Bambusicolous Fungi in Pleosporales: Introducing Four Novel Taxa and a New Habitat Record for Anastomitrabeculia didymospora

While conducting a survey of bambusicolous fungi in northern Thailand and southwestern China, several saprobic fungi were collected from dead branches, culms and twigs of bamboos, which were preliminarily identified as species belonging to Pleosporales (Dothideomycetes) based on a morphological approach. Multigene phylogenetic analyses based on ITS, LSU, SSU, rpb2, tef1-α and tub2 demonstrated four novel taxa belonging to the families Parabambusicolaceae, Pyrenochaetopsidaceae and Tetraploasphaeriaceae. Hence, Paramultiseptospora bambusae sp. et gen. nov., Pyrenochaetopsis yunnanensis sp. nov. and Tetraploa bambusae sp. nov. are introduced. In addition, Anastomitrabeculia didymospora found on bamboo twigs in terrestrial habitats is reported for the first time. Detailed morphological descriptions and updated phylogenetic trees of each family are provided herein.


Introduction
Bamboo is one of the most useful perennial woody grasses that contains the highest amount of living biomass [1]. It belongs to the subfamily Bambusoideae, family Poaceae, comprising about 127 genera, with approximately 1680 species, covering around 25 million hectares in tropical, subtropical, and mild temperate regions of Africa, America, Asia and Oceania, but it is rarely found in Europe [1][2][3][4][5]. Bamboo is important for ecological and socioeconomic sustainability throughout the world. Bamboo forests are crucial for the environmental benefits and climate change mitigation; they are sustainable in soil erosion control, carbon sequestration, soil and water conservation, windbreaks and shelterbelts, land rehabilitation, as well as releasing negative oxygen ions [4,[6][7][8][9]. Bamboo has also been utilized for traditional Chinese medicine, food sources, furniture and building construction, musical instruments, paper and textile industries, reinforcing fibers, as well as feedstock Samples were collected from the dead branches, culms, and twigs of bamboo in Chiang Mai and Chiang Rai Provinces of Thailand in 2011 and Yunnan Province of China in 2021. The samples were stored in paper bags and brought to the laboratory for observation and examination. Fungal fruiting bodies on host substrates were observed under an Olympus SZ61 series stereo microscope, and a centrum was mounted in sterilized distilled water on a clean slide for examination and captured under a Nikon ECLIPSE Ni compound microscope connected to a Nikon DS-Ri2 camera. Cotton blue was added to observe the fungal centrum, and Indian ink was used to check the mucilaginous sheath covering the ascospores. Morphological features were measured using Tarosoft (R) Image FrameWork version 0.9.7. Photographic plates were edited and combined in Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, USA). The permanent slides were prepared by adding lacto-glycerol and sealed by nail polish and deposited with herbarium specimens at the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (KUN-HKAS), China and the herbarium of Mae Fah Luang University, Chiang Rai, Thailand (MFLU).
Pure cultures were obtained from single-spore isolation based on a spore suspension technique [82]. Germinated ascospores were aseptically transferred to potato dextrose agar (PDA) and cultivated under normal light at 20-25 • C. Fungal colonies were observed and recorded after one week and four weeks. The asexual morph that sporulated in vitro was observed and examined after two months. Axenic living cultures were deposited in the Mae Fah Luang University Culture Collection (MFLUCC) and the Culture Collection of Kunming Institute of Botany (KUMCC). The newly described taxa were registered in Index Fungorum (http://www.indexfungorum.org/names/IndexFungorumRegister.htm; accessed on 13 May 2022).

DNA Extraction, Amplification, and Sequencing
Fresh mycelia were scraped from fungal colonies growing on PDA for a month and stored in a 1.5 mL sterilized microcentrifuge tube in an aseptic condition. Fungal genomic DNA was extracted by using Biospin Fungus Genomic DNA Extraction Kit (BioFlux ® , Hangzhou, China) following the manufacturer's instructions (Hangzhou, China). Fungal genomic DNA was also extracted from fruiting bodies directly in case the fungi could not germinate on PDA using a Forensic DNA Kit (Omega ® , Norcross, GA, USA). The generated fungal genomic DNA was stored at 4 • C for PCR amplification and duplicated at −20 • C for long-term storage.
Fungal genomic DNA was amplified by polymerase chain reaction (PCR) using informative phylogenetic markers of each family, including the internal transcribed spacers (ITS1-5.8S-ITS2), the 28S large subunit rDNA (LSU), the 18S small subunit rDNA (SSU), the partial RNA polymerase second largest subunit (rpb2), the translation elongation factor 1-alpha (tef1-α) and β-tubulin (tub2). The forward and reverse primer pairs ITS5 and ITS4 [83], LR0R and LR5 [84], NS1 and NS4 [83], fRPB2-5F and fRPB2-7cR [85], EF1-983F and EF1-2218R [86], and T1 and BT2B [87,88] were used to amplify the PCR fragments of these genes, respectively. Components of the PCR reaction mixture and the PCR thermal cycle program for ITS, LSU, SSU, rpb2, and tef1-α genes followed the condition described in Jiang et al. [50]. The PCR thermal cycle program for tub2 was set up initially at 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 52 • C for 40 s, elongation at 72 • C for 1 min, a final extension at 72 • C for 10 min, before being held at 4 • C. PCR products were sent to TsingKe Biological Technology (Beijing) Co., Ltd., Beijing, China for purification and sequencing. The quality of the Sanger DNA sequences and sequence consensus from forward and reverse directions were checked and compiled manually in BioEdit v. 7.2.3 [89].

Sequence Alignment and Phylogenetic Analyses
The generated ITS sequences of the new isolates were used to search the related fungal group via the nucleotide BLAST search tool in the NCBI website (https://blast.ncbi.nlm. nih.gov/Blast.cgi; accessed on 5 December 2021). The nucleotide BLAST searches of the ITS sequence showed that the newly generated sequences had the closest similarity with species in families Anastomitrabeculiaceae, Parabambusicolaceae, Pyrenochaetopsidaceae and Tetraploasphaeriaceae. Thus, sequences generated from this study were analyzed with representative taxa in Anastomitrabeculiaceae, Parabambusicolaceae, Pyrenochaetopsidaceae and Tetraploasphaeriaceae, which were retrieved from GenBank based on recent publications (Table 1). Individual gene alignments were performed and improved manually where necessary using MEGA 7 [90]. Ambiguous sites were excluded from the alignment. Improved individual gene alignments were prior analyzed by maximum likelihood (ML) analysis using RaxmlGUI version 7.3.0 [91]. After checking the tree topologies of every individual gene alignment for congruence, the combined gene dataset of each family was analyzed based on Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) analyses. Table 1. GenBank accession numbers used in the phylogenetic analyses. The ex-type cultures are indicated with superscript " T ", and the newly generated sequences are indicated in bold. n/a n/a n/a MG976037 Neohendersonia kickxii CBS 112403 T Neohe NR_154248 NG_058264 n/a n/a n/a n/a Neohendersonia kickxii CBS 122938 Neohe KX820257 KX820268 n/a n/a n/a n/a Neohendersonia kickxii CPC 24865 Neohe KX820259 KX820270 n/a n/a n/a n/a n/a n/a n/a n/a Tetraploa sasicola FU31019 Tetra MN937236 MN937218 n/a n/a n/a n/a Tetraploa sp. KT 1684 Tetra n/a AB524628 AB524487 n/a n/a n/a Tetraploa sp. KT 2578 Tetra n/a AB524629 AB524488 n/a n/a n/a Tetraploa sp. CY112 Tetra HQ607964 n/a n/a n/a n/a n/a The evolutionary model of nucleotide substitution analysis was selected independently for each locus using MrModeltest 2.3 [92]. The best-fit model under the Akaike Information Criterion (AIC) of each locus was shown in Table 2. Bayesian inference (BI) was analyzed using MrBayes on XSEDE v. 3.2.7a via the CIPRES Science Gateway v. 3.3 [93]. Posterior probabilities (PP) [94,95] were determined by Markov Chain Monte Carlo sampling (MCMC). Two parallel runs with six simultaneous Markov chains were run for 1-2 million generations and stopped automatically when the average standard deviation of split frequencies reached below 0.01. Trees were sampled every 100th generation. The MCMC heated chain was set with a "temperature" value of 0.15. All sampled topologies beneath the asymptote (25%) were discarded as part of the burn-in procedure, and the remaining trees were used for calculating posterior probabilities in the majority rule consensus tree. Maximum likelihood (ML) was analyzed in RaxmlGUI version 7.3.0 [91] using the default algorithm of the program from a random starting tree for each run that was adjusted by setting up the GTR + GAMMAI model of nucleotide substitution with 1000 rapid bootstrap replicates. Maximum parsimony was analyzed by PAUP v. 4.0b10 [96] using the heuristic search function with 1000 random stepwise addition replicates and tree bisection-reconnection (TBR) as the branch-swapping algorithm. Maxtrees were set up to 1000, and a zero of maximum branch length was collapsed. All characters were unordered and of equal weight, and gaps were treated as missing data. Significant parsimonious trees were determined by Kishino-Hasegawa tests (KHT) [97]. All equally parsimonious trees were saved. Clade stability was estimated by bootstrap (BS) support values with 1000 replicates, each with 10 replicates of random stepwise addition of taxa [98]. Descriptive tree statistics viz. tree length (TL), consistency index (CI), retention index (RI), relative consistency index (RC) and homoplasy index (HI) were calculated.

Phylogeny
Four phylogenetic analyses were conducted to resolve phylogenetic relationships of taxa in Anastomitrabeculiaceae (Analysis 1), Parabambusicolaceae (Analysis 2), Pyrenochaetopsidaceae (Analysis 3), and Tetraploasphaeriaceae (Analysis 4) as follows: Analysis 1: Taxa   The phylogenetic results based on maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference analyses ( Figure 1) showed overall similar tree topologies. Two new strains of Anastomitrabeculia didymospora (MFLUCC 11-0197, MFLUCC 11-0200) shared the same branch length with 72% ML support and grouped with the type strains of A. didymospora (MFLUCC 16-0412, MFLUCC 16-0416) with high support (100% ML, 100% MP, 1.00 PP) in Anastomitrabeculiaceae.  The phylogenetic results based on maximum likelihood (ML) and Bayesian inference analyses ( Figure 2) showed overall similar tree topologies. A novel genus, Paramultiseptospora formed a stable subclade, clustered with the genera Multiseptospora, Neomultiseptospora, and Scolecohyalosporium with low support. These four genera formed a well-resolved clade (100% ML, 1.00 PP) within Parabambusicolaceae.    Phylogram generated from RAxML analysis of a concatenated LSU-ITS-SSU-tub2-tef1-α sequence dataset to represent the phylogenetic relationships of the novel taxon in Tetraploasphaeriaceae. Bootstrap support values for ML and MP equal to or greater than 70% and the Bayesian posterior probabilities equal to or higher than 0.95 PP are indicated above the nodes as ML/MP/PP. Support values lower than 70% ML/MP and 0.95 PP are indicated by a hyphen (-). Ex-type strains are in bold, and the new species is indicated in blue.

Taxonomy
Anastomitrabeculiaceae Bhunjun, Phukhams. and K.D. Hyde Bhunjun et al. [71] introduced the novel family Anastomitrabeculiaceae to accommodate a monotypic genus Anastomitrabeculia based on morphological characteristics and phylogenetic analyses of a combined LSU, SSU and tef1-a dataset coupled with divergence time estimates using molecular clock methodologies. The novel taxa were isolated from bamboo culms submerged in freshwater in southern Thailand. The genus is characterized by gregarious, uni-loculate, globose to subglobose, coriaceous ascomata, immersed under a clypeus to semi-immersed, with short, carbonaceous ostiolar neck, bitunicate, fissitunicate, cylindric-clavate asci, embedded in a hyaline, trabeculate pseudoparaphyses, and hyaline, fusiform, septate ascospores with longitudinally striate wall ornamentation, surrounded by a distinct, mucilaginous sheath [71]. According to Bhunjun et al. [71], Anastomitrabeculiaceae has a close phylogenetic relationship with Halojulellaceae. However, Halojulellaceae can be distinguished from Anastomitrabeculiaceae in having cellular pseudoparaphyses and pigmented ascospores. In this study, we collected Anastomitrabeculia didymospora from bamboo branches in terrestrial habitats in northern Thailand reported for the first time. Culture characteristics: Ascospores germinated on PDA within 12 h. Colonies on PDA reaching 29-33 mm diam after 2 weeks at room temperature (30-35 • C). Colonies medium dense, irregular in shape, flat to slightly raised, surface smooth with an undulate edge, floccose to fluffy; colonies from above white at the margin, pale gray at the middle, with white hyphal turfs at the center; from below white to cream at the margin, yellowish-brown at the middle, dark greenish-gray to black at the center, slightly radiating inwards colony; not producing pigmentation on PDA. Known distribution: Krabi Province, southern Thailand [71], Chiang Mai and Chiang Rai Provinces, northern Thailand (this study).
Known host and habitats: saprobic on bamboo in freshwater [71] and terrestrial environments (this study).
Notes: The nucleotide BLAST search of ITS, LSU and tef1-α sequences resulted in the two newly generated strains (MFLUCC 11-0197 and MFLUCC 11-0200) being similar to Anastomitrabeculia didymospora MFLU 20-0694 (100% similarity). A nucleotide pairwise comparison of ITS, LSU and tef1-α sequences also indicated that strains MFLUCC 11-0197 and MFLUCC 11-0200 are consistent (less than 1.5% different base pair) with A. didymospora MFLU 20-0694 (type strain). We, therefore, identified our strains as A. didymospora. Morphologically, the new collection (MFLU 11-0233) is slightly larger in ascomata, asci, and ascospores than those of the type of A. didymospora [71]. The differences in the size range may be affected by environmental factors. Bhunjun et al. [71] mentioned that A. didymospora (MFLU 20-0694) has one-septate ascospores; however, we found that the species has 1(-3)-septate ascospores in this study. The host preference of A. didymospora is currently restricted to bamboo. However, the species is reported from terrestrial habitats for the first time. Notes: The nucleotide BLAST search of ITS, LSU and tef1-α sequences resulted in the two newly generated strains (MFLUCC 11-0197 and MFLUCC 11-0200) being similar to Anastomitrabeculia didymospora MFLU 20-0694 (100% similarity). A nucleotide pairwise comparison of ITS, LSU and tef1-α sequences also indicated that strains MFLUCC 11-0197 and MFLUCC 11-0200 are consistent (less than 1.5% different base pair) with A. didymospora MFLU 20-0694 (type strain). We, therefore, identified our strains as A. didymospora. Morphologically, the new collection (MFLU 11-0233) is slightly larger in ascomata, asci, and ascospores than those of the type of A. didymospora [71]. The differences in the size range may be affected by environmental factors. Bhunjun et al. [71] mentioned that A. didymospora (MFLU 20-0694) has one-septate ascospores; however, we found that the species has 1(-3)-septate ascospores in this study. The host preference of A. didymospora is currently restricted to bamboo. However, the species is reported from terrestrial habitats for the first time.  Parabambusicolaceae Kaz. Tanaka and K. Hiray. Parabambusicolaceae was introduced by Tanaka et al. [27] to accommodate the genera Aquastroma, Multiseptospora, Parabambusicola, and the other two "Monodictys sp.". Later, a monotypic genus Multilocularia was included in this family by Li et al. [100], while Wanasinghe et al. [101] and Phukhamsakda et al. [102] addressed both sexual and coelomycetous asexual species of Neoaquastroma in this family. Phukhamsakda et al. [102] also included Pseudomonodictys in Parabambusicolaceae. Subsequently, many genera were introduced in this family, including Lonicericola, Neomultiseptospora, Paramonodictys, Paratrimmatostroma, and Scolecohyalosporium [63,103,104]. Presently, 11 genera are accepted in this family based solely on the morpho-molecular approach. We follow the latest treatment of Xie et al. [104] and introduce the new genus Paramultiseptospora to accommodate a single species P. bambusae sp. nov. in this study.
Paramultiseptospora Phookamsak, H.B. Jiang and Chomnunti, gen. nov. Index Fungorum number: IF 554966 Etymology: Referring to relations with phylogenetically close genus Multiseptospora. Saprobic on dead stems of bamboo. Sexual morph: Ascomata gregarious, scattered to clustered, immersed in dark brown longitudinal clypeus, visible as raised, becoming superficial, lying along the host surface, uni-loculate, hemispherical to flattened ellipsoidal, or quadrilateral, glabrous, with apapillate ostiole. Peridium thin-to thick-walled, slightly thick at the sides, thinner at the apex, poorly developed at the base, composed of several layers of brown to dark brown, pseudoparenchymatous cells, paler brown to hyaline toward the inner layers, arranged in a textura angularis, outer layers intermixed with host tissues. Hamathecium composed of dense, branched, septate cellular pseudoparaphyses, anastomosed above the asci, embedded in a hyaline gelatinous matrix. Asci eight-spored, bitunicate, fissitunicate, cylindric-clavate to clavate, shortly pedicellate, apically rounded, with a well-developed ocular chamber. Ascospores overlapping one to three-seriate, hyaline, fusiform to oblong, with rounded ends, septate, constricted at the septa, smooth-walled, surrounded by a thick, mucilaginous sheath, with small guttules. Asexual morph: Undetermined.
Type species: Paramultiseptospora bambusae Phookamsak and H.B. Jiang, sp. nov. Notes: A monotypic genus Paramultiseptospora is introduced herein due to the differences in morphological characteristics with the other related genera (viz. Multiseptospora, Neomultiseptospora and Scolecohyalosporium), although the phylogenetic affinity of the genus does not support in this study. Paramultiseptospora formed a stable clade, closely related to Multiseptospora and Scolecohyalosporium in both BI and ML analyses and clustered with Neomultiseptospora. These four genera formed a well-resolved clade (100% ML, 1.00 PP; Figure 2) within Parabambusicolaceae. Paramultiseptospora can be easily distinguished from Multiseptospora and Scolecohyalosporium in having hemispherical to flattened ellipsoidal, glabrous, ascomata, immersed in longitudinal clypeus, visible as raised, lying along the host surface, cylindric-clavate to clavate asci with short pedicel and fusiform to oblong ascospores, with rounded ends. Meanwhile, Multiseptospora has globose to subglobose ascomata, immersed in the host, covered by dark, hair-like hyphae, broadly cylindrical, subsessile asci, and fusiform to vermiform ascospore, with acute ends [33]. Scolecohyalosporium is different in having conical to ovoid, black, rough-walled ascomata, erumpent to superficial on the host, long cylindrical asci, with short pedicel and filiform ascospores [104]. Paramultiseptospora morphologically resembles Neomultiseptospora in having hemispherical to subconical, glabrous ascomata, immersed in the host, with apapillate ostiole, clavate asci, with short pedicel and fusiform or oblong, septate ascospores, surrounded by a thick mucilaginous sheath. However, these two genera are slightly different in the characteristics of ascomata on the host. Paramultiseptospora formed gregarious, scattered to clustered ascomata, immersed in dark brown longitudinal clypeus, lying along the host surface whereas Neomultiseptospora formed solitary, scattered, immersed, visible as raised, black dome-shaped on the host surface [104]. Phylogenetically, Paramultiseptospora always formed a separate branch from Neomultiseptospora. Therefore, we consider Paramultiseptospora as a distinct genus with Neomultiseptospora based on morphology coupled with the phylogenetic relationship.
Paramultiseptospora bambusae Phookamsak and H.B. Jiang, sp. nov. Index Fungorum number: IF 554968, Figure 6 Etymology: Referring to the host, bamboo, of on which the species was collected. Holotype: KUN-HKAS 122241 Saprobic on a dead stem of bamboo. Sexual morph: Ascomata 115-150 µm high, 340-470 µm diam, gregarious, scattered to clustered, immersed in dark brown longitudinal clypeus, visible as raised, becoming superficial, lying along the host surface, uni-loculate, hemispherical to flattened ellipsoidal, or quadrilateral, glabrous, indistinct apapillate ostiole. Peridium 30-90 µm wide at the sides toward the apex, 10-25 µm wide at the base, thin-to thick-walled, slightly thick at the sides, thinner at the apex, poorly developed at the base, composed of several layers of brown to dark brown, pseudoparenchymatous cells, paler brown to hyaline toward the inner layers, arranged in a textura angularis, outer layers intermixed with host tissues. Hamathecium composed of dense, 1-2.5 µm wide, branched, septate cellular pseudoparaphyses, anastomosed above the asci, embedded in a hyaline gelatinous matrix. Asci The species is also different from Neomultiseptospora yunnanensis strain KUMCC 21-0411 (ex-type strain) in 102/606 bp of ITS (16.83%), 21/832 bp of LSU (2.52%), and 52/979 bp of tef1-α (5.31%). Paramultiseptospora bambusae is morphologically similar to N. yunnanensis but differs in having fusiform to oblong, six-septate ascospores with rounded ends, narrower toward the end cells, and constricted at the septa, whereas N. yunnanensis has fusiform to ellipsoidal, or oblong, (four to) five-septate ascospores, with rounded ends, slightly constricted at the central septum, which are less constricted at the other septa [104].
(2.52%), and 52/979 bp of tef1-α (5.31%). Paramultiseptospora bambusae is morphologically similar to N. yunnanensis but differs in having fusiform to oblong, six-septate ascospores with rounded ends, narrower toward the end cells, and constricted at the septa, whereas N. yunnanensis has fusiform to ellipsoidal, or oblong, (four to) five-septate ascospores, with rounded ends, slightly constricted at the central septum, which are less constricted at the other septa [104].  [105] to accommodate the asexual genera Pyrenochaetopsis (type genus), Neopyrenochaetopsis and Xenopyrenochaetopsis. The family is characterized by pycnidial, pale brown to brown, solitary or confluent, glabrous or setose, subglobose to ovoid conidiomata, with apapillate or papillate ostiole, acropleurogenous conidiophores, phialidic, hyaline, discrete or integrated, septate conidiogenous cells, and aseptate, hyaline, smooth-and thin-walled, ovoid, cylindrical to allantoid ascospores [105]. Mapook et al. [106] introduced a novel species, Pyrenochaetopsis chromolaenae collected on Chromolaena odorata in Thailand and reported the sexual morph of Pyrenochaetopsis for the first time. The sexual morph is characterized by brown to dark brown solitary or scattered, globose ascomata, superficial on the host, with  [105] to accommodate the asexual genera Pyrenochaetopsis (type genus), Neopyrenochaetopsis and Xenopyrenochaetopsis. The family is characterized by pycnidial, pale brown to brown, solitary or confluent, glabrous or setose, subglobose to ovoid conidiomata, with apapillate or papillate ostiole, acropleurogenous conidiophores, phialidic, hyaline, discrete or integrated, septate conidiogenous cells, and aseptate, hyaline, smooth-and thin-walled, ovoid, cylindrical to allantoid ascospores [105]. Mapook et al. [106] introduced a novel species, Pyrenochaetopsis chromolaenae collected on Chromolaena odorata in Thailand and reported the sexual morph of Pyrenochaetopsis for the first time. The sexual morph is characterized by brown to dark brown solitary or scattered, globose ascomata, superficial on the host, with short papillate ostiole, with reddish-brown setae covering the papilla, thin-walled peridium, fissitunicate, cylindric-clavate asci, with a short, bulbous pedicel, and hyaline to pale brown or yellowish-brown, cylindrical to broadly fusiform, three to four-septate ascospores [106]. Species in Pyrenochaetopsidaceae have been isolated from various substrates as saprobes and also opportunistic pathogens on humans as well as on cysts of plant-parasitic nematodes. [81,105,106].
Pyrenochaetopsis was treated as the generic type of Pyrenochaetopsidaceae and is typified by P. leptospora. The genus was introduced by de Gruyter et al. [107] to accommodate phoma-like taxa. Recently, 19 species are accepted in this genus [108]. In this study, we introduce a holomorph species, P. yunnanensis, which occurred on bamboo in Yunnan, China.
Pyrenochaetopsis yunnanensis C.F. Liao, H.B. Jiang and Phookamsak, sp. nov. Index Fungorum number: IF 554979, Figure 7 Etymology: Referring to the locality, Yunnan Province of China, of which the species was collected.
Culture characteristics: Ascospores germinated on PDA within 24 h. Colonies on PDA reach 25-28 mm diam after one week at room temperature (15-20 • C). Colonies medium dense, irregular in shape, flat to slightly raised, surface smooth with a lobate edge, floccose to cottony; colony from above pale gray to white-gray at the margin, white at the middle toward the center, sectored; from below white at the margin, pale yellowish-brown at middle toward the center; not producing pigmentation on PDA. Sporulation on PDA after two months. Based on a nucleotide pairwise comparison, Pyrenochaetopsis yunnanensis (KUMCC 21-0843) is consistent with P. terricola strain HGUP1802 in LSU nucleotide pairwise comparison but differs from P. terricola in 11/519 bp of ITS (2.12%), 53/1002 bp of rpb2 (5.29%), and 7/363 bp of tub2 (1.93%). Pyrenochaetopsis yunnanensis (KUMCC 21-0843) grouped with P. terricola strain HGUP1802 with high support (100% ML, 100% MP, 1.00 PP; Figure 3) in the present study. Pyrenochaetopsis yunnanensis (KUMCC 21-0843) morphological resembles P. terricola but the conidial size is slightly longer than P. terricola (2-3 × 1-2 µm) [109]. Wang et al. [109] isolated P. terricola from the soil in Guizhou Province, China and determined only the asexual morph sporulated on OA, while our novel species was found as a saprobe on bamboo and both sexual and asexual morph.
Tetraploa (= Tetraplosphaeria), generic type of Tetraplosphaeriaceae, was introduced by Berkeley and Broome [115] with T. aristata as the type species. The asexual morph of Tetraploa is characterized by lacking conidiophores, monoblastic conidiogenous cells, and brown, short-cylindrical, verrucose conidia, composed of four columns with four setose appendages at the apex [12,81]. The sexual morph is characterized by scattered to gregarious, immersed to erumpent, globose to subglobose, glabrous ascomata, with short-papillate to cylindrical ostiole, fissitunicate, cylindrical to clavate, short-pedicellate asci, and hyaline, narrowly fusiform, septate, smooth-walled ascospores, surrounded by a mucilaginous appendage-like sheath [12,81]. Species in Tetraploa mostly occurred on bamboos and other herbaceous plants or rotten wood as well as isolated from soil or raindrops [81]. In this study, the novel species, T. bambusae, isolated from bamboo in Yunnan, China is introduced based on morphological characteristics and multigene phylogenetic analyses.    18-23(-26) µm (x = 24.1 × 19.8 µm, n = 30), muriform, obovoid to turbinate, with obtuse end, brown to dark brown, composed of four columns of cells, four-septate in each column, coarsely verruculose, with four apical appendages, sometimes, a small piece of the denticle remains attached to the base of the conidium. Appendages 15-40 µm long, 2.5-4.5 µm wide at the base, wider at the base, tapering toward the apex, divergent, brown, one to three-septate, straight or slightly flexuous, smooth-walled.
Culture characteristics: Ascospores germinated on PDA within 24 h. Colonies on PDA reach 22-25 mm diam after two weeks at room temperature (15-20 • C). Colonies dense, irregular in shape, raised to umbonate, surface smooth with an undulate edge, velvety; colony from above white-gray to pale gray at the margin, gray at the middle toward the center; from below white to cream at the margin, orange-brown at the middle, brown to dark brown at the center, slightly radiated outwards colony with concentric rings; not producing pigmentation on PDA. Phylogenetic analyses based on a combined LSU-ITS-SSU-tub2-tef1-α sequence dataset demonstrated that Tetraploa bambusae (KUMCC 21-0844) is sister to Tetraploa sp. KT 1684 and clustered with T. endophytica CBS 147114 and T. obpyriformis KUMCC 21-0011 with high support (99% ML, 98% MP, 1.00 PP; Figure 4). Based on a nucleotide pairwise comparison, T. bambusae (KUMCC 21-0844) is consistent with Tetraploa sp. KT 1684 in LSU nucleotide pairwise comparison (differs in 1 bp), but it could not be compared for the other informative gene regions (viz. ITS, tub2, and tef1-α) due to the lack of sequence data of Tetraploa sp. KT 1684. Tanaka et al. [12] included Tetraploa sp. KT 1684 in their analyses when they introduced the new family Tetraplosphaeriaceae; however, the morphological characteristics of Tetraploa sp. KT 1684 were not described. Thus, we could not compare the morphology of the novel species with Tetraploa sp. KT 1684, while T. obpyriformis KUMCC 21-0011 is an unpublished species. Tetraploa endophytica CBS 147114 was isolated from the roots of Microthlaspi perfoliatum (Brassicaceae) as an endophyte. The strain did not sporulate in any of the different culture media [116]. Therefore, the species also could not compare their morphology.
Anastomitrabeculia didymospora is a putative species accommodated in Anastomitrabeculiaceae. Bhunjun et al. [71] reported that the species that occurred on the bamboo host was submerged in freshwater. In this study, we also found the species occurring on a bamboo host in the terrestrial habitat near the waterfall. There are few studies concerning relationships between freshwater and terrestrial fungi [134][135][136][137]. Boonyuen et al. [136] mentioned that fungal species partially overlap between freshwater and terrestrial habitats, of which the submerged samples yielded the most fungal diversity. Boonyuen et al. [136] also suggested that the diversity of wood-inhabiting fungi depended on tree species, geography, and exposure period. There is no clear evidence to prove that terrestrial fungi will continue to thrive when submerged in water [137]. Kodseub et al. [137] attempted to investigate the differences in fungal communities that occurred in Magnolia liliifera wood from freshwater and terrestrial habitats. Kodseub et al. [137] mentioned that dominant fungi in the terrestrial environment were significantly different from fungi submerged in freshwater, and few species have been found in both freshwater and terrestrial habitats, suggesting that most fungi occurring on wood in terrestrial habitats did not thrive in freshwater habitats. According to Kodseub et al. [137], we hypothesized that A. didymospora is one of the few species that can survive in both freshwater and terrestrial habitats. The species may initially occur on a bamboo host in the terrestrial environment and continue to thrive in submerged freshwater.
Parabambusicolaceae shows to be heterogeneous, and it currently contains 12 genera, including the new genus introduced in this study. Even though most genera of Parabambusicolaceae contain a single species, they showed high genetic heterogeneity, which can be interpreted by their phylogenetic relationships. Most genera in Parabambusicolaceae are only represented by their sexual or asexual morph, except for Neoaquastroma. Hence, the morphology of some sexual and asexual genera could not be compared, which led to the generic status becoming questionable. More taxa sampling is required for a better understanding of each genus in Parabambusicolaceae.
Pyrenochaetopsis was introduced to accommodate phoma-like taxa that occurred on various host substrates [81,105,106,138]. The genus was previously treated in Cucurbitariaceae [72,107,139]. Later, Valenzuela-Lopez et al. [105] introduced the new family Pyrenochaetopsidaceae to accommodate this genus together with Neopyrenochaetopsis and Xenopyrenochaetopsis. Mapook et al. [106] determined the sexual morph of Pyrenochaetopsis, P. chromolaenae, for the first time. In the present study, the holomorph of P. yunnanensis sp. nov. is also determined. The sexual morph of P. yunnanensis can be distinguished from P. chromolaenae in having subglobose to subconical, or quadrilateral, glabrous ascomata and hyaline, fusiform, one to three-septate ascospores, whereas P. chromolaenae has globose ascomata with setose papilla and hyaline to pale brown or yellowish-brown, cylindrical to broadly fusiform, three to four-septate ascospores [106]. Pyrenochaetopsis yunnanensis is reported as a saprobe on bamboo host in Yunnan Province, China for the first time. Species of Pyrenochaetopsis are well-studied based on molecular analyses coupled with morphological characteristics of their asexual morph. Nevertheless, the sexual morph of this genus is still rarely detected.