A Taxonomic Appraisal of Bambusicolous Fungi in Occultibambusaceae (Pleosporales, Dothideomycetes) with New Collections from Yunnan Province, China

During our ongoing studies of bambusicolous fungi in southwest China and Thailand, three saprobic pleosporalean taxa were discovered on bamboos in Yunnan Province of China. Occultibambusa hongheensis and Seriascoma bambusae spp. nov. are introduced based on morphological characteristics coupled with multi-locus phylogenetic analyses of combined LSU, SSU, TEF1-α, RPB2 and ITS sequence data. Occultibambusa kunmingensis is also reported from a terrestrial habitat for the first time. Comprehensive descriptions, color photo plates of micromorphology, and a phylogenetic tree showing the placements of these three taxa are provided. In addition, synopsis tables of Occultibambusa and Seriascoma with morphological features are also provided.


Collection, Examination, Isolation and Preservation
Dead bamboo branches and culms were collected from Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province, China in January 2019 and Honghe County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province, China, in October 2020. Samples were stored in plastic Ziploc bags and taken to the laboratory at Kunming Institute of Botany, CAS, Kunming, Yunnan Province, China for observation and examination following the method described by Senanayake et al. [20]. Fungal fruiting bodies on host substrates were visualized under a Motic SMZ 140 series dissecting stereoscope and photographed by digital camera. Vertical sections of ascostromata and conidiomata and other micro-morphological characteristics (e.g., peridium, pseudoparaphyses, asci, ascospores, conidiogenous cells and conidia) were observed and captured with a Nikon ECLIPSE Ni compound microscope connected with a Canon EOS 600D digital camera. The Tarosoft (R) Image FrameWork version 0.9.7 program was used to measure the size (10-20 measurements of each structure) of fungal characteristics. Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, USA) was used to edit and combine photographic plates. Ex-type living culture of Occultibambusa fusispora (MFLUCC 11-0127) was also loaned from Mae Fah Luang University Culture Collection, Chiang Rai, Thailand (MFLUCC). It was aseptically sub-cultured in a laminar flow and incubated at room temperature (20)(21)(22)(23)(24)(25)  Facesoffungi and Index Fungorum numbers have been registered for the newly described taxa [21,22]. New species have been established based on the guidelines of Jeewon and Hyde [23].

Alignment and Phylogenetic Analyses
The nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 10 April 2021)) was applied to discover taxa closely related to our three new isolates (KUMCC 21-0019, KUMCC 21-0020 and KUMCC 21-0021). Similarity indices from the BLAST search indicated that KUMCC 21-0019 and KUMCC 21-0020 belong to Occultibambusa (Occultibambusaceae) and KUMCC 21-0021 belongs to Seriascoma (Occultibambusaceae). Therefore, to reveal accurate phylogenetic placements of our three new isolates, multi-gene phylogeny of Occultibambusaceae and the closely related family Nigrogranaceae (Pleosporales, Dothideomycetes) were done based on maximum-likelihood and Bayesian inference methods. DNA sequences of representative taxa in Occultibambusaceae and Nigrogranaceae are shown in Table 1. Sequence alignments and phylogenetic analyses were carried out following methods described by Dissanayake et al. [29]. Preliminarily individual DNA sequence matrixes were aligned via the online platform, MAFFT v. 7.475 [30]. Aligned sequence datasets were trimmed by TrimAl v. 1.3 via the web server phylemon 2 (http://phylemon.bioinfo.cipf.es/utilities.html (accessed on 20 April 2021)) and then improved where necessary using BioEdit v. 6.0.7 [31], i.e., complementing the missing bases at the start and end of the consensus sequence. Individual gene datasets were analyzed by maximum likelihood criteria in order to compare the congruence of tree topologies.
Maximum-likelihood (ML) analysis was performed via the online portal CIPRES Science Gateway v. 3.3 [32], with RAxML-HPC v.8 on XSEDE (8.2.12) tool, using default settings but following the adjustments: the GAMMA nucleotide substitution model and 1000 rapid bootstrap replicates. The evolutionary model of nucleotide substitution for Bayesian inference (BI) analysis was selected independently for each locus using MrModeltest 2.3 [33]. GTR+I+G was the best-fit for LSU, TEF1-α, and RPB2 loci under the Akaike Information Criterion (AIC), while the GTR+G substitution model was the best-fit for the ITS locus and HKY+I+G was the best-fit for the SSU locus. BI analysis was performed via MrBayes v. 3.1.2 [34]. Markov chain Monte Carlo sampling (MCMC) was used to determine posterior probabilities (PP) [35,36]. Six simultaneous Markov chains were run for 1,000,000 generations and trees were sampled every 100th generation. The 0.15 "temperature" value was set in MCMC heated chain. All sampled topologies beneath the asymptote (20%) were discarded as part of a burn-in procedure and the remaining 8000 trees were used for calculating posterior probabilities (PP) in the 50% majority rule consensus tree (when split frequency lower than 0.01).
The tree topologies generated in this study were visualized on FigTree v. 1.4.0 (http: //tree.bio.ed.ac.uk/software/figtree/ (accessed on 20 April 2021)). The phylogram was edited and redrawn by using Microsoft Office PowerPoint 2016 (Microsoft Inc., Redmond, WA, USA) and converted to tiff file on Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, USA). New sequences generated from the present study were deposited in GenBank ( Table 1). The final alignment and phylogram were submitted to TreeBASE (submission ID: 28553, https://www.treebase.org/ (accessed on 20 July 2021)).

Discussion
Species of Occultibambusa have been discovered in both freshwater and terrestrial habitats (Table 2). Presently, all Occultibambusa species have been reported as saprobes on dead bamboo, indicating that the host preference of the genus is restricted to bamboo. Occultibambusa has currently been reported from China and Thailand (Table 2). More than 1500 bamboo species are distributed worldwide [39], especially in subtropical and tropical regions [40] Therefore, there is a high potential to discover more new species of the genus from bamboos in other regions [41]. Most species in Occultibambusa have similar morphology, but they can be distinguished by dimensions of ascostromata, asci and ascospores and color of ascospores (Table 2). In addition, significant phylogenetic distances of ITS, TEF1-α, and RPB2 can also be used.

Discussion
Species of Occultibambusa have been discovered in both freshwater and terrestrial habitats (Table 2). Presently, all Occultibambusa species have been reported as saprobes on dead bamboo, indicating that the host preference of the genus is restricted to bamboo. Occultibambusa has currently been reported from China and Thailand (Table 2). More than 1500 bamboo species are distributed worldwide [39], especially in subtropical and tropical regions [40] Therefore, there is a high potential to discover more new species of the genus from bamboos in other regions [41]. Most species in Occultibambusa have similar morphology, but they can be distinguished by dimensions of ascostromata, asci and ascospores and color of ascospores (Table 2). In addition, significant phylogenetic distances of ITS, TEF1-α, and RPB2 can also be used.
The phylogenetic placement of Occultibambusa fusispora is unstable in several previous publications. Occultibambusa fusispora was separated from all Occultibambusa species and Versicolorisporium triseptatum in Dong et al. [13] and Wanasinghe et al. [15], while Phukhamsakda et al. [7] showed that Occultibambusa fusispora clustered with O. maolanensis and Versicolorisporium triseptatum. Therefore, in order to resolve this issue, we re-sequenced the ex-type living culture of Occultibambusa fusispora. Previously, Dai et al. [4] did not sequence the SSU region of this species, while we sequenced SSU, ITS, LSU, TEF1-α, and RPB2 regions. In our phylogeny, the newly generated sequences of O. fusispora (MFLUCC 11-0127II) are consistent with MFLUCC 11-0127 (100% ML, 1.00 PP; Figure 1) and separated well from all Occultibambusa species and Versicolorisporium triseptatum with high statistical support (98% ML, 1.00 PP; Figure 1).
Occultibambusa fusispora matches the typical morphology of sexual morph of Occultibambusa; however, it cannot be compared with asexual morphs of other Occultibambusa species because O. fusispora is the only species of this genus known in its holomorph, as the asexual morph was induced on bamboo pieces in vitro. In addition, our phylogeny showed O. fusispora is basal to Occultibambusa and Versicolorisporium clade. Therefore, in order to give a more reliable explanation for the placement of Occultibambusa fusispora, further studies on Occultibambusa species had better be focused on the induction of asexual morph sporulation in vitro. Induction of asexual morph sporulation in vitro can be performed by following the method described in Phookamsak et al. [42].
In the present study, Occultibambusa maolanensis and O. hongheensis clustered with Versicolorisporium triseptatum and were separated from the main Occultibambusa clade with low statistical support (73% ML, 0.71 PP; Figure 1). In addition, the nucleotide BLAST search of SSU sequence of V. triseptatum indicated that V. triseptatum has consistent base pairs with O. maolanensis. The phylogenetic position of O. maolanensis and V. triseptatum concurs with the studies of Dong et al. [13] and Wanasinghe et al. [15]. Occultibambusa maolanensis and O. hongheensis cannot be compared with Versicolorisporium triseptatum as they are known from different morphs. Occultibambusa maolanensis and O. hongheensis have the typical morphology of the sexual morph of Occultibambusa. The asexual morph of Occultibambusa is very different from Versicolorisporium. Therefore, the congeneric status of Occultibambusa and Versicolorisporium is pending further studies.
Versicolorisporium is a poorly known coelomycetous genus with V. triseptatum collected in Japan on dead culms of Pleioblastus chino and Sasamorpha borealis (bamboo) [14]. Fresh collections and sequencing of Versicolorisporium are needed in order to solve its confusing phylogenetic placement.
Serisacoma is presently known as saprobic on bamboo and dead and decaying wood in the terrestrial or freshwater habitats distributed in China and Thailand [4,12,13,38]. The genus accommodates only three species, suggesting that more taxa await discovery [41]. The sexual morphs of Seriascoma can be distinguished based on dimensions of ascostromata and ascospores, and the number of locules. The asexual morphs of Seriascoma can be distinguished based on dimensions of conidiomata and conidia, the number of locules, and the shape of conidia (Table 3).    thank Mae Fah Luang University for his PhD scholarship. Chiang Mai University is thanked for partially supporting this research work.

Conflicts of Interest:
The authors declare no conflict of interest.