New Insights into Tetraplosphaeriaceae Based on Taxonomic Investigations of Bambusicolous Fungi and Freshwater Fungi

Species within Tetraplosphaeriaceae have been frequently documented in recent years with the extensive investigations of microfungi along a latitudinal gradient from north to south in the Asian/Australian region. Both bamboo substrates and freshwater habitats serve as extensive reservoirs, hosting a rich diversity of fungi that exhibit broad geographical distributions. The most common fungi in these two environments are generally distributed in distinct families. However, our statistics have revealed an intriguingly distinct preference of Tetraplosphaeriaceae species for inhabiting both bamboo substrates and freshwater habitats. The genera Pseudotetraploa (100%) and Triplosphaeria (100%) exhibit a strong preference, followed by Shrungabeeja (71%) and Quadricrura (67%). Our taxonomic and phylogenetic study of microfungi in southern China have identified four additional novel species, viz., Aquatisphaeria bambusae sp. nov., Pseudotetraploa phyllostachydis sp. nov., Pseudotetraploa yangjiangensis sp. nov., and Tetraploa submersa sp. nov. from bamboo substrates and freshwater habitats. In addition, Aquatisphaeria thailandica has previously been documented from freshwater habitats in Thailand; however, we have once again isolated this species from decaying bamboo substrates in Guangdong, China. The new findings substantiate our hypothesis that the preference of Tetraplosphaeriaceae species for colonizing bamboo substrates and freshwater habitats will be more evident through more extensive investigations conducted in such environments.

We are studying the freshwater microfungi along a latitudinal gradient from north to south in the Asian/Australian region [19].As bamboo grows alongside rivers, the fungi found on both are very much intertwined [20].In the present study, we investigate the tetraploa-like taxa from decaying bamboo and submerged wood in Southern China, and revise the taxon diversity within Tetraplosphaeriaceae. A total of five specimens were subjected to morphological and phylogenetic analyses, and the preliminary results indicated that they belong in Aquatisphaeria, Pseudotetraploa, and Tetraploa.A phylogenetic analysis was conducted using a combined dataset consisting of large subunit ribosomal RNA (LSU), small subunit ribosomal RNA (SSU), internal transcribed spacer (ITS), betatubulin (tub2) and RNA polymerase II largest subunit 2 (rpb2) to infer their phylogenetic relationships.

Sampling, Isolation, and Morphological Examination
In 2023, samples of decaying culms of bamboo and submerged wood were collected from the Guangdong and Yunnan provinces in China.The specimens were placed in a zip-lock bag and taken to the laboratory for morphological examination.A stereomicroscope (Chongqing Optec Instrument Co., Ltd., Chongqing, China) was utilized to examine the colonies developed on the natural substrate.The micro fungal structures were photographed through a compound microscope (Nikon Eclipse Ni-U, Tokyo, Japan) fitted with a digital camera (Canon 750D, Tokyo, Japan).The measurement of fungal structures (conidiophores, conidiogenous cells and conidia) was conducted using the TaroSoft (R) Image Frame Work program v. 0.9.0.7.The single spore isolations were made following the method described by Senanayake et al. [21].Herbarium specimens were deposited at the Mycological Herbarium of Zhongkai University of Agriculture and Engineering, Guangzhou, China (MHZU).The living cultures were deposited into the Zhongkai University of Agriculture and Engineering Culture Collection, Guangzhou, China (ZHKUCC).The novel species were registered in the databases of the Index Fungorum (http://www.indexfungorum.org/names/names.asp,accessed on 24 February 2024) [15] and Facesoffungi (http://www.facesoffungi.org,accessed on 24 February 2024) [22].

DNA Extraction, PCR Amplification, and Sequencing
The fungal mycelia were cultivated on a potato dextrose agar (PDA) medium at 28 • C for 30 days.A Maglso plant DNA isolation kit (Magen, Guangzhou, China) was employed to extract the genomic DNA following the instructions provided by the manufacture.DNA amplification was conducted using the polymerase chain reaction (PCR) technique, employing a 2720 Thermal Cycler instrument (Applied Biosystems, Foster City, CA, USA).LSU, ITS, SSU, tub2, and rpb2 were amplified using the primer pairs LR0R/LR5 [23], ITS5/ITS4 [24], NS1/NS4 [24], T1/BT2b [25,26], and fRPB2-5f/fRPB2-7cR [27], respectively.The amplifications were carried out in a 25 µL reaction volume containing 9.5 µL of ddH 2 O, 12.5 µL of 2 × FastTaq PCR Master Mix (Vazyme Co., Nanjing, China), 1 µL of DNA template, and 1 µL of each forward and reverse primer (10 µM).The PCR thermal cycles program for the amplification of LSU, ITS, SSU, and tub2 commenced with an initial denaturation step at 94 • C for 3 min, followed by 35 cycles consisting of denaturation at 94 • C for 30 s, annealing at 53 • C for 30 s, elongation at 72 • C for 60 s, and a final extension step at 72 • C for 10 min.For rpb2 amplification, the annealing temperature was adjusted to 56 • C. PCR products were checked on 1% agarose electrophoresis gels stained with Gel Red.The products with bright bands were transferred to Tianyi Huiyuan Biotechnology Co., Ltd.(Guangzhou, China) for sequencing reactions.

Phylogenetic Analyses
The quality of the sequences was initially evaluated using SeqMan v. 7.0.0[28].To evaluate the sequence similarities between the new collections and other species, the newly generated sequences were subjected to BLASTn searches utilizing the powerful Basic Local Alignment Search Tool (BLAST) (https://www.ncbi.nlm.nih.gov/,accessed on 10 October 2023).The reference sequences were obtained from the GenBank (Table 1) through BLASTn search results and relevant publications [4,7,12,29].Each dataset of the LSU, ITS, SSU, tub2, and rpb2 was aligned using the online version of MAFFT v. 7.0362 (https://mafft.cbrc.jp/alignment/server/,accessed on 10 October 2023) [30] with default settings, and manually adjusted using BioEdit v. 7.0 [31].The aligned datasets were merged using Mesquite v. 3.81 [32].The Alignment Transformation Environment online program (https://sing.ei.uvigo.es/ALTER/,accessed on 10 October 2023) was used to convert the FASTA file to PHYLIP and NEXUS formats for phylogenetic analyses.The phylogenetic analyses were conducted using maximum likelihood (ML) [33] and Bayesian inference (BI) approaches [34].
The ML analysis was conducted using RAxML-HPC2 on XSEDE v. 8.2.8 [33] within the CIPRES Science Gateway platform [35], employing the GTR+I+G model of evolution.For BI analysis, the evolutionary model of nucleotide substitution analysis was selected independently for each locus using MrModeltest v2.3 [36].The best-fit model for each gene selected by Akaike Information Criterion (AIC) is GTR+I+G.Markov Chain Monte Carlo sampling (MCMC) was used to evaluate Bayesian posterior probabilities (BYPP) [37].MCMC sampling was run for 1,000,000 generations, and the trees were sampled every 100th generation.The first 25% of the trees that represented the burn-in phase were discarded, and the remaining 75% of the trees were used for calculating the posterior probabilities (PP) for the majority rule consensus tree.The resulting trees were viewed in FigTree v. 1.4.0 (Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK) [38], and edited using Microsoft Office Power Point 2007 (Microsoft Corporation, Redmond, WA, USA).
Table 1.Taxa used for phylogenetic analyses and their corresponding GenBank accession numbers.Newly generated sequences are indicated in red, ex-type strains are indicated in bold, and missing sequences are indicated with "N/A".

Results
We base our new species following the principle of multiple evidence, as in the guidelines of Chethana et al. [39].

Conidial secession schizolytic.
Culture characteristics: The colonies on the PDA reach 22 mm in diam.after two weeks at 25 • C, circular, pale grey, grey at the margin, with a dense aerial mycelium, velvety; from below, pale brown, with a white entire edge.
Culture characteristics: The colonies on the PDA reach 50 mm in diam.after five weeks at 25 • C, circular, dry, with dense mycelium, off-white; from below, dark brown in the middle, white to pale brown outwards, white at the margin, with a filiform edge.

Culture characteristics:
The colonies on the PDA reach 52 mm in diam.after five weeks at 25 • C, circular, cottony, flat, pale brown; from below, dark brown, with an undulate, pale brown edge.
Regarding the DNA sequence data comparison, there is a discrepancy of 0.5% (4 out of 865), 2.4% (13 out of 539, 3 gaps) and 4.8% (20 out of 413) in nucleotide variations within the LSU, ITS, and tub2 genes, respectively, between T. submersa ZHKUCC 24-0009 and T. pseudoaristata NFCCI 4624; there is a discrepancy of 0.5% (4 out of 852, 1 gap) and 2.0% (11 out of 553, 2 gaps) in nucleotide variations within the LSU and ITS genes, respectively, between T. submersa ZHKUCC 24-0009 and T. puzheheiensis KUMCC 20-0151.To the best of our knowledge, T. submersa does not correspond to any known species within the genus.Therefore, it is introduced as a novel species of Tetraploa.

Discussion
Bambusicolous fungi are a diverse group associated with various bamboo substrates, including leaves, culms, branches, rhizomes, and roots.The extensive research from articles and monographs has confirmed the global distribution and high fungal diversity of bambusicolous fungi [1,7,[50][51][52].Freshwater fungi is another group that partially or completely resides in freshwater habitats, with numerous novel species having been discovered over the past decades [4,8,20,[53][54][55][56][57][58][59].The most common fungi in the two groups are generally clustered into distinct families.Hyde et al. [50] and Dai et al. [51] proposed that the common families of fungi on bamboo are Hypocreaceae, Phyllachoraceae and Xylariaceae, Bambusicolaceae, Tetraplosphaeriaceae, and Roussoellaceae.Dong et al. [4] summarized that freshwater fungi predominantly reside in Aliquandostipitaceae, Dictyosporiaceae, Morosphaeriaceae, Tetraplosphaeriaceae, and Tubeufiaceae.It is likely that the species within Tetraplosphaeriaceae exhibit a preference for inhabiting both bamboo substrates and freshwater habitats.To investigate this assumption, our study presents a compilation of Tetraplosphaeriaceae species that have been isolated from bamboo substrates and freshwater habitats, accompanied by notes for each genus explaining the isolation source for those particular species.Interestingly, we found that the species within the two genera, viz., Pseudotetraploa and Triplosphaeria, exclusively inhabit bamboo substrates and freshwater habitats, indicating their strong preference for these specific environments.The species identified within Aquatisphaeria and Pseudopolyplosphaeria have also been isolated from such environments; however, due to the limited sampling size thus far, it remains difficult to determine their definitive environmental preferences.The taxa within Ernakulamia, Polyplosphaeria, and Tetraploa, on the other hand, exhibit a relatively low percentage of species that inhabit such environments at 25%, 29%, and 31%, respectively.The species within Quadricrura and Shrungabeeja exhibit a moderate percentage at 67% and 71%, respectively.Four species, viz., Aquatisphaeria thailandica, Pseudotetraploa yunnanensis, Shrungabeeja vadirajensis, and Triplosphaeria acuta, are capable of thriving in both environments.In total, Tetraplosphaeriaceae comprises 48% (38/79) of species that have been isolated from bamboo substrates and freshwater habitats.In other words, 41 species in this family were isolated from other substrates or habitats, while 27 of them (66%) belong to the Tetraploa.Tetraploa species are frequently documented from herbaceous plants [1,[60][61][62][63].This prompts us to question whether the majority of species within Tetraploa have experienced migration from bamboo substrates or freshwater habitats to other herbaceous plants in response to significant environmental pressures.Numerous matters are still pending in the field of freshwater fungal biology.
The taxonomic investigations of microfungi have been conducted by various researchers, with an emphasis on their respective habitats, such as marine habitats [64], freshwater habitats [56], or an emphasis on their hosts, such as mangrove fungi [65], teak fungi [66], grass fungi [67], and entomopathogenic fungi [68].Alternatively, researchers may focus on particular fungal groups, such as annulatascaceae-like taxa [57,69], tubeufialike taxa [55], pestalotiopsis-like taxa [70,71].However, the taxonomic studies on fungal groups, simultaneously providing valuable insights into their hosts and habitats, have been rarely investigated.The compilation presented in this study, along with our new findings, have suggested that the Tetraplosphaeriaceae is likely to exhibit a high degree of host and habitat specificity.This can be clearly observed from examples of Aquatisphaeria, Pseudotetraploa, Quadricrura, Shrungabeeja, and Triplosphaeria.The species within Tetraploa have not exhibited a distinct preference for bamboo substrates and freshwater habitats.Due to the rich fungal diversity and wide distribution of species in bamboo substrates and freshwater habitats, we believe that numerous novel species and genera will be discovered within Tetraplosphaeriaceae through extensive investigations into fungi from these specific substrates and habitats.In this study, five new collections were collected from decaying bamboo substrates in terrestrial habitats and submerged wood in freshwater habitats.Morphological and phylogenetic analyses have identified four novel species, viz., Aquatisphaeria bambusae sp.nov., Pseudotetraploa yangchunensis sp.nov., Pseudotetraploa yangjiangensis sp.nov., and Tetraploa submersa sp.nov., as well as a previously documented species Aquatisphaeria thailandica within Tetraplosphaeriaceae. Aquatisphaeria thailandica was initially discovered from a freshwater habitat on submerged wood in Thailand, and we first report finding this species in a terrestrial habitat on decaying bamboo substrate in China.
The phylogenetic relationships between genera in Tetraplosphaeriaceae have been extensively investigated utilizing DNA sequence data [1,[5][6][7]10].However, the classification of several species, such as Polyplosphaeria thailandica, remains uncertain.Polyplosphaeria thailandica was introduced within Polyplosphaeria based on morphological and phylogenetic analysis [42].However, subsequent phylogeny revealed that P. thailandica does not cluster with other species in the genus Polyplosphaeria but instead shows closer phylogenetic affinities with Aquatisphaeria.The BLASTn searches on P. thailandica using the LSU sequence reveals P. thailandica to have the closest relationships to Aquatisphaeria thailandica (98.44%), followed by Quadricrura bicornis (97.58%), and Triplosphaeria acuta (97.58%).The BLASTn searches of the ITS sequence reveals P. thailandica to have the closest relationships to Hermatomyces sphaericus (94.68%), followed by Aquatisphaeria thailandica (94.13%), and Shrungabeeja fluviatilis (94.09%).The results from both LSU and ITS sequence Blast analyses showed that P. thailandica exhibited phylogenetic affinities with Aquatisphaeria.However, based on its morphology, P. thailandica should be classified under Polyplosphaeria rather than Aquatisphaeria [1,5,42].Considering the contradiction between the morphology and phylogeny, the identification of P. thailandica is pending, to be resolved with new collections.
Abbreviations: BCC: Biotec Culture Collection, Bangkok, Thailand; BCRC: FU: Bioresource Collection and Research Center Collection, Taiwan, China; CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; CGMCC: China General Microbiological Culture Collection Centre, Beijing, China; DLUCC: Dali University Culture Collection, Yunnan, China; GZCC: Guizhou Culture Collection, Guizhou, China; HHUF: Herbaria of Hirosaki University; IFRDCC: Culture Collection, International Fungal Research and Development Centre, Chinese Academy of Forestry, Kunming, China; KUNCC: Kunming Institute of Botany Culture Collection; KT: Kazuaki Tanaka; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; MFLU: Mae Fah Luang University Herbarium Collection; NFCCI: National Fungal Culture Collection of India NFCCI-A National Facility; PRC: the Herbarium of the Charles University, Prague, Czech Republic; UESTCC: University of Electronic Science and Technology Culture Collection, Chengdu, China; ZHKUCC: Zhongkai University of Agriculture and Engineering Culture Collection, Guangzhou, China.