Four novel species of Pleurotheciaceae collected from freshwater habitats in Jiangxi Province, China

During an investigation of fungal diversity from freshwater environments in different regions in Jiangxi Province, China, four interesting species were collected. Morphology coupled with combined gene analysis of an ITS, LSU, SSU, and rpb2 DNA sequence data showed that they belong to the family Pleurotheciaceae. Four new species, Pleurotheciella ganzhouensis, Pla. irregularis, Pla. verrucosa, and Pleurothecium jiangxiense are herein described. Pleurotheciella ganzhouensis is characterized by its capsule-shaped conidia and short conidiophores, while Pla. irregularis has amorphous conidiophores and 3-septate conidia. Pleurotheciella verrucosa has cylindrical or verrucolose conidiogenous cells, 1-septate, narrowly fusiform, meniscus or subclavate conidia. Pleurothecium jiangxiense characterized in having conidiogenous cells with dense cylindrical denticles and short conidiophores. Pleurothecium obovoideum was transferred to Neomonodictys based on phylogenetic evidence. All species are compared with other similar species and comprehensive descriptions, micrographs, and phylogenetic data are provided.


Introduction
Freshwater fungi refer to fungi that depend on aquatic environments for their entire or partial life cycle (Shearer, 1993;Calabon et al., 2023).They play a vital role in maintaining the balance of the freshwater ecosystem.Freshwater fungi are involved in the nutrient cycling of the ecosystem, they can decompose plant litter and other carbon sources that are difficult to degrade, such as insect bones, fish scales, and some animal hair (Palmer et al., 1997;Yuen et al., 1998;Vijaykrishna et al., 2005;Cai et al., 2006;Hyde et al., 2016).There have been some studies on lignicolous freshwater fungi in Jiangxi Province, China (Chen et al., 2022;Liu et al., 2022;Peng et al., 2022;Zhai et al., 2022;Yan et al., 2023).However, Pleurotheciaceae Réblová species have not been reported in Jiangxi Province so far.
Pleurotheciella was introduced by Réblová et al. (2012) to accommodate two species, Pla.rivularia and Pla.centenaria, which have nonstromatic peridium, unitunicate asci, persistent paraphyses and hyaline, and 3-septate ascospores.They have dactylaria-like asexual morph characterized by holoblastic, denticulate conidiogenous cells, subhyaline conidiophores and hyaline, septate conidia.Based on morphology and phylogenetic analyses, Réblová et al. (2015) transferred Dactylaria uniseptata Matsushima to Pleurotheciella as Pla.uniseptata.The species subsequently introduced later in Pleurotheciella are mainly asexual morphs.The morphological characteristics of most species in Pleurotheciella are similar to Pleurothecium in terms of conidiophores and denticulate conidiogenous cells, but they usually have conidia with a single septum (Hyde et al., 2018;Luo et al., 2018;Abdel-Aziz et al., 2020;Réblová et al., 2020;Dong et al., 2021;Shi et al., 2021).In this study, we introduce three new species of Pleurotheciella based on morphological characters and analyses of ITS, LSU, SSU, and rpb2 sequence data which are isolated from aquatic habitats in Jiangxi, China.
In this study, we introduce four new species collected from Jiangxi province, China.Detailed descriptions and illustrations of morphological characteristics are provided for the new taxa.

Samples collection, morphological observation, and isolation
Submerged decaying wood were collected from streams and rivers in Jiangxi province, China, and brought back to the laboratory in sealed plastic bags.The samples were incubated at room temperature (25°C) for 2 weeks in plastic boxes, spraying sterile water for moisturizing during the incubation.The samples were viewed under a Nikon SMZ-1270 microscope (Nikon Corporation, Japan) to observe fungi.Micro-morphological characteristics were observed and captured using a Nikon ECLIPSE Ni-U compound microscope (Nikon Corporation, Japan), equipped with a Nikon DS-Fi3 camera.All measurements were calculated using PhotoRuler Ver.1.1 software. 1Figures were processed using Adobe Photoshop CS6 Extended version 10.0 software (Adobe Systems, United States) (Zhai et al., 2022).
Pure cultures of fungi were obtained by single spore isolation method described by Chomnunti et al. (2014).The germinated conidia were individually transferred to potato dextrose agar (PDA) and incubated at 25°C for 2 weeks.The fungal cultures were deposited in the Jiangxi Agricultural University Culture Collection (JAUCC) and the herbarium specimens were deposited in the Herbarium of Fungi Jiangxi Agricultural University (HFJAU).

Phylogenetic analyses
The combined dataset consists of 39 taxa including our newly generated taxa.Dematipyriforma aquilariae was used as the out-group taxon.Taxa with the highest similarities to our strains were determined with standard nucleotide BLASTn searches in GenBank. 2  The other sequences used in the analyses were obtained from the recent publications (Luo et al., 2018;Réblová et al., 2020).Detailed information on fungal strains used in this paper is provided in Table 1.
The aligned matrix for the combined analysis, ITS + LSU + SSU + rpb2, comprised 3,322 bp, including 599 bp of ITS,  Etymology: Referring to the collecting place, Ganzhou city, Jiangxi Province, China.

Discussion
Pleurotheciaceae members are mainly distributed in the tropics and subtropics, with a few in central and southern Europe, such as P. semifecundum, P. recurvatum, Pla.erumpens, and Pla.rivularia (Réblová et al., 2012;Réblová et al., 2020).Most species of Pleurotheciella and Pleurothecium have been reported in aquatic habitat from Yunnan province, China, and Thailand (Luo et al., 2018).Inaddition, two species, Pla.nilotica and Pla.obovoideum, have been reported in Japan and and Egypt, respectively, (Arzanlou et al., 2007;Abdel-Aziz et al., 2020).Currently there are no reports of Pleurotheciella or Pleurothecium from Jiangxi Province, China.
Ten strains including four new species introduced in this paper are all from the freshwater environment in Jiangxi Province.Pleurotheciaceae species observed mostly from submerged decaying woods (Luo et al., 2018;Réblová et al., 2020).The 10 strains examined 10.3389/fmicb.2024.1452499 Frontiers in Microbiology 10 frontiersin.org in this study were all recovered from decaying wood within a freshwater ecosystem.These strains were observed and isolated from the wood over a period of time.Upon observing a substantial collection of specimens, no sexual reproductive patterns have been identified.Consequently, it appears that species within the genera Pleurotheciella and Pleurothecium may predominantly exist in an asexual form within their natural habitat.
According to phylogenetic analyses, species of Saprodesmium and Neomonodictys have longer genetic distance from other species of Pleurotheciaceae.This observation underscores the need for additional research and discovery to enhance the taxonomic classification within the Pleurotheciaceae.Sterigmatobotrys has not been definitively identified as a genus within the Pleurotheciaceae family, but Sterigmatobotrys groups together with species of Pleurotheciaceae with a strong statistical support (Figure 1).Previously, P. obovoideum was reported with a separate phylogenetic tree not fully integrated into other species sequences of Pleurotheciaceae, which have some problems in its taxonomic position (Arzanlou et al., 2007).According to phylogenetic analysis, we adjusted the taxonomic position of Pleurothecium obovoideum to Neomonodictys obovoideum by its strong clade support (PP/BS = 1.00/100) clades with Neomonodictys aquatica and Neomonodictys muriformis.

FIGURE 1
FIGURE 1Phylogenetic tree of Pleurotheciaceae, inferred from the combined regions (ITS-LSU-SSU-rpb2) using Bayesian Inference (BI) analysis.The Dematipyriforma aquilariae was used as the outgroup.The lineages with new species were shown in blue font.The lineages with adjusted species were shown in yellow background.PP ≥ 0.90 and BS ≥ 70% were indicated around the branches.Supported clade (PP/BS = 1.00/99) with the lineage(Continued) consisting of Pleurothecium brunius, Pleurothecium aquaticum, and Pleurothecium guttulatum.Species of Saprodesmium and Neomonodictys have longer genetic distance from other species of Pleurotheciaceae.Sterigmatobotrys groups together with species of Pleurotheciaceae with a strong statistical support.

TABLE 1
Sequences used in this study.

TABLE 1 (
Continued) LSU, 974 bp of SSU, and 890 bp of rpb2.The combined ITS, LSU, SSU, and rpb2 dataset consisted of 57 sequences representing 36 species of the Pleurotheciaceae, two sequences representing two species of Sterigmatobotrys, and one sequence representing one species of Dematipyriforma.The topologies of trees generated by ML and BI analyses are congruent.The Bayesian tree with BS and PP is shown in Figure1.All species of Pleurotheciaceae form a monophyletic group.Pleurotheciella verrucosa groups together with Pla.krabiensis, Pla.tropica, and Pla.sympodia (PP/BS = 0.94/71), which four collections are from two different regional freshwater habitats with a strongsupported clade (BS/PP = 100/1.00).The strains of Pleurotheciella ganzhouensis form a strong-supported clade (PP/BS = 1.00/100), group together with Pla.rivularia,Pla.erumpens,Pla.irregularis,  Pla.centenaria, and Pla.submersa.Collections of Pleurotheciella irregularis form a strong-supported clade (PP/BS = 1.00/99) with Pla.centenaria.Collections of Pleurothecium jiangxiense form a strongly supported clade (PP/BS = 1.00/99) with the lineage consisting of P. brunius, P. aquaticum, and P. guttulatum.Species of Saprodesmium and Neomonodictys have longer genetic distance from other species of Pleurotheciaceae.Sterigmatobotrys groups together with species of Pleurotheciaceae with a strong statistical support.
and more obvious cylindrical conidiogenous site and 3-septate conidia, which significantly distinguished from Neta. denticles