Biodiversity of Lignicolous Freshwater Hyphomycetes from China and Thailand and Description of Sixteen Species

Freshwater hyphomycetes are a highly diverse group of fungi with a worldwide distribution and have been mostly reported from tropical and subtropical regions. During investigations of freshwater fungi from the Greater Mekong subregion in China and Thailand, sixteen freshwater hyphomycetes (three of them belong to the class Dothideomycetes while thirteen belong to the class Sordariomycetes) were collected. Based on morphology and multi-gene phylogenetic analyses, Neospadicoides thailandica, Pseudodactylaria aquatica, Sporidesmium nujiangense, Tetraploa thailandica, Vamsapriya aquatica and Wongia fusiformis are described as new species; Aquapteridospora bambusinum is proposed as a new combination; Acrodictys liputii, Chloridium gonytrichii, Pseudoberkleasmium chiangmaiense, Pleomonodictys capensis, Sporidesmium aturbinatum and Vamsapriya indica are reported as new country records; and Sporidesmium tropicale, Sporoschisma chiangraiense and Sporoschisma longicatenatum are introduced as three new collections. In addition, a checklist of freshwater fungi from China over the last five years is also provided.


Introduction
Freshwater hyphomycetes are anamorphic (asexual) fungi, typically with relatively large branched stauroform or scolecoform conidia [1]. These taxa commonly live on submerged woody debris, decaying tree leaves and roots of riparian vegetation in lotic habitats [1][2][3][4]. Freshwater hyphomycetes are an ecologically defined group that can be divided into four biological groups, namely aero-aquatic hyphomycetes, terrestrial-aquatic hyphomycetes, submerged-aquatic (amphibious) hyphomycetes and Ingoldian fungi [4][5][6][7]. In this study, we focused on submerged-aquatic hyphomycetes, which are defined as fungi growing on submerged decaying wood [6]. Freshwater hyphomycetes are cosmopolitan with most species reported from temperate, tropic and subtropic regions [2]. They play important roles in the ecosystem, decay when submerged and waterlogged, have woody debris and release nutrients, which are imperative in ecosystem functioning [3].
Traditional identification of freshwater hyphomycetes was based on morphological characters, especially conidiophores, conidiogenous cells and conidia [6]. However, the morphology of some species is quite similar and some morphological characters overlap between taxa and, therefore, morphology alone is not sufficient for reliable identification at lower taxonomic levels [31][32][33]. Molecular data has significantly solved this problem, allowing a better species classification of fungi [31][32][33][34]. For example, species of the freshwater hyphomycetes genus Cancellidium, are quite similar in morphology and previous studies have identified them as the same species. However, Hyde et al. [35] showed that they are not one species, and there are at least four species based on phylogenetic analyses. Molecular data has significantly improved our understanding on the phylogenetic relationships and taxonomy of freshwater fungi. Phylogenetic analyses showed that freshwater hyphomycetes are polyphyletic and distributed in different phyla, such as Ascomycota and Basidiomycota, the dominant phylum being Ascomycota [6,29,30,36], with most species reported in Dothideomycetes and Sordariomycetes [29,30,37], and a few species belonging to Eurotiomycetes [38][39][40]. Some freshwater hyphomycetes have been linked with their sexual morphs [6,29,[41][42][43][44][45].
During an investigation of freshwater fungi from the Greater Mekong subregion, sixteen freshwater hyphomycetes were collected from Thailand and southern China (Yunnan Province and Tibet autonomous region), of which six were new species, one new combination, six new geographic records and three new collections are introduced based on both morphology and phylogeny.

Collection, Isolation and Morphology
Samples of submerged decaying wood were collected from streams or rivers in Yunnan and Tibet autonomous region, China and Thailand during 2015-2020. Sample incubation, examination and morphological studies followed the methods provided by Luo et al. [65]. Single spore isolations were made on potato dextrose agar (PDA) and germinating conidia were transferred to fresh PDA [74]. Herbarium materials were deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand and herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (Herb. HKAS). Cultures were deposited in Mae Fah Luang University Culture Collection (MFLUCC) and Dali University (DLUCC) China. Faces of Fungi and Index Fungorum numbers were registered as outlined in Jayasiri et al. [75] and Index Fungorum (2021). A check list of freshwater fungi from China has been made based on published data.

Phylogenetic Analyses
The taxa used in the phylogenetic analyses were obtained from previous studies and downloaded from GenBank. SEQMAN v. 7.0.0 (DNASTAR, Madison, WI, USA). MAFFT v.7 online program (http://mafft.cbrc.jp/alignment/server/, 12 August 2021) was used to assemble the consensus sequences and align the sequences respectively [79]. BioEdit was used to manually adjust the alignments and the alignment fasta file was converted to Phylip format by phylogeny website tool "ALTER" [80].
Maximum likelihood (ML) analysis was performed in RAxML-HPC v.8 [81,82] on the XSEDE Teragrid of the CIPRES Science Gateway (https://www.phylo.org, 12 August 2021) [83] with rapid bootstrap analysis, followed by 1000 bootstrap replicates. The final tree was selected amongst suboptimal trees from each run by comparing likelihood scores under the GTR-Gamma substitution model. Bayesian analyses were performed by MrBayes v. 3.2 [84], best fit model of DNA evolution for the Bayesian inference analysis were estimated by MrModeltest v. 2.2 [85]. Posterior probabilities (PP) [86,87] were defined by the Bayesian Markov Chain Monte Carlo (BMCMC) sampling method in MrBayes v. 3.0b4 [88]. Phylogenetic trees were visualized with FigTree v. 1.4.2 [89] and modified in Adobe Illustrator CS5 software (Adobe Systems, San Jose, CA, USA). Newly obtained sequences in this study were deposited in GenBank.  Culture characteristics: Conidia germinating on PDA within 24 h. Colonies grew on PDA, reaching 20-30 mm in three weeks at 25 • C. Mycelia superficial, circular, with the entire margin flat and smooth from above, white at the center, pale grey at the edge, greyish brown from below and not producing pigmentation in culture.
Notes: Pseudoberkleasmium chiangmaiense was introduced by Hyde et al. [90] from Thailand. Phylogenetic analyses showed that our new isolate (DLUCC 1655) clustered with the ex-type strain of P. chiangmaiense (MFLUCC 17-1809) with strong bootstrap support (100 ML/1.00 PP, Figure 2). Morphology of our new isolate is almost identical to the holotype of P. chiangmaiense, except the basal cell of conidia in our isolate are hyaline to brown, while in the holotype they are hyaline. Based on both phylogeny and morphology, we identified our isolate as P. chiangmaiense, which is new to China.  Saprobic on submerged decaying wood. Sexual morph: Undetermined. Asexual morph: Colonies effuse and brown or dark greyish brown. Mycelium mostly immersed, composed of branched, septate, subhyaline and hyphae. Conidiophores indistinct. Conidiogenous cells holoblastic, monoblastic or occasionally polyblastic, integrated, determinate, terminal or intercalary and cylindrical. Conidia solitary, dry, straight, septate, verrucose and composed of a conidial body at the base with 2-4 brown to pale brown apical appendages. Conidial body 23-37 × 17-22.5 µm (x = 30 × 20 µm, n = 30) narrowly ovate or ovate, greyish-brown, pale brown to subhyaline at the apex when young, and brown to dark brown at maturity, verrucose and composed of 2-4 closely-adhered vertical columns of cell, with each column 3-5-septate. Appendages 73-136 µm long, 2-3.5 µm wide at the apex and 5.5-8 µm at the base (n = 35). Setose brown to greyish brown, 5-10-pseudoseptate and smooth-walled.
Culture characteristics: Conidia germinating on PDA within 24 h. Germ tubes were produced from the basal cell of conidia. Colonies grew on MEA, reaching 25-35 mm in three weeks at 25 • C. Mycelia was superficial and circular with the entire margin flat and smooth, greyish brown from above, dark brown to black from below and not producing pigmentation in culture. Notes: species of Tetraploa are quite similar and the asexual morphs are characterized by micronematous conidiophores, monoblastic conidiogenous cells and tetraploate conidia composed of four columns, which are short-cylindrical, euseptate, brown, verrucose at the base and with 2-4-setose and septate appendages at the apex [91][92][93]. Tetraploa thailandica is morphologically most similar to T. aquatica. They share some characteristicss, such as monoblastic or occasionally polyblastic conidiogenous cells and conidia composed of four closely-adhered vertical columns of cells, with 2-4 apical appendages. However, T. aquatica has a shorter but wider conidial body (23-37 × 17-22.5 vs. 22.5-27 × 20-24).
Culture characteristics: Conidia germinating on PDA within 24 h. Colonies grew on MEA, reaching 25-30 mm in four weeks at 25 • C, with rough surface, dense mycelia, velvety, dry, dark brown from above, dark brown to black from below; not producing pigmentation in culture. Notes: Pleomonodictys was established for two monodictys-like species, Pleomonodictys capensis and P. descalsii [94]. The genus is characterized by micronematous or semi-macronematous conidiophores, often reduced to conidiogenous loci on the hyphae and blastic conidia which are solitary or in short chains, variable in shape, muriform, dark brown to black. Our species fits well within the species concept of Pleomonodictys.
Culture characteristics: Conidia germinating on PDA within 24 h. Colonies grew on PDA, reaching 20-25 mm in two weeks at 25 • C. Colonies were medium sparse, circular, flat, a slightly rough surface with edge, entire margin well-defined, grey to brown from above, dark brown to black from below and not producing pigmentation in culture.
Culture characteristics Conidia germinating on PDA within 24 h. Colonies growing on PDA, reaching 15-20 mm in two weeks at 25 • C, surface rough, with dense mycelia, dry, rigid, from above greyish white at the center, brown at the edge, from below yellowish brown at the center, greyish brown at the edge and not producing pigmentation in culture.
Culture characteristics: Conidia germinating on PDA within 24 h, germ tubes produced from the basal and apical cell of conidia. Colonies growing on MEA, reaching 20-25 mm in two weeks at 25 • C, surface rough, with dense mycelia, dry, flat, rugose, from above dark brown, from below dark brown to black and not producing pigmentation in culture.
Notes: Sporidesmium tropicale was described by Ellis [99]. The species has a wide distribution, known from Bolivia, China, India, Ghana, Jamaica, Malaya, Nigeria, Sierra Leone, Sri Lanka, Thailand and USA [97,[99][100][101]. Yang et al. [101] has provided sequence data for this species. In our phylogenetic analyses (Figure 9), our new collection clustered with two strains of S. tropicale (HKUCC 10838 and MFLU 17-0850) with strong support (100% ML and 1.00 PP). Morphology of our collection is almost the same as S. tropicale [101]. We, therefore, identified our species as S. tropicale.
Culture characteristics: Conidia germinating on PDA within 24 h, with germ tubes produced from the apical cell of conidia. Colonies grew on PDA, reaching 10-15 mm in one week at 25 • C, rough surface, with dense mycelia, dry, flat, rigid, umbonate from the side view and entire edge, greyish brown from above, dark brown from below andnot producing pigmentation in culture.
Morphologically, Sporidesmium nujiangense is similar to S. appendiculatum and S. chiangmaiense in having obclavate to fusiform conidia with a mucilaginous sheath over the apex. However, conidia of both S. appendiculatum and S. chiangmaiense have a filamentous appendage at the apex, while S. nujiangense lacks this character. Moreover, our phylogenetic analysis showed that S. nujiangense is placed in a different clade to S. appendiculatum and S. chiangmaiense.
Culture characteristics: Conidia germinated on PDA within 24 h and germ tubes produced from the basal cell of conidia. Colonies growing on PDA, reached 20-25 mm in two weeks at 25 • C, rough surface, dense, circular, slightly raised to umbonate with the entire edge, floccose, brown from above, dark brown from below and not producing pigmentation in culture.
Material examined: Sakon Nakhon, Tao  Notes: Neospadicoides was introduced by Luo et al. [28] with three new species: N. lignicola, N. aquatica and N. yunnanensis. The genus is characterized by macronematous, mononematous, septate conidiophores, holoblastic, terminal, integrated conidiogenous cells and acrogenous or acropleurogenous, fusiform, obovoid and septate conidia. Neospadicoides thailandica fits well with the generic concept. However, N. thailandica is unique in the genus by its branched conidiophores, sometimes with a hyaline to brown sheath over the branches and conidia. Other species in the genus lack a sheath and the conidiophores are unbranched. Phylogenetic analyses showed that our collections formed a distinct and stable lineage within Neospadicoides ( Figure 16). Therefore, we introduce our collections as a new species. All species in the genus were reported from freshwater habitats in China [28], while N. thailandica was collected from Thailand. Saprobic on submerged decaying wood. Sexual morph: Undetermined. Asexual morph: Colonies on superficial substratum, effuse, scattered, hairy and brown to dark brown. Mycelium partly superficial and partly immersed, composed of branched, septate, brown to dark brown and with smooth hyphae. Conidiophores 97-180 × 4.5-6 µm (x = 138.5 × 5.5 µm, n = 20), macronematous, mononematous, simple, cylindrical, erect, 4-8-septate, straight or slightly flexuous, pale brown to brown and smooth-walled. Conidiogenous cells holoblastic, monoblastic, terminal, integrated, cylindrical and truncate, brown. Conidia 11.5-15 × 3.4-5 µm (x = 13 × 4 µm, n = 30), acrogenous, dry, solitary, muriform, subglobose, obovoid to pyriform, truncated at base, rounded at apex, pale brown to subhyaline at the basal cell, brown to greyish brown at other parts, with 3-4-transverse septa and 2-longitudinal septa, constricted at the septa, with conspicuous pores in the septa and smooth. Culture characteristics: Conidia germinating on PDA within 24 h, germ tubes produced from the basal cell of conidia. Colonies grew on PDA, reaching 20-25 mm in two weeks at 25 • C, surface rough, circular, with dense mycelia, velvety, slightly raised to umbonate, with edge entire, floccose, white at center from above, yellow at the edge, dark brown at center from below, pale yellow to greyish yellow at the edge and not producing pigmentation in culture.
Material  Notes: In the phylogenetic analyses, our isolate clustered with Acrodictys liputii (HSAUPmlg 2137) with strong bootstrap support (100% ML/1.00 PP, Figure 18). Morphology of our isolate and the holotype of A. liputii are indistinguishable. Acrodictys liputii was introduced by Cai et al. [106] and collected on submerged bamboo culm from the Philippines. Xia et al. [107] provided sequence data for this species and first reported this species from China. Our collection is the first record from Thailand. Saprobic on decaying wood submerged in freshwater habitats. Sexual morph: Undetermined. Asexual morph: Colonies effuse, hairy and dark green to dark brown. Mycelium superficial. Conidiophores 190-336 × 4.5-6.5 µm, (x = 262.5 × 5.5 µm, n = 15), macronematous, mononematous, single, unbranched, septate, gradually becoming narrower towards the apex, with 3-4 long branches at the upper part, with 2-5 whorls of phialides in the midsection to lower section and a single phialide at the apex, dark brown and paler towards the apex. Conidiogenous cells 9-16.5 × 3-4 µm, (x = 12.5 × 3.5 µm, n = 25), cylindrical to lageniform, phialides, producing conidia from multiple entero-blastic conidiogenous loci and phialides borne on collar hyphae around the conidiophore. Conidia 3.5-4.5 × 2.5-3.0 µm, (x = 4 × 2.5 µm, n = 35), globose to subglobose, aseptate and hyaline to subhyaline. Culture characteristics: Conidia germinated on PDA within 24 h. Colonies grew on PDA, reaching 20-25 mm in two weeks at 25 • C, with rough surface and dense mycelia, dry, rigid, umbonate from the side view and entire edge, greyish brown to pale brown at the margin from above, white to grey at the center, dark grey to brown from below and not producing pigmentation in culture.
Material examined: Sakon Nakhon, Tao  Notes: our new collection is identified as Chloridium gonytrichii based on both phylogeny and morphology. Chloridium gonytrichii is characterized by single, unbranched conidiophores with 5-8 whorls of phialides in the midsection, a single phialide at the apex, phialide and cylindrical to lageniform conidiogenous cells ellipsoid conidia [28,108]. Our collection is almost the same as the holotype of C. gonytrichii [108]. Therefore, we identified our new collection as C. gonytrichii.
Chloridium gonytrichii was described by Fernández et al. [108] with its sexual and asexual morphs. It was originally placed in Melanopsammella. Crous et al. [109] provided sequence data for this species and phylogenetic analysis showed that C. gonytrichii clustered with Melanopsammella vermicularioides. Réblová et al. [110] transferred it to Chloridium, synonymized C. gonytrichii under Melanopsammella gonytrichii. Phylogenetic analyses of Luo et al. [28] and Lin et al. [111] showed that gonytrichii formed a stable and well-supported lineage within Chloridium. In our analyses, C. aseptatum  Chloridium aseptatum was introduced by Wei et al. [113], based on morphology and ITS sequence data, Yuan et al. [112] synonymized C. macrocladum instead C. aseptatum, our phylogenetic analysis also obtained the same result, C. aseptatum (MFLUCC 11-0216) clustered with C. gonytrichii ( Figure 20). Hence, we are in agreement with Yuan et al. [112], and synonymized both C. macrocladum and C. aseptatum under C. gonytrichii, based on phylogeny and morphology.  Saprobic on decaying wood submerged in freshwater habitats. Sexual morph: Undetermined. Asexual morph: Colonies were effuse, hairy and black, with long chains of conidia. Mycelium immersed, composed of brown to dark brown and unbranched hyphae. Setae 99−162 × 4-6 µm, capitate, scattered or in groups mixed with conidiophores, straight or slightly flexuous, apex swollen, surrounded by hyaline mucilage, median brown, subhyaline to pale brown near the apex and septate. Conidiophores 120-170 µm long, 12-16 µm wide at venter, 5-8 µm wide below venter, 13-15 µm wide above, macronematous, mononematous, solitary or in a small group of 2-3, erect, unbranched, straight or slightly flexuous, arising from dark brown to black bulbous base, dark brown to black, paler at the torn apex, composed of a cylindrical stipe and a swollen venter with a long cylindrical neck, sometimes proliferating percurrently and smooth. Conidiogenous cells monophialidic, integrated, percurrent, lageniform, brown and frayed at the apex. Conidia 27-32 × 11-13 µm (x = 29 × 12 µm, n = 30), catenate, cylindrical, hyaline and smooth-walled when young, becoming olivaceous brown to dark brown and verrucose at maturity, with a big guttule in each cell when mature, 1-septate, conspicuously darkened and slightly constricted at the septa and darkened at both ends.
Culture characteristics: Conidia germinated on PDA within 24 h and colonies grew on MEA, reaching 15-25 mm in two weeks at 25 • C, withrough surface and dense mycelia, velvety, dry, umbonate in the middle from the side view, edge undulate, white to pale grey from above, dark grey to brown at the margin from below andnot producing pigmentation in culture.
Notes: Sporoschisma chiangraiense was introduced by Hyde et al. [90], it is unique in Sporoschisma by having 1-septate, verrucose conidia, while conidia of other species are multi-septate with smooth-walled. Phylogenetic analyses showed that our new isolate MFLUCC 18-0336 clustered with the ex-type strain of S. chiangraiense (MFLUCC 18-0703) with high bootstrap support ( Figure 19). Morphology of our isolate and holotype of S. chiangraiense are almost the same, except the wall of conidia; conidia of our isolate are verrucose at maturity, while this character was not observed in holotype. Based on both phylogenetic analyses and morphology characters, we identified our new isolate as S. chiangraiense.
Culture characteristics: Conidia germinated on PDA within 24 h and colonies grew on MEA, reaching 25-30 mm in three weeks at 25 • C, with rough surface and dense mycelia, dry, raised from the side view and entire edge, dark grey at the margin from above, pale grey at the middle, dark brown at the margin from below, greyish brown at the middle and not producing pigmentation in culture.
Notes: Species of Pseudodactylaria are characterized by single, unbranched, septate, hyaline conidiophores, polyblastic, denticulate conidiogenous cells and solitary, fusoidellipsoid, hyaline conidia [69,114,115]. Pseudodactylaria aquatica fits well with the generic concept of Pseudodactylaria, such as, single, unbranched, septate conidiophores, polyblastic, denticulate conidiogenous cells and solitary, fusoid-ellipsoid and hyaline conidia. However, our species can be distinguished from other Pseudodactylaria species in having brown to dark brown conidiophores, which are in groups of 3-5, and cylindrical, narrowly fusiform conidia with a hyaline appendage at the base. While other Pseudodactylaria species have single conidiophores and conidia are lacking an appendage. Phylogenetic analysis showed that P. aquatica formed a distinct lineage within the genus ( Figure 24) [69,114,116]. Pseudodactylaria species were reported as saprobes from freshwater or terrestrial habitats in Australia [116], China [55,69] and Thailand [114]. Our new species was collected from freshwater habitats in Thailand.
Culture characteristics: Conidia germinated on PDA within 24 h and colonies grew on MEA, reaching 25-30 mm in three weeks at 25 • C, dense, floccose, dry, raised from the side view and entire edge, greyish yellow at the middle from above, pale yellow at the margin, dark yellow at the middle from below, pale yellow at the margin and not producing pigmentation in culture.
Culture characteristics: Conidia germinated on PDA within 24 h and colonies grew on MEA, reaching 30-35 mm in three weeks at 25 • C, dense, floccose, dry, raised from the side view with entire edge, pale grey at the middle from above, white to greyish white at the margin, dark yellow at the middle from below, pale yellow at the margin and not producing pigmentation in culture.
Material Notes: The new collection is identified as Vamsapriya indica based on morphology and phylogeny. In the phylogenetic analysis, our new isolate clustered with the ex-type of V. indica (MFLUCC 12-0544) with strong bootstrap support (95 ML/1.00 PP, Figure 25). Morphology of our collection is almost identical to the holotype of V. indica, except for the conidial size; the conidia of holotype are much longer than our collection (DLUCC 2062).
Vamsapriya indica was introduced by Dai et al. [117] and was collected on bamboo culms in Thailand and India. While our collection was collected from freshwater habitats in China, this is the first report of this species from China.

Biodiversity of Freshwater Fungi in China
Hu et al. [63] documented the biodiversity of aquatic fungi in China and reported 782 freshwater fungi from 24 provinces/districts of China. In recent years, many new species have been introduced from China and over the last five years, studies of freshwater fungi in China have been rapidly increasing. In this paper, a checklist of freshwater fungi in China from 2015-2020 is provided (Table S1). A total 243 freshwater fungi have been described in China during last five years, of which 170 species are new species ( Figure 28). Thus, until 2020, about 1025 freshwater fungi have been reported from China. Freshwater fungi reported in China are highly diverse in Ascomycota, a few are Zygomycetes [63]. All the 243 freshwater fungi reported in China belong to Ascomycota as follows: 241 species belong to 56 families and 25 orders, of which 25 families and 5 orders belong to Dothideomycetes, one family and one order belong to Eurotiomycetes, 30 families and 19 orders belong to Sordariomycetes, two species have been referred to as ascomycota incertae sedis (Table S1). The most common orders in Dothideomycetes are Pleosporales, followed by Tubeufiales, while the common orders in Sordariomycetes are Chaetosphaeriales, followed by Pleurotheciales and Hypocreales (Table S1).
During the last five years, studies of freshwater fungi in China have mainly been focused on southern China (Yunnan, Guizhou, Guangxi, Hainan, Jiangxi, Sichuan and Tibet province, Figure 29), while other regions are poorly reported. In southern China, most species were reported from Yunnan province (194 species), flowed by Guangxi (21 species), Guizhou (10 species), Hainan (1species), Jiangxi (3 species), Sichuan (1 species) and Tibet provinces (1 species). Few species were reported from other regions of China, e.g., Hubei, Jiangxi, Xinjiang provinces and Taiwan (Figure 29), and this points out the regional limitations in research on freshwater fungi in China. Freshwater fungi in many regions of China are still to be studied and the current trend suggests that a large proportion of new species awaits discovery in China. In the present study, we report sixteen freshwater hyphomycetes from Thailand and China. Six new species, six new country records (four new records for China and two new records for Thailand), one new combination and three new collections are introduced. Of the sixteen freshwater hyphomycetes, thirteen of them are Sordariomycetes and three are Dothideomycetes. These taxa are from twelve genera, namely. Aquapteridospora, Acrodictys, Chloridium, Neospadicoides, Pseudoberkleasmium, Pseudodactylaria, Pleomonodictys, Sporidesmium, Sporoschisma, Tetraploa, Vamsapriya and Wongia, of which two genera, Aquapteridospora and Neospadicoides were exclusively from freshwater habitats and were known only from China and Thailand [28,29]. Acrodictys, Chloridium, Pseudoberkleasmium, Pseudodactylaria, Sporidesmium, Sporoschisma and Tetraploa are commonly found freshwater habitats. Pleomonodictys and Vamsapriya were first reported from freshwater habitats and China in this study [94,95,[117][118][119][120][121].

Discussion
Freshwater hyphomycetes are a highly diverse group, with a worldwide distribution. They can adapt to different habitats and environments. They have unique adaptions to adapt and survive in the freshwater habitats with the distinct conidia morphologies [122].
The conidia of freshwater hyphomycetes have diverse shapes and sizes. For example, most Ingoldian fungi have tetraradiate, branched or filiform conidia and these can help attach to surfaces and colonize substrates [6,123,124]. When a tetraradiate spore attaches to the surface of a substrate, it attaches at three points, with the spore acting as a tripod, representing a very stable attachment form [123]. Some species have conidia with prominent sheaths or appendages and these characters strengthen adhesion to surfaces of substrate, which also helps to prevent conidial detachment by strong water currents [124,125]. Freshwater hyphomycetes are dominant fungi on submerged leaves and woody substrates. The main advantage of these fungi on submerged leaves and woody substrates is that they have the ability to maintain activity at low temperatures and degrade submerged organic matter under various climatic conditions [126][127][128].
Traditional identification of freshwater hyphomycetes was mainly based on morphological characters but with the advent of DNA sequence analyses, the identification and classification of freshwater hyphomycetes have greatly improved with better insights into species complexes and linking sexual-asexual morphs as well resolving problems associated with dual nomenclature. Freshwater hyphomycetes are distributed in different phyla, classes and orders. Despite some species having similar morphologies, phylogenetic analyses showed that they are polyphyletic and associated with different orders, families, and classes, e.g., Acrodictys-like taxa and Sporidesmium-like taxa [101,124]. Sporidesmium is a typical example to show the polyphyletic nature of freshwater hyphomycetes, the genus was redescribed by Ellis [97] with a broad generic concept, characterized by solitary or gregarious conidiophores, monoblastic, determinate or percurrent conidiogenous cells, cylindrical, fusiform, obclavate, obpyriform and sometimes rostrate conidia [71,97,99,129]. Later studies have segregated Sporidesmium in several different genera, namely Ellisembia, Imimyces, Linkosia, Penzigomyces, Polydesmus, Repetophragma, Sporidesmiella and Stanjehughesia based on morphological characters [130,131]. Recent studies found that Sporidesmium is polyphyletic and related to different orders and families in Dothidieomycetes and Sordariomycetes [71,101,102,132]. Our study herein reports similar results.
In this study, three Sporidesmium species are introduced. Phylogenetic analyses showed that the three species grouped in three different clades within Diaporthomycetidae S. aturbinatum were within Sporidesmiaceae with strong bootstrap support (Figure 13), while S. tropicale and S. nujiangense were phylogenetically distant from Sporidesmiaceae in two other clades. S. tropicale clustered as a sister taxon to Bullimycetaceae with low support, which is consistent with the analyses of Yang et al. [101]. The placement of this species is still questionable and further phylogenetic analyses are needed to resolve its placement. The novel species, Sporidesmium nujiangense clustered with S. olivaceoconidium, S. aquaticivaginatum and S. guizhouense in a stable and well-supported clade sister to Distoseptisporales. Yang et al. [101] showed that S. olivaceoconidium and S. aquaticivaginatum are basal to Sporidesmiaceae with low support, while, in the analyses of Liu et al. [102], S. olivaceoconidium and S. aquaticivaginatum and S. guizhouense formed a stable clade close to Jobellisiales. Morphology of these taxa are not that different from other taxa in the Sporidesmiaceae; however, they are phylogenetically distinct.
Freshwater fungi in Thailand have been studied for several decades [18,47,48,50,53,133]. Calabon et al. [134] summarized the studies of freshwater fungi in Thailand during 2015-2020 with a total 129 new species. Fungal numbers have been estimated between 2.2 and 3.8 million; however, only around 2.6 and 4.5% species of fungi and fungus-like taxa have been accepted [135]. Over the past five years, 298 new species have been reported from China and Thailand, which indicates a high diversity of freshwater fungi, and many new species are still not yet discovered. Recently, the new generation sequencing (NGS) method has been used to better assess fungal diversity, and this greatly improved our understanding on the diversity, ecology and distribution of fungi [136][137][138][139][140]. Studies of freshwater fungi are mainly focused on lignicolous freshwater fungi but fungi on other hosts are poorly reported. The diversity of freshwater fungi is much higher than previously thought. Using NGS method to investigate freshwater fungal diversity may help for a better understanding of their ecology and distribution.