Morphological and multi-gene phylogenetic analyses reveal five new hyphomycetes from freshwater habitats

During the survey on freshwater hyphomycetes in Guangxi, Guizhou and Hainan Provinces, China, five fresh collections were encountered. Based on their morphology, these five isolates were identified as belonging to Hermatomyces, Kirschsteiniothelia, Paramonodictys, Pleopunctum and Sparticola. Multi-gene phylogenetic analyses were performed for each genus, which resulted in the identification of five new species, namely Hermatomyces hainanensis, Kirschsteiniothelia ramus, Paramonodictys globosa, Pleopunctum guizhouense, and Sparticola irregularis. Detailed descriptions and illustrations of the morphological characteristics of these new taxa were provided. This research enriches the biodiversity of freshwater dematiaceous hyphomycetes.


Introduction
Freshwater fungi are a diverse and heterogeneous group that can be classified into different classes (Marvanová, 1980;Goh and Hyde, 1996;Shearer et al., 2009;Baschien et al., 2013;Luo et al., 2019;Dong et al., 2020;Yang et al., 2023).Calabon et al. (2022) listed 3,870 species occurring in freshwater habitats.They play essential roles, such as decomposers for submerged woody debris, in freshwater ecosystems (Wong et al., 1998;Grossart et al., 2019), and many of them possess unique biochemical properties that have great potential for various applications (Krauss et al., 2011;El-Elimat et al., 2021).Therefore, the study of fungal biodiversity in freshwater habitats is important, and five genera are involved in the present paper.
Sparticola was introduced by Phukhamsakda et al. (2016) to accommodate S. forlicesenae, S. junci (type species) and S. triseptata.A fourth species, S. muriformis, was introduced by Karunarathna et al. (2017).All four of these terrestrial species have been found to exhibit sexual morphs in nature.Only S. junci produces a hyphomycetous asexual morph in culture, characterized by semi-macronematous to macronematous, pale brown to brown conidiophores, holoblastic conidiogenous cells, and irregular, brown to dark brown conidia (Phukhamsakda et al., 2016).
In this study, we introduce five new species collected from freshwater habitats in Guangxi, Guizhou and Hainan Provinces, China.Based on morphological characteristics and phylogenetic analyses, they are identified as Hermatomyces hainanensis sp.nov., Kirschsteiniothelia ramus sp.nov., Paramonodictys globosa sp.nov., Pleopunctum guizhouense sp.nov., and Sparticola irregularis sp.nov.Detailed descriptions and illustrations are provided for these five new taxa.

Collections and examination of specimens
Fresh samples were collected from May 2021 to July 2022 in Guangxi, Guizhou and Hainan Provinces, China.The samples were incubated in moist plastic boxes at room temperature for 14 days.A Motic SMZ 168 Series dissecting microscope was used to check the specimen.Fruiting bodies of the new collections were examined and photographed with a Nikon ECLIPSE Ni compound microscope fitted with a Canon 90D digital camera.The software Tarosoft (R) Image Frame Work was used to take measurements of fungal structures, and Adobe Photoshop CC 2019 (Adobe Systems, USA) was used to prepare the photo-plates.
Single conidium isolations were carried out on potato dextrose agar (PDA) media (Senanayake et al., 2020).Germinated conidia were individually transferred to fresh PDA media plates and incubated in a constant temperature incubator at 25°C.Dried specimens were deposited in the Herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China, and the herbarium of Guizhou Academic of Agriculture Sciences (GZAAS), Guiyang, China.Pure cultures were deposited in the Guizhou Culture Collection (GZCC), Guiyang, China.Fungal Names numbers were applied in Fungal Names (2023).1

Phylogenetic analyses
Sequences obtained from different primers were analyzed with related taxa determined by blastn search in NCBI.Alignments for different gene loci were automatically performed by online MAFFT version 7. 2 Trimal v1.2 (Capella-Gutierrez et al., 2009) was used to remove ambiguously aligned regions and uninformative positions with gappyout option.Multi-gene alignments were combined using SequenceMatrix 1.7.8 (Vaidya et al., 2011).Alignments were checked visually using AliView (Larsson, 2014).Sequences derived in this study were deposited in GenBank (Table 1).Maximum likelihood (ML) analyses were performed using IQ-TREE web server (Trifinopoulos et al., 2016).Substitution model was automatically tested.Ultrafast bootstrap (BS) analysis was implemented with 1,000 replicates.Maximum likelihood bootstrap values (ML-BS) equal or greater than 75% are marked near each node.
Bayesian inference (BI) analyses were carried out in MrBayes 3.2.6 (Ronquist et al., 2012) using a Markov Chain Monte Carlo (MCMC) algorithm.The best-fit substitution model GRT + I + G was decided for all four gene regions by MrModeltest 2.3 (Nylander, 2008) under the Akaike Information Criterion (AIC).Two parallel runs of four simultaneous Markov chains were performed for 1,000,000 generations.Trees were sampled every 1,000th generations.Burn-in phase was set at 25% and the remaining trees were used for calculating posterior probabilities (PP).PP values equal or greater than 0.95 are marked near each node.
Trees were visualized with FigTree v1.4.4, 3 and the layouts were edited using Adobe Illustrator CS6 software (Adobe Systems, USA).
Saprobic on decaying wood in freshwater habitat.Sexual morph: Undetermined.Asexual morph: hyphomycetous.Colonies on natural substrate superficial, brown, sporodochial, punctiform.Mycelium mostly immersed, composed of hyaline to pale brown,   Notes: Pleopunctum guizhouense (GZCC 23-0595) clusters together with Pl. menglaense (KUMCC 21-0025 and KUMCC 21-0026) with a weak support in the phylogenetic analyses (Figure 8).However, it can be differentiated from Pl. menglaense by its monomorphic conidia, which are brown and oval to ellipsoidal with a basal cell, while the latter has two types of conidia: spatulate to obovate, hyaline conidia, and brown, ellipsoidal to oblong conidia with 1-3 basal cells (Wanasinghe et al., 2022).The ITS (464 bp) sequence variation between Pl. guizhouense (GZCC 23-0595) and Pl.menglaense (KUMCC 21-0026) occurs in 16   Notes: Sparticola species are typically identified by their sexual morphology (Phukhamsakda et al., 2016;Karunarathna et al., 2017).Sparticola junci is the only species known to produce a hyphomycetous asexual morphology in culture, which is similar to that of our new collection in terms of conidial morphology (Phukhamsakda et al., 2016).Although our collection has larger conidiophores (97-162 × 9-11.5 μm vs. up to 35 × 4-7 μm) than those of S. junci, this difference might be attributed to variations in growth conditions (nature vs. culture).Unfortunately, we were unable to observe sporulation in our culture.Phylogenetically (Figure 10), Sparticola irregularis (GZCC 23-0593) forms a basal clade to the Sparticola group (ML-BS = 74%, PP = 0.93), and could represent a new genus because the LSU (823 bp) sequences comparison between S. irregularis (GZCC 23-0593) and S. junci (MFLUCC 15-0030) shows there are 28 position differences, including 2 gaps.However, considering that we only have one isolate, the evidence is insufficient to propose a new genus.Thus, we provisionally assign our new collection to Sparticola and introduce the new species Sparticola irregularis.Further fresh collections of Sparticola species or the discovery of the sexual morphology of S. irregularis may provide better resolution for its taxonomic identification.

Discussion
In this study, we introduce five new species, namely Hermatomyces hainanensis, Kirschsteiniothelia ramus, Paramonodictys globosa, Pleopunctum guizhouense, and Sparticola irregularis.The discovery of these five new taxa enriches the freshwater fungi resources of China and further reveals the diverse morphology for this group of fungi.Hermatomyces, Kirschsteiniothelia, Paramonodictys and Pleopunctum have been reported from both terrestrial and freshwater habitats (Sun et al., 2021;Calabon et al., 2022;Xu et al., 2023).Among them, Hermatomyces and Kirschsteiniothelia have a worldwide distribution, while Pleopunctum are reported from China and Thailand (Liu et al., 2019;Phukhamsakda et al., 2020;Senwanna et al., 2021;Xu et al., 2023).To date, all published Paramonodictys species are described from China (Hyde et al., 2020;Yang et al., 2022;Xu et al., 2023). However, occurring 10.3389/fmicb.2023.1253239Frontiers in Microbiology 15 frontiersin.orgat different altitudes suggests that Paramonodictys is highly adaptable to different environments and thus may also exist in other countries.Sparticola species mainly occur in Europe except for S. muriformis from China (Phukhamsakda et al., 2016;Karunarathna et al., 2017).In this study, we report Sparticola from freshwater habitat for the first time.
The classification of species in Hermatomyces, particularly H. sphaericus, has been widely debated.Koukol et al. (2018) considered H. chromolaenae, H. saikhuensis and H. tectonae as synonyms for H. sphaericus, a species that they regarded as monomorphic.Using the GCPSR method, Phukhamsakda et al. (2020) further supported this conclusion.Conversely, Tibpromma et al. (2018) identified H. biconisporus as a distinct species that produces two types of conidia, and clusters within H. sphaericus clade.They rejected Koukol's treatment and suggested that H. sphaericus might be a species complex.The taxonomic statuses of two other species in the H. sphaericus clade, H. biconisporus and H. pandanicola, remain unresolved.However,  Koukol and Delgado (2019) speculated that contamination during single spore isolation may have led to a mixture of conidia from H. sphaericus and H. biconisporus, while H. pandanicola might be a hybrid species, or the sequences in GenBank could have been provided erroneously (Koukol et al., 2018).Therefore, it is essential to collect fresh samples of H. biconisporus and H. pandanicola to resolve their taxonomic controversies.

FIGURE 4
FIGURE 4 ML tree (−ln = 9010.980915)based on the combined LSU-ITS rDNA sequences.The combined dataset comprises 32 strains, including the new collection.The alignment comprises 1,461 characters (LSU: 1-872, ITS: 873-1461) including gaps.Among them, number of constant sites are 849, and number of parsimony informative sites are 474.Bootstrap support values for ML greater than 75% and PP greater than 0.95 are given near nodes as ML-BS/PP.The tree is rooted with Phyllobathelium anomalum (MPN242) and Phyllobathelium firmum (ERP 3175).The new taxon is indicated in bold and blue.

FIGURE 6
FIGURE 6ML tree (−ln = 11817.931619)based on the combined LSU-ITS-tef1-α rDNA sequences.The combined dataset comprises 26 strains, including the new collection.The alignment comprises 2,548 characters (LSU: 1-847, ITS: 848-1366, tef1-α: 1367-2548) including gaps.Among them, number of constant sites are 1,802, and number of parsimony informative sites are 515.Bootstrap support values for ML greater than 75% and PP greater than 0.95 are given near nodes as ML-BS/PP.The tree is rooted with Corynespora cassiicola (CBS 100822) and Corynespora torulosa (CPC 15989).The new taxon is indicated in bold and blue.

FIGURE 10 ML
FIGURE 10 ML tree (−ln = 5776.431900)based on the combined LSU-ITS rDNA sequences.The combined dataset comprises 26 strains, including the new collection.The alignment comprises 1,364 characters (LSU: 1-847, ITS: 848-1364) including gaps.Among them, number of constant sites are 1,039, and number of parsimony informative sites are 265.Bootstrap support values for ML greater than 70% and PP greater than 0.90 are given near nodes as ML-BS/PP.The tree is rooted with Paradictyoarthrinium diffractum (MFLUCC 12-0557) and Paradictyoarthrinium tectonicola (MFLUCC 12-0556).The new taxon is indicated in bold and blue.

TABLE 1
Taxa used in this study and GenBank accession numbers. A

TABLE 2
Conidial size of accepted Paramonodictys species.