﻿Unveiling fungi associated with Castanopsis woody litter in Yunnan Province, China: Insights into Pleosporales (Dothideomycetes) species

﻿Abstract During a survey of the diversity of lignicolous fungi in Yunnan Province, China, we collected and identified five microfungi species from dead woody litters of Castanopsis trees in terrestrial habitats. Through both morphological comparisons and phylogenetic analyses of multi-gene sequences, we identified two taxa as new species and three collections as new host records within Pleosporales. Pseudolophiostomalincangensesp. nov. is introduced as a sexual morph in Lophiostomataceae, Pleopunctumbaoshanensesp. nov. is introduced as a hyphomycetous fungi in Phaeoseptaceae, and Paraphomaaquatica as a first report of sexual morph in Paraphoma. In addition, Occultibambusakunmingensis and Pleopunctummegalosporum were isolated for the first time from the dead twigs of Castanopsisdelavayi and C.calathiformis, respectively. Comprehensive morphological descriptions, illustrations, and phylogenetic analysis results are provided for the above-mentioned species.


Introduction
Castanopsis is an evergreen tree belonging to Fagaceae and represents one of the largest genera with approximately 134 species, predominantly distributed across tropical and subtropical regions of Asia (Tan et al. 2023).Notably, 58 species are native to China, with 30 being endemic.Castanopsis species are valued for their timber and edible nuts, contributing significantly to the economy (Huang et al. 1999;Tang et al. 2005).Fungal diversity associated with Castanopsis has been extensively documented across various countries, including China, Korea, India, Indonesia, Japan, Nepal, Papua New Guinea, Thailand, and the United States (Crawford et al. 1987;Tang et al. 2005;Duong et al. 2008;Osono et al. 2020;Jayawardena et al. 2020).The presence of fungi on Castanopsis trees has been widely introduced, with approximately 360 records worldwide representing 220 species across 35 different Castanopsis species (Duong et al. 2008).Tang et al. (2005) reported 38 fungal taxa during a study on decaying leaves of Castanopsis fissa in Hong Kong, China highlighting the rich fungal diversity within this genus.These fungi encompass endophytes and saprobes found on different parts of Castanopsis trees, such as ectomycorrhizal, woody branches, fallen trunks, bark, and leaves (Inácio et al. 2005;Gao et al. 2016;Hyde et al. 2016;Ren et al. 2022;Pang et al. 2023).
Yunnan Province, located in southwestern China, boasts significant biological diversity attributed to its complex topography, highly variable climate, and lush vegetation (Feng and Yang 2018).This region covers an extensive area of 394,000 square with approximately 94% comprising mountainous terrain (Asian Development Bank 2012).Over the past decade, there has been a surge in interest in studying microfungi in Yunnan Province, with numerous studies focusing on leaf litter fungi and lignicolous freshwater fungi (Cai et al. 2002;Luo et al. 2018;Hapuarachchi et al. 2019;Dong et al. 2020).Recent discoveries have unveiled several new taxa from Dothideomycetes and Sordariomycetes inhabiting woody litter in terrestrial habitats, such as Diatrypaceae, Didymosphaeriaceae, Hermatomycetaceae, Hysteriaceae, Monoblastiaceae and Phaeoseptaceae (Maharachchikumbura et al. 2021;Mortimer et al. 2021;Ren et al. 2022;Wanasinghe and Mortimer et al. 2022).However, many studies lack proper identification and phylogenetic data, underscoring the need to re-evaluate various species in this region (Mortimer et al. 2021).
Pleosporales was established by Barr (1987) and is recognized as the largest order within the class Dothideomycetes, constituting a quarter of all species (Wijayawardene et al. 2022).This order has a remarkable diversity, comprising 91 families and 614 genera (Wijayawardene et al. 2022).Taxonomically, pleosporalean taxa exhibit versatility in ecological niches, being found as epiphytes, endophytes, parasites, hyperparasites, lichenized organisms, or saprobes across a wide range of habitats worldwide (Wanasinghe et al. 2020;Yang et al. 2023).Studies have highlighted the discovery of numerous new pleosporalean species from freshwater, marine, and terrestrial environments (Brahmanage et al. 2020).Morphologically, the sexual morph of Pleosporales is characterized by perithecial ascomata, typically with a papillate apex, ostiolate, cellular pseudoparaphyses, and bitunicate asci.The asexual morphs encompass both coelomycetes and hyphomycetes (Hongsanan et al. 2020).
The present study aims to describe two novel fungal species and three new host records collected from dead woody litter in Baoshan and Lincang of Yunnan Province, China.This involves morphological illustrations and multi-gene phylogenetic analyses utilizing ML and BI methods to confirm the phylogenetic placement.The study aims to contribute to fungal diversity and ecology in Yunnan Province while providing valuable insights into the taxonomy and phylogenetics of woody litter fungi.

Sample collection, observation, and isolation
Decayed woody samples were collected from mixed forest areas in China (Yunnan Province) during the rainy season (July) and brought to the laboratory in separate zip-lock plastic bags.Specimens were examined using a stereomicroscope (Olympus SZ61, Tokyo, Japan).Micro-morphological characteristics were photographed using a Canon EOS 600D (Tokyo, Japan) digital camera mounted on a Nikon ECLIPSE 80i (Tokyo, Japan) compound microscope.All microscopic measurements were taken using the Tarosoft (R) Image Frame Work v.09, and the measurements were reported as minimum-maximum values and average values.Images were processed with Adobe Photoshop CS6 software v.13 (Adobe Systems, San Jose, CA, USA).Single-spore isolation was used to obtain pure cultures, following the methods described by Ren et al. (2022).Herbarium materials were deposited at the Herbarium of Cryptogams Kunming Institute of Botany, Academia Sinica (HKAS), Kunming, China, and living cultures were deposited at the Kunming Institute of Botany Culture Collection (KUNCC), Kunming, China.Faces of fungi (Jayasiri et al. 2015) and Index Fungorum (2024) numbers were obtained for the new taxa.

Phylogenetic analyses
Sequences exhibiting high similarities (>90%) were identified through BLASTn searches to determine the closest match to the taxa.Representative sequences were individually blasted and the initial results from BLASTn searches show our five taxa belong to Pseudolophiostoma in Lophiostomataceae, Occultibambusa in Occultibambusaceae, Pleopunctum in Phaeoseptaceae, and Paraphoma in Phaeosphaeriaceae.Thus, four different datasets were prepared and analysed in this study based on recent publications (Gomzhina et al. 2020;Phukhamsakda et al. 2020;Magaña-Dueñas et al. 2021;Guarnaccia et al. 2022;Yu et al. 2022;Xu et al. 2023)  The newly generated sequences are indicated in bold.T refers to ex-type strains, and NA refers to "no data in GenBank".
The newly generated sequences were assembled by BioEdit 7.2.3 (Hall 1999).
The individual gene regions were separately aligned in the MAFFT v.7 web server (http://mafft.cbrc.jp/alignment/server/)(Katoh et al. 2019).The alignments of each gene were improved by manually deleting the ambiguous regions plus gaps and combined using BioEdit 7.2.3.The final alignments were converted to NEXUS format (.nxs) using Clustal X version 1.81 (Thompson et al. 1997) and processed for Bayesian and maximum parsimony analysis.The FASTA format was changed into PHY format via the Alignment Transformation Environment (ALTER) online program (http://www.sing-group.org/ALTER/)and used for maximum likelihood (ML) analysis.
Analyses 3, Phaeoseptaceae phylogeny, was based on combined SSU, LSU, ITS, tef1-α, and rpb2.The final alignment contained 3,195 characters used for the phylogenetic analyses, including alignment gaps.The alignment contained  37 strains, and the tree was rooted with Angustimassarina acerina (MFLUCC 14-0505) and A. populi (MFLUCC 13-0034).The RaxML analysis of the combined dataset yielded a best-scoring tree with a final ML optimization likelihood value of -12325.441217.The matrix had 882 distinct alignment patterns with 30.9% undetermined characters or gaps.The estimated base frequencies were as follows: A = 0.249369, C = 0.247445, G = 0.266516, T = 0.236670; substitution rates AC = 1.406931,AG = 4.076513, AT = 1.212607,CG = 1.303216,CT = 8.242374, GT = 1.000000; gamma distribution shape parameter α = 0.201687 and tree-length = 3.128705.The tree topologies of combined sequence data  2023).Bootstrap values for ML are equal to or greater than 75%, and posterior probability values are equal to or greater than 0.95 from the BYPP analysis labelled on the nodes.Strains of the newly described species are in red, while type strains are in bold.The tree is rooted with Angustimassarina acerina (MFLUCC 14-0505) and A. populi (MFLUCC 13-0034).

Taxonomy
The present study introduces two new species and three new records.These taxa belong to the order Pleosporales and are described below.
Culture characteristics.Ascospores germinated on PDA within 24 h at room temperature (25 °C).Germ tubes produced from the apical or the second cell of ascospore.Colonies on PDA, reaching 25 mm diameter after two weeks at 20-25 °C, mycelia superficial, circular, flat, fimbriate, undulate edge, gray with white gray at the center; reverse, dark green, pale yellow at the center.
Culture characteristics.Ascospores germinated on PDA within 24 h at room temperature (25 °C).Germ tubes produced from the basal and apical cells of ascospore.Colonies on PDA, reaching 25 mm diameter after 2 weeks at 20-25 °C, mycelia superficial, dense, circular, raised, entire edge, velvety, flossy; reverse black.Known host, habitats, and distribution.Bamboo and Castanopsis delavayi from freshwater and terrestrial in China (Dong et al. 2020;Jiang et al. 2021;this study).
Notes.According to the multi-gene phylogenetic analyses of combined SSU, LSU, ITS, tef1-α, and rpb2 sequence dataset, our new isolate (KUNCC 21-0506) nested with Occultibambusa kunmingensis (HKAS 102151), which was isolated from decaying bamboo submerged in freshwater in China (Dong et al. 2020) with 100% ML and 1.00 BYPP bootstrap support (Fig. 2).Our new isolate fits well with the morphological characteristics of the holotype of O. kunmingensis in having semi-immersed to superficial ascomata with short papillate, clavate or cylindric-clavate asci, fusiform, 1-septate, brown ascospores without mucilaginous sheaths and appendages (Dong et al. 2020).Sequence comparison for the ITS and tef1-α region between our isolates (KUNCC 21-0506) and O. kunmingensis (HKAS 102151) showed no significant base pair differences.Therefore, we identified our taxon as a new host record of O. kunmingensis from Castanopsis delavayi (Fagaceae) in China, and it is the first record from woody litter.
Culture characteristics.Conidia germinated on PDA within 24 h at room temperature (25 °C).Germ tubes produced from the basal cells of conidia.Colonies on PDA, reaching 30 mm diameter after 2 weeks at 20-25 °C, mycelia superficial, irregular, umbonate at the center, fimbriate, undulate entire, grey, radially furrowed, with hyaline, glistening, rough, wrinkled, granular droplets of oil; reverse, grey, radially furrowed, grey white at the margin.

Pleopunctum megalosporum
Culture characteristics.Conidia germinated on PDA within 24 h at room temperature (25 °C).Germ tubes produced from the basal cells of conidia.Colonies on PDA, reaching 30 mm diameter after 2 weeks at 20-25 °C, mycelia superficial, irregular, slightly umbonate at the center, fimbriate, undulate edge, grey at the margin, grey white at the center, with hyaline, glistening, granular droplets of oil; reverse, atrovirens, notably radially furrowed, golden brown at the margin.

Discussion
This study introduces two new species of woody litter fungi: Pseudolophiostoma lincangense, and Pleopunctum baoshanense from Yunnan Province, China.We also report new host records of Occultibambusa kunmingensis on Castanopsis delavayi, Pleopunctum megalosporum on Castanopsis calathiformis for the first time in China, and Paraphoma aquatica is the first sexual morph of Paraphoma on Castanopsis delavayi.
Pseudolophiostoma was introduced by Thambugala et al. (2015), with P. vitigenum designated as the type species.Currently, this genus comprises seven accepted species (Species Fungorum 2024).Pseudolophiostoma species are typically saprobes found on various herbaceous, woody, or vine plants such as Bidens pilosa var.radiata, Clematis fulvicoma, Livistona boninensis, Stachytarpheta jamaicensis, and Vitis coignetiae, and are distributed across China (Taiwan), Japan, and Thailand (Thambugala et al. 2015;Hashimoto et al. 2018;Tennakoon et al 2018;Phukhamsakda et al. 2020).Pseudolophiostoma species are known only by their sexual morph, and their asexual morph has not been discovered yet (Thambugala et al. 2015).Therefore, the asexual morph of Pseudolophiostoma remains uncertain and thus, further studies with additional collections are needed to understand the asexual morph.
Occultibambusa, introduced by Dai et al. (2017) with O. bambusae as the type species, is commonly found on bamboo culms.These species have a wide distribution, particularly in China and Thailand, where they inhabit both freshwater and terrestrial habitats (Hyde et al. 2016;Dai et al. 2017;Zhang et al. 2017;Dong et al. 2020;Jiang et al. 2021;Yu et al. 2022).Currently, Species Fungorum (2024) recognizes 10 Occultibambusa species.Most of these species have been reported based on their sexual morphs, with only O. fusispora known from its holomorph, and the coelomycetous asexual morph discovered in culture (Dai et al. 2017;Jiang et al. 2021).In this study, our collection also reports sexual morph and thus, further studies are needed to understand the asexual morph with additional fresh collections.

Figure 1 .
Figure 1.Phylogram generated from ML analysis based on SSU, LSU, ITS, tef1-α, and rpb2 sequence data representing the family Lophiostomataceae.Related sequences were obtained from Phukhamsakda et al. (2020).Bootstrap values for ML are equal to or greater than 75%, and posterior probability values are equal to or greater than 0.95 from the BYPP analysis labelled on the nodes.Strains of the newly described species are in red, while type strains are in bold.The tree is rooted with Teichospora rubriostiolata (TR7) and T. trabicola (C134).

Figure 2 .
Figure 2. Phylogram generated from maximum likelihood analysis based on the combined SSU, LSU, ITS, tef1-α, and rpb2 dataset of Occultibambusaceae species.Related sequences were obtained from Yu et al. (2022).Bootstrap values for ML are equal to or greater than 75%, and posterior probability values are equal to or greater than 0.95 from the BYPP analysis labelled on the nodes.Strains of the newly described species are in red, while type strains are in bold.The tree is rooted with Ernakulamia krabiensis (MFLUCC 18-0237) and E. xishuangbannaensis (KUMCC 17-0187).

Figure 3 .
Figure 3. Phylogram generated from maximum likelihood analysis based on the combined SSU, LSU, ITS, tef1-α, and rpb2 dataset of Phaeoseptaceae species.Related sequences were obtained from Xu et al. (2023).Bootstrap values for ML are equal to or greater than 75%, and posterior probability values are equal to or greater than 0.95 from the BYPP analysis labelled on the nodes.Strains of the newly described species are in red, while type strains are in bold.The tree is rooted with Angustimassarina acerina (MFLUCC 14-0505) and A. populi (MFLUCC 13-0034).

Figure 4 .
Figure 4. Phylogram generated from maximum likelihood analysis based on the combined LSU, ITS, tef1-α, rpb2 and tub2 dataset of Paraphoma species.Related sequences were obtained from previous publications (Magaña-Dueñas et al. 2021,Guarnaccia et al. 2022, andGomzhina et al. 2020).Bootstrap values for ML are equal to or greater than 75%, and posterior probability values are equal to or greater than 0.95 from the BYPP analysis labelled on the nodes.Strains of the newly described species are in red, while type strains are in bold.The tree is rooted with Setophoma terrestris (CBS 335.29).