarded as burn-in.
Tree topologies were visualised and exported using FigTree v. 1.4.0 [50]. The phylogram was edited and annotated using Microsoft Office PowerPoint 2016 (Microsoft Inc., Redmond, WA, USA) and Adobe Photoshop CS6 Extended version 13.0.1 (Adobe Systems, San Jose, CA, USA). Finally, the newly generated sequences were deposited in the GenBank database ( Table 2). Table 2. Names, Index Fungorum strain numbers, and corresponding GenBank accession numbers of the taxa used for phylogenetic analyses in this study.

Species Name
Strain Numbers GenBank Accession Numbers ITS LSU SSU tub2 rpb2    The decision as to whether the new species should be introduced followed the polyphasic guidelines of Chethana et al. [51] and Maharachchikumbura et al. [52].
Holotype: HKAS 124668 Culture characteristics: Ascospores germinated on PDA within 20 h, and a germ tube was initially produced from the middle. Colonies on PDA reaching 15 mm at two weeks at room temperature, circular, slightly raised, curled, floccose, pale yellow from above, dark brown in the centre, gradually pale yellow towards the edges from below, grows towards the filamentous edge.
Culture characteristics: Ascospores germinated on PDA within 20 h, and germ tube initially produced from both ends of the ascospores. Colonies on PDA reaching 20 mm at four weeks at room temperature, irregular, flat, centre is slightly raised, panniform, mycelium grows on the surface of PDA, white from above, brown in the centre gradually pale yellow towards the edges from below. Asexual spores and sexual spores were not formed on PDA within 60 days.   Notes: Plenodomus zhaotongensis is introduced as a new species based on its distinct morphology and phylogenetic analysis of combined SSU, LSU, ITS, tub2, and rpb2 sequence data. Plenodomus zhaotongensis is closely related to Pl. agnitus strains with 91% ML and 1.00 BYPP statistical support (Figure 1) [13,55]. Therefore, based on the guidelines for new species boundaries delimitation [51,52], we introduce Pl. zhaotongensis as a novel taxon.

Discussion
In this study, we introduce four new species, viz. L. yunnanensis, L. zhaotongensis, Pa. kunmingensis, and Pl. zhaotongensis, associated with grasses from Zhaotong and Kunming in Yunnan Province, southwestern China, based on polyphasic approaches [56,57] through multilocus analyses of five gene loci (SSU, LSU, ITS, tub2, and rpb2) and their morphological characteristics. Although Leptosphaeria is a speciose genus with 1682 species epithets, many are likely to belong to other genera [2] and need recollecting and sequencing. However, in speciose genera, many novel taxa can still be found [58], as in this study.
Paraleptosphaeria and Leptosphaeria have similar morphologies. In our study, Pa. kunmingensis fits within the generic concept of Paraleptosphaeria and is phylogenetically closely related to Pa. macrospora (Figure 1), whereas L. yunnanensis and L. zhaotongensis clustered distinctly in Leptosphaeria ( Figure 1). All species differ in morphology (Figures 2, 3 Table S2). Using molecular data, Piątek et al. [20] also showed that Paraleptosphaeria and Leptosphaeria are phylogenetically distinct. We also described Pl. zhaotongensis based on the morphological characteristics ( Figure 5) and molecular phylogeny ( Figure 1). Nevertheless, in Plenodomus, it is challenging to have well-resolved species delimitation because many species lack molecular data and detailed morphological descriptions [5,21]. Therefore, in future studies, precise morphological characteristics, molecular data, and phylogenetic analyses should be provided for all newly introduced and existing Plenodomus species.
The four described species in this study were collected from climate-contrasting grasslands in Yunnan Province. In Zhaotong, subtropical and warm temperate zones coexist, with an annual average temperature of 12.6 • C [68,69], while Kunming has distinct wet and dry seasons [70,71]. In terms of fungi, many new fungal species have been reported in Yunnan Province in the last two decades. More than 1300 new fungal species have been described, accounting for nearly 25% of the total fungal species described in China [72].
This scenario is consistent with Hyde et al. [66,67], who stated that continued exploration of new environments would result in undescribed taxa.
In Yunnan's grasslands, many grass species are the primary source of carbohydrates and feed for livestock, and fungi play a pivotal role in maintaining and shaping grass communities. Each grass-associated fungal community is responsible for specific ecological properties of the environment [29]. Our study fills some gaps in the research on Leptosphaeriaceae species in grasslands by providing detailed information on four new species from China and insights into the number of grass-associated Leptosphaeria. In addition, fungi have never been reported in Zhaotong City; thus, future studies are needed to reveal the actual fungal diversity, especially those associated with grasses.