Morpho-Phylogenetic Evidence Reveals Novel Pleosporalean Taxa from Sichuan Province, China

Pleosporales is the largest and most morphologically diverse order in Dothideomycetes, including a large proportion of saprobic fungi. During the investigation of microfungi from decaying wood in Sichuan Province, several novel fungal taxa of asexual and sexual morphs were collected, identified, and well-described. Phylogenetic analyses based on SSU, ITS, LSU, RPB2 and TEF1α gene sequences suggested that these new taxa were related to Pleosporales and distributed in five families, viz. Amorosiaceae, Bambusicolaceae, Lophiostomataceae, Occultibambusaceae and Tetraplosphaeriaceae. The morphological comparison and molecular phylogeny evidence justify the establishment of six new taxa, namely Bambusicola guttulata sp. nov., Flabellascoma sichuanense sp. nov., Neoangustimassarina sichuanensis gen. et sp. nov., Occultibambusa sichuanensis sp. nov. and Pseudotetraploa bambusicola sp. nov. Among them, Neoangustimassarina was introduced as the second sexual morph genus in Amorosiaceae; Bambusicola guttulata, O. sichuanensis and P. bambusicola were isolated from bamboos, which contributed to the diversity of bambusicolous fungi. The detailed, illustrated descriptions and notes for each new taxon are provided, as well as a brief note for each family. The potential richness of fungal diversity in Sichuan Province is also discussed.


Introduction
Pleosporales is the largest order in the class Dothideomycetes [1], and its members are found worldwide on a variety of host plants as epiphytes, endophytes, saprobes and parasites [2][3][4]. In addition, they are commonly found in terrestrial, marine and freshwater habitats [5][6][7]. Some of them produce secondary metabolites that can serve as a basis for developing new antimicrobials, agrochemical pesticides and other useful compounds [8].
Fungi have a broad geographical distribution and diversity comparable to plants and other organisms [17,18]. However, the fungal kingdom, in general, is less well-documented than the plant kingdom in terms of the number of species [19]. As one of the biodiversity hotspots in China, Sichuan Province (located in southwestern China) has a variety of

Phylogenetic Analyses
Notes: The monotypic genus was introduced to accommodate Neoangustimassarina sichuanensis, which formed a distinct clade within Amorosiaceae (Figure 1). Neoangustimassarina resembles Angustimassarina in forming globose to subglobose ascomata, hyaline, and septate ascospores surrounded by mucilaginous sheaths [38]. However, Neoangustimassarina differs from the latter in having immersed ascomata without a pore opening, broader asci (broad clavate to cylindric-clavate vs. cylindrical to cylindric-clavate), and the septa of the ascospores (1-septate vs. 1-3-septate). We, hereby, introduce the new genus based on the distinctiveness of morphology and multi-gene phylogeny.
Lophiostomataceae Sacc., Sylloge Fungorum 2: 672 (1883) Notes: Nitschke [53] introduced "Lophiostomeae" based on the type species of Lophiostoma macrostomum (Tode) Ces. and De Not. Saccardo formally established the family Lophiostomataceae and placed "Lophiostomeae" in the order Pleosporales [54]. Members of this family have crest-like ostioles in most cases and easily to be recognized. They are characterized by immersed to erumpent ascomata, mostly clavate asci, hyaline to dark brown ascospores with appendages or mucilaginous sheaths [38,55]. With the continuous increase of new members, the family currently comprises 30 genera [16]. A new species added to the genus Flabellascoma is identified and described.

MycoBank: MB 843719
Etymology: The epithet refers to Sichuan Province where the fungus was collected.
Culture characteristics: Colonies on PDA reaching 40-50 mm after 7 weeks at 25 • C, circular, with dense mycelium on the surface, dark grayish of the inner ring, and brown of the outer ring; in reverse black of the inner ring, and brown of the outer ring. Notes: The phylogenetic result based on SSU, ITS, LSU, RPB2 and TEF1α sequence data showed that the new collection Flabellascoma sichuanense nested in Flabellascoma (Figure 1) and formed a distinct lineage. Morphologically, it fits well with the genus Flabellascoma in having immersed ascomata, bitunicate, fissitunicate, cylindrical, clavate asci and fusiform, hyaline, 1-septate ascospores with a narrow bipolar sheath [56]. However, the dimensions of asci and ascospores distinguish F. sichuanense from other species (Table 3).  Notes: Species of Occultibambusaceae are mostly saprobic and frequently found on monocotyledons or hardwood trees in terrestrial and aquatic habitats [47,58]. Dai et al. [47] established this family to accommodate Neooccultibambusa, Occultibambusa, Seriascoma and Versicolorisporium. Brunneofusispora, typified by Brunneofusispora sinensis, was subsequently introduced to this family by Phookamsak et al. [59]. Phylogenetically, the coelomycetous genus Versicolorisporium appeared to be a close relationship with Occultibambusa in previous studies [21,52,60,61]. However, they continue to be treated as two distinct genera because the known asexual morph of Occultibambusa is different from Versicolorisporium [60]. In this study, a new Occultibambusa species is introduced.

MycoBank: MB 843720
Etymology: The epithet refers to Sichuan Province where the fungus was collected.
Culture characteristics: Colonies on PDA reaching 40-50 mm after 7 weeks at 25 • C, circular, with sparse mycelium on the surface, light gray of the inner ring, and brown of the outer ring; in reverse olive green.
Notes: The blast search based on LSU sequence data of our new collection showed that the closest hits were Versicolorisporium triseptatum (HHUF 28815 = JCM14775, identity 99.18%; NMX1222, identity 99.03%), and Occultibambusa bambusae (MFLUCC 11-0394, identity 98.01%); the closest hits based on ITS sequence were Versicolorisporium triseptatum (JCM 14775, identity 93.95%; NMX1222, identity 93.72%), and Occultibambusa hongheensis (KUMCC 21-0020, identity 90.95%); the closest hits based on TEF1α sequence were Occultibambusa hongheensis (KUMCC 21-0020, identity 97.02%), and O. maolanensis (KUMCC 21-0020, identity 96.91%). Multi-gene phylogeny showed that the new collection grouped with Occultibambusa and Versicolorisporium. It formed a sister clade with V. triseptatum with high statistical support (100% ML/1.00 BYPP, Figure 1). However, the morphology of our collection fits well with Occultibambusa. Further morphological evidence of its association with Versicolorisporium is somewhat difficult due to the lack of asexual morph in our collection. Therefore, we recognize our new collection as a new species of Occultibambusa, namely, O. sichuanensis. The morphological comparison of Occultibambusa species was listed in Table 4.    [64], and typified by Tetraplosphaeria. The latest taxonomic treatment of the family contains nine genera [1]. Pseudotetraploa is a genus with only known asexual forms, which were commonly associated with Poaceae (Dendrocalamus stocksii, Pleioblastus chino, Pleioblastus chino, Sasa kurilensis) distributed in Japan or India [64,65]. In this study, a new Pseudotetraploa species associated with bamboos from China is introduced.

MycoBank: MB 843721
Etymology: Refers to the bamboo host.
Holotype: HKAS 123092  type B. massarinia compared to the anamorphic species in the genus Bambusicola [43]. However, B. guttulata has broader conidia than that of B. massarinia (14-21 × 4-6 μm vs. 14-20 × 2-3 μm). The establishment of the new species B. guttulata is justified by morphological and phylogenetic evidence.  increase of new members, the family currently comprises 30 genera [16]. A new species added to the genus Flabellascoma is identified and described.  in previous studies [21,52,60,61]. However, they continue to be treated as two distinct genera because the known asexual morph of Occultibambusa is different from Versicolorisporium [60]. In this study, a new Occultibambusa species is introduced.

MycoBank: MB 843721
Etymology: Refers to the bamboo host.

Discussion
The genus Flabellascoma was introduced by Hashimoto et al. [56] to accommodate two terrestrial species F. cycadicola A. Hashim., K. Hiray and Kaz. Tanaka and F. minimum. Subsequently, two species F. aquaticum D.F. Bao, Z.L. Luo, K.D. Hyde and H.Y. Su and F. fusiforme D.F. Bao, Z.L. Luo, K.D. Hyde and H.Y. Su from freshwater habitats were introduced based on multi-gene phylogeny [57]. Members of Flabellascoma have similar morphological features [56,57] and it is difficult to distinguish Flabellascoma species by the size and shape of asci and ascospores [57]. Bao et al. [57] proposed that the ascomatal features appear to be remarkable features to distinguish taxa in this genus. Molecular data were found to be more supportive for the identification of the new species in this study, and we believed that DNA data provided more objective evidence for the species distinction of Flabellascoma.
In previous studies, the relationship between Occultibambusa and Versicolorisporium has not been well resolved due to the asexual morphs of Occultibambusa and Versicolorisporium being inconsistent [60]. In our phylogenetic tree, however, the genus Occultibambusa is not monophyletic (Figure 1), of which O. fusispora formed an independent lineage in Occultibambusaceae; this is consistent with recent relevant studies [60,61]. Occultibambusa fusispora is the only species in the genus reported with its holomorph [47]; we cannot solve the problem between Occultibambusa and Versicolorisporium due to the type-of-species issue, although O. fusispora has an asexual morph. Therefore, further studies are needed to provide sexual and asexual links of the type of species of O. bambusae and V. triseptatum towards the classification of Occultibambusa and Versicolorisporium with more sampling and taxa population included in the analysis.
During the investigation of microfungi in Sichuan Province, we randomly sampled three times in the vicinity of Chengdu city from July to August 2021. Morphological and phylogenetic results showed that these newly collected interesting taxa were distributed in five different pleosporalean families. It is worth noting that three new species found on bamboo are typical bambusicolous fungi. Bamboo is a gramineous plant with economic and ornamental value, and its culms and leaves are abundant in saprobic fungi [44,[66][67][68]. China has the richest bamboo resources, with a total of 861 species distributes in 43 genera [69]. Among them, Sichuan has a large area of bamboo forests, with an area of 592,800 ha, ranking fifth in the country after Fujian, Jiangxi, Zhejiang and Hunan [69]. In recent years, an increasing number of new species of bambusicolous fungi have been reported and discovered in China [60,67,70,71]. Therefore, the unique natural conditions in Sichuan are of great potential for the excavation and identification of bamboo fungi.