Taxonomy and Phylogeny of Hyphomycetous Muriform Conidial Taxa from the Tibetan Plateau, China

During the investigation of lignicolous freshwater fungi in the Tibetan Plateau habitat, fifteen collections were isolated from submerged decaying wood. Fungal characteristics are commonly found as punctiform or powdery colonies with dark pigmented and muriform conidia. Multigene phylogenetic analyses of combined ITS, LSU, SSU and TEF DNA sequences showed that they belong to three families in Pleosporales. Among them, Paramonodictys dispersa, Pleopunctum megalosporum, Pl. multicellularum and Pl. rotundatum are established as new species. Paradictyoarthrinium hydei, Pleopunctum ellipsoideum and Pl. pseudoellipsoideum are reported as new records on the freshwater habitats in Tibetan Plateau, China. The morphological descriptions and illustrations of the new collections are provided.


Introduction
The Tibetan Plateau locates in Central Asia with a mean elevation of more than 4000 m above sea level and an area of about 2,300,000 km 2 [1]. The Tibetan Plateau encompasses remarkable endemic biodiversity as one of the largest and most unique geographical units on earth [2][3][4][5]. The Tibetan Plateau is rich in water resources, from which almost all major rivers in Asia originate and these rivers serve nearly 1.4 billion people [6]. Furthermore, the Tibetan Plateau, acting as an environmental and ecological barrier [7], has an important effect on ecological security in Asia [8]. It is called the "third pole" of the world [9]. Therefore, it is important to investigate and study the Tibetan Plateau.
Freshwater fungi are a critical component of aquatic ecosystems, playing key roles in the cycling of carbon, nutrients and, energy and are involved in the decomposition of dead organic matter [10][11][12][13]. Despite their ecological importance, freshwater fungi have been often overlooked in studies of freshwater ecosystems [10,12]. The unique environmental conditions of the Tibetan Plateau provide an ideal and unmatched setting for the diversity and distribution of freshwater fungi. The plateau's high altitude, cold temperatures, and low precipitation create a distinctive set of environmental conditions that shape the uniqueness of the fungal diversity communities and their interactions with other organisms [7,14].

Collection, Morphological Examination and Isolation
Submerged decaying wood samples were collected from freshwater habitats in Hengduan Mountains of the Tibetan Plateau, China. Fresh specimens were studied following the methods of Senanayake et al. [38]. Microscopic structures were examined by using a stereomicroscope (SteREO Discovery.V12, Carl Zeiss Microscopy GmBH, Göttingen, Germany), photographed by using a Nikon ECLIPSE 80i compound microscope fitted with a Nikon DS-Ri2 digital camera, and measured by using the Tarosoft (R) Image Framework program. Illustrated figures were processed by using Adobe Photoshop CS6 version 10.0 software (Adobe Systems, San Jose, CA, USA).
Single spore isolation was performed on potato dextrose agar (PDA) plates following the methods described in Senanayake et al. [38]. Fungal herbarium specimens and axenic living cultures were deposited into the Herbarium of Cryptogams of the Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS) and Kunming Institute of Botany Culture Collection (KUNCC), Kunming, China. The novel species were registered in the Faceoffungi [39] and Index Fungorum databases (Index Fungorum 2023).

DNA Extraction, PCR Amplification and Sequencing
Fresh mycelia scraped from colonies growing on PDA plates were used for DNA extraction by using a total DNA extraction kit according to the manufacturer's instructions (TOLOBIO Plant Genomic DNA Extraction Kit, Shanghai Co., Ltd., Shanghai, China). Polymerase Chain Reaction (PCR) amplifications were performed by using primer pairs ITS5/ITS4 for internal transcribed spacer rDNA region and covered 5.8S ribosomal (ITS), LR0R/LR5 for the nuclear ribosomal large subunit 28S rDNA gene (LSU), NS1/NS4 for the nuclear ribosomal small subunit 18S rDNA gene (SSU) and TEF1-983F/TEF1-2218R for TEF1, respectively [40,41]. The DNA template was carried out in 25 µL reaction volume containing 21 µL of 1 × Power Taq PCR Master Mix, 1 µL of each primer (10 µL stock) and 2 µL of genomic DNA template. Amplifications were carried out by using the BioTeke GT9612 thermocycler (Beijing City, China). The PCR amplification conditions for ITS, LSU and SSU consisted of initial denaturation at 98 • C for 3 min, followed by 35 cycles of denaturation at 98 • C for 20 s, annealing at 53 • C for 10 s, extension at 72 • C for 20 s and the final extension at 72 • C for 5 min. The PCR amplification conditions for TEF1 consisted of initial denaturation at 98 • C for 3 min, followed by 35 cycles of denaturation at 98 • C for 20 s, annealing at 64 • C for 10 s, extension at 72 • C for 20 s, final extension at 72 • C for 5 min. PCR products were visualized by using 1% agarose gel electrophoresis and distinct bands were checked in Gel documentation system (Compact Desktop UV Transilluminator analyzer GL-3120). The PCR products were sequenced by Tsingke Company, Beijing, China.
Maximum likelihood (ML) analysis was performed by RAxML-HPC2 v.8.2.12 [45] in the CIPRES Science Gateway web server [46] http://www.phylo.org/portal2, accessed on 6 Match 2023, by using 1000 rapid bootstrap replicates and the GTRGAMMA+I model. Bootstrap support values for ML equal to or greater than 75% were given above the nodes in the phylogenetic tree ( Figure 1).
The model of evolution for the Bayesian inference (BI) analysis was performed by using MrModeltest v2.3 [47]. GTR+I+G was selected as the best-fitting model for LSU, TEF1, SSU and ITS dataset. The Markov chain Monte Carlo sampling (BMCMC) was carried out to assess posterior probabilities (PP) by using MrBayes v.3.2.7 [48]. Six simultaneous Markov chains were run for random trees for 1,000,000 generations, and trees were sampled every 200th generation. Bayesian posterior probabilities (PP) equal to or greater than 0.95 were given above the nodes in the phylogenetic tree ( Figure 1). Phylograms were visualized by using FigTree v1.4.0 [49] and rearranged in Adobe Photoshop CS6 software (Adobe Systems, USA). The new sequences were deposited in GenBank (Table 1), and the final alignments and phylogenetic tree were registered in TreeBASE under the submission ID: 30133 (http://www.treebase.org/, accessed on 6 Match 2023). RAxML tree based on analysis of a combined LSU, TEF1, SSU and ITS sequence dataset from three families in Pleosporales, viz. Parabambusicolaceae, Paradictyoarthriniaceae and Phaeoseptaceae. Bootstrap support values for ML equal to or greater than 75% were given above the nodes (black). PP values equal to or greater than 0.95 were given above the nodes (yellow). The tree was rooted to Ceratostomella cuspidata (ICMP 17629) and C. pyrenaica (CBS 129343). The type strains were shown in bold, and the newly generated isolates were shown in red.
The model of evolution for the Bayesian inference (BI) analysis was performed by using MrModeltest v2.3 [47]. GTR+I+G was selected as the best-fitting model for LSU, TEF1, SSU and ITS dataset. The Markov chain Monte Carlo sampling (BMCMC) was carried out to assess posterior probabilities (PP) by using MrBayes v.3.2.7 [48]. Six simultaneous Markov chains were run for random trees for 1,000,000 generations, and trees were sampled every 200th generation. Bayesian posterior probabilities (PP) equal to or greater than 0.95 were given above the nodes in the phylogenetic tree ( Figure 1). Phylograms were visualized by using FigTree v1.4.0 [49] and rearranged in Adobe Photoshop CS6 software (Adobe Systems, USA). The new sequences were deposited in GenBank (Table 1), and the final alignments and phylogenetic tree were registered in TreeBASE under the submission ID: 30133 (http://www.treebase.org/, accessed on May 2023). Bootstrap support values for ML equal to or greater than 75% were given above the nodes (black). PP values equal to or greater than 0.95 were given above the nodes (yellow). The tree was rooted to Ceratostomella cuspidata (ICMP 17629) and C. pyrenaica (CBS 129343). The type strains were shown in bold, and the newly generated isolates were shown in red.  The newly generated sequences are indicated in red and the ex-type strains are bold and "-" indicated unavailable sequences.
Culture characters: Conidium germinated on PDA within 24 h. Germ tubes are produced from around. Mycelia circular, gray and dense, with the entire edge. brown to dark brown, black on the reverse. Notes: Pleopunctum ellipsoideum was introduced by Liu et al. [36], which is isolated from decaying woods in Guizhou Province, China. Based on phylogenetic analyses, our new isolate KUNCC 21-10784 was placed among two species, i.e., Pl. ellipsoideum and Pl. pseudoellipsoideum [36] with 100% ML/1.00 PP support (Figure 1). However, our new isolate shares identical morphological characters to the holotype of Pl. ellipsoideum. Thus, we identified the two strains as Pl. ellipsoideum, and as a new record to the Tibetan Plateau, China.  Saprobic on decaying wood submerged in freshwater habitats. Sexual morph: Undetermined. Asexual morph: Hyphomycetous. Colonies on wood substrate superficial, sporodochial, brown, scattered, gregarious, punctiform. Mycelium immersed in the substratum, composed of septate, branched, subhyaline to grayish brown hyphae. Conidiophores macronematous, mononematous, cylindrical, branched, septate, medium brown, smooth and thick-walled. Conidiogenous cells monoblastic, terminal, integrated, medium brown. Notes: Pleopunctum ellipsoideum was introduced by Liu et al. [36], which is isolated from decaying woods in Guizhou Province, China. Based on phylogenetic analyses, our new isolate KUNCC 21-10784 was placed among two species, i.e., Pl. ellipsoideum and Pl. pseudoellipsoideum [36] with 100% ML/1.00 PP support (Figure 1). However, our new isolate shares identical morphological characters to the holotype of Pl. ellipsoideum. Thus, we identified the two strains as Pl. ellipsoideum, and as a new record to the Tibetan Plateau, China.
Pleopunctum multicellularum R.J. Xu, Q. Zhao and Boonmee, sp. nov., Figure 6.  Culture characteristics: Conidium germinated on PDA within 48 h. Germ tubes are produced from basal cells. Mycelia superficial, irregular circular, flat, entire, dark brown in the center and grayish white near the edge from above. Dark brown in the center with paler towards the edge.
Culture characteristics: Conidium germinated on PDA within 48 h. Germ tubes are produced from basal cells. Mycelia superficial, irregular circular, gray to pale brown in the central cycle from above and pale brown to yellowish to brown from below.  muriform, oval to ellipsoidal conidia. However, Pl. rotundatum differs from Pl. ellipsoideum in having smaller basal cells (5-10 × 8-13 µm vs. 8-20 × 8.5-18.5 µm), and differs from Pl. pseudoellipsoideum in Pl. pseudoellipsoideum has a short, septate conidiophores [36]. Furthermore, multi-gene phylogenetic analyses have shown that Pl. rotundatum can be classified into a distinct clade, which is the sister group of Pl. ellipsoideum and Pl. pseudoellipsoideum with high bootstrap support (100% ML/1.00 PP, Figure 1).

Discussion
The Tibetan Plateau is a region known for its unique biological diversity. Our investigation focuses on the freshwater fungal diversity in the Hengduan Mountains Range, because it is an almost virgin field for the discovery of freshwater fungal diversity. We found seven species from the collections from Hengduan Mountains into Pleosporales based on morphological and multi-gene phylogenetic analyses. The colonies of Pleopunctum are gregarious and widely distributed on the substrate [32,[35][36][37], while Paramonodictys are dispersed and scattered [20,23]. In Paradictyoarthrinium, colonies are generally a powdery mass [26]. Thus far, only asexual morphs have been documented in these three genera. Therefore, to gain a more comprehensive understanding of their systematic relationships, it is imperative to obtain further collections and conduct research on their sexual morphs.
The Tibetan Plateau harbors a wide range of aquatic habitats, including lakes, rivers, and wetlands, which support different fungal communities [7]. Although freshwater fungi play an essential role in the ecosystem, they have been understudied in this region due to few researchers have studied freshwater fungi in this region. It is necessary to continue more studies on their diversity, distribution and adaptation to the harsh environmental conditions of the Tibetan Plateau, which has significant implications for our understanding of the functioning in freshwater ecosystems and for the conservation and sustainable use of freshwater resources in this critical region.