Freshwater fungi are a diverse and heterogeneous taxonomic group occurring saprobically on partially or fully submerged organic substrates in aquatic habitats. Their life cycle, in whole or part, relies on free freshwater and submerged substrates (Calabon et al., 2020, 2023a). Freshwater environments are categorized into three types: 1) lentic, any natural aquatic environment lacking continuous flow but exhibiting static, low, or slow movement of water as in lakes, ponds, swamps, and pools; 2) lotic, any natural aquatic environment with a continuous flow of water such as rivers, streams, creeks, and brooks; and 3) other habitats, which include artificial water bodies such as in cooling towers and tree holes (Luo et al., 2004). Fungi found in freshwater habitats were grouped into several morphological and ecological entities, viz., freshwater ascomycetes, freshwater hyphomycetes (i.e., Ingoldian fungi, aero-aquatic hyphomycetes or asexual ascomycetes, terrestrial–aquatic hyphomycetes, and submerged aquatic hyphomycetes), freshwater basidiomycetes, coelomycetes, microsporidia, zoosporic fungi, and zygomycetes (Tsui et al., 2016; Schuster et al., 2022). Furthermore, freshwater fungi subsist as saprobes, mutualists, or parasites and have also been isolated as endophytes (Tsui et al., 2016). However, freshwater fungi have an important ecological function in decomposing wood by breaking down complex organic compounds into simpler inorganic materials and facilitating the passage of energy and nutrients across all trophic levels in the food chain (Bärlocher et al., 2011; Sridhar and Sudheep, 2011; Tsui et al., 2016). Two crucial biological characteristics of these fungi include the ability to sporulate underwater and thrive on decaying deciduous leaves and twigs in streams and rivers (Krauss et al., 2011; Wurzbacher et al., 2014; Tsui et al., 2016). In addition, the biotechnological potential of freshwater fungi as producers of bioactive metabolites, with promising values in drug discovery, is becoming evident (Duarte et al., 2013; Shen et al., 2022; El-Elimat et al., 2021; Calabon et al., 2023a). Lignicolous freshwater fungi can degrade indigestible lignocellulose in submerged wood, releasing nutrients into the water. However, their precise ecological role and economic value remain less understood (Bucher et al., 2004; Shen et al., 2022).

Freshwater fungi have been documented since the mid-19th century, and a wealth of information is now available (de Wildeman, 1895; Ingold, 1942, 1955; Calabon et al., 2021). Over the recent past decades, these fungi have been relatively well-studied in Asia, particularly in China, India, and Thailand (Sridhar and Sudheep, 2011; Mehboob et al., 2021; Luo et al., 2019; Dong et al., 2020; Shen et al., 2022; Calabon et al., 2023a, b). In the early stages of studies on freshwater fungi, identification has primarily relied on morphology (Ingold, 1975; Krauss et al., 2011; Calabon et al., 2023a). In the case of yeasts, additional methods such as biochemical, fermentation, and assimilation tests were included. Presently, in addition to morphology, molecular data analyses were applied to taxonomic studies of freshwater fungi (Ranghoo et al., 1999; Nikolcheva and Bärlocher, 2002; Luo et al., 2019; Dong et al., 2020; Calabon et al., 2023a, b). Recent studies revealed that lignicolous freshwater fungi constitute a highly diverse taxonomic group with a substantial population. To date, more than 3,870 species of freshwater fungi have been documented from various substrates and geographical locations (Luo et al., 2019; Dong et al., 2020; Shen et al., 2022; Calabon et al., 2023a, b). Lignicolous freshwater fungi are complex assemblages of mostly filamentous fungi, single-celled chytrids, and yeasts (Tsui et al., 2016). Freshwater fungi belong to Ascomycota, Basidiomycota, Chytridiomycota, and Rozellomycota; in class levels within the Ascomycota, most species are accommodated in Dothideomycetes and Sordariomycetes (Calabon et al., 2022).

Chaetopsina (family Nectriaceae) was established by Rambelli (1956), with the type species Chaetopsina fulva, which was isolated from decaying leaves in northern Italy. Chaetopsina is a dematiaceous hyphomycete genus, the members of which occur on decaying wood, leaves, pine needle litter, and bark, as well as on ascomycetous stromata and soil (Seifert et al., 2011). Most Chaetopsina species are found in tropical and subtropical areas (Lechat and Fournier, 2019). Chaetopsina is initially characterized by reddish-brown setose conidiophores turning yellow in lactic acid, and hyaline, smooth, fusiform ameroconidia, and fertile regions situated terminally or along the axes of the setiform conidiophores (Samuels, 1985; Kirk and Sutton, 1985). Later, some species with dark brown conidiophores or lateral branches on the conidiophores also have been accommodated (Bakhit and Abdel-Aziz, 2021). According to Luo and Zhuang (2010), Chaetopsinectria chaetopsinae was distinct as the sexual morph of C. fulva. Lombard et al. (2015) and Rossman et al. (2016) subsequently recommended using Chaetopsina despite Chaetopsinectria in respect to the 1F = 1N policy, and hitherto, Chaetopsinectria was treated as a synonym of Chaetopsina. The sexual morph of Chaetopsina is seen featuring superficial, non-stromatic, reddish brown to bright red, oval ascomata with an acute ostiolar apex, with walls composed of cells of textura epidermoidea, with paler papilla; unitunicate, clavate, short-stipitate asci containing 8-spored, fusiform, 0–1-septate, hyaline ascospores, and growing in association with upright conidiophores (Lechat and Fournier, 2020). So far, a total of 33 species epithets are listed under Chaetopsina, and four epithets are listed under Chaetopsinectria in Index Fungorum (https://indexfungorum.org/Names/Names.asp; accessed on 21 June 2024). However, eight previously described Chaetopsina epithets have been synonymized under other genera in Nectriaceae or incertae sedis (Index Fungorum, 2024), and the genus now embodies only 25 accepted species wherein molecular data of only 13 species are available (Lechat and Fournier, 2019; Bakhit and Abdel-Aziz, 2021). There are four species in Chaetopsina that have so far been recorded from freshwater habitats, including Chaetopsina aquatica, C. fulva, Chaetopsina hongkongensis, and Chaetopsina polyblastia (Sivichai et al., 2000; Ho et al., 2002; Luo et al., 2019; Bakhit and Abdel-Aziz, 2021). C. fulva and C. polyblastia were found on woody test blocks of Xylia dolabriformis in a freshwater stream of Khao Yai National Park, Thailand (Sivichai et al., 2000). C. hongkongensis was found on decaying submerged wood in Tai Po Kau Forest Stream, Hong Kong, China (Ho et al., 2002). Chaetopsina beijingensis (treated herein as a synonym of C. fulva) was isolated from decaying wood submerged in a freshwater stream in Yunnan, China (Luo et al., 2019). C. aquatica was collected on decaying submerged stems of Phragmites australis (Poaceae) in the River Nile, Sohag, Egypt (Bakhit and Abdel-Aziz, 2021).

Parafuscosporella (family Fuscosporellaceae), typified by Parafuscosporella moniliformis, was introduced by Yang et al. (2016). Yang et al. (2016) also introduced the new order Fuscosporellales to accommodate a new single family Fuscosporellaceae and to which six genera, viz., Bactrodesmiastrum, Fuscosporella, Mucispora, Parafuscosporella, Plagiascoma, and Pseudoascotaiwania were initially accommodated. Parafuscosporella is one of four new genera established by Yang et al. (2016) when the new order Fuscosporellales and the new family Fuscosporellaceae were established. The genus is characterized by spherical to cushion-shaped, black, gelatinous sporodochia, with a jelly-like cover, semi-macronematous, mononematous, compact, flexuous, simple or branched, mostly moniliform, globose to subglobose, ellipsoidal or clavate celled conidiophores, monoblastic, integrated, sometimes discrete, terminal, globose or subglobose, ellipsoidal or clavate conidiogenous cells and acrogenous, ellipsoidal to broadly obpyriform, smooth, dark brown to black conidia with a septum near the base, sometimes with a small protuberance and a pale brown basal cell (Boonyuen et al., 2016; Yang et al., 2016). Ten species are listed in Parafuscosporella, comprising Parafuscosporella aquatica, Parafuscosporella ellipsoconidiogena, Parafuscosporella garethii, Parafuscosporella lignicola, Pa. moniliformis, Parafuscosporella mucosa, Parafuscosporella nilotica, Parafuscosporella obovata, Parafuscosporella pyriformis, and Parafuscosporella xishuangbannaensis (Yang et al., 2016, 2020; Boonyuen et al., 2021; Boonmee et al., 2021; Wang et al., 2023; Li et al., 2023). Species of the genus have been reported from freshwater habitats in China and Thailand (Yang et al., 2016, 2020; Boonmee et al., 2021; Boonyuen et al., 2021). Goh et al. (2023) introduced two novel freshwater fungi from Taiwan, namely, Vanakripa oblonga and Vanakripa taiwanensis. Based on the morphological resemblance between Parafuscosporella and Vanakripa, Goh et al. (2023) treated Parafuscosporella as a synonym of Vanakripa and transferred all Parafuscosporella species to Vanakripa. In contrast, Vanakripa chiangmaiensis and Vanakripa minutiellipsoidea clustered with Conioscypha species were excluded from Vanakripa (Goh et al., 2023). Unfortunately, the type species of Vanakripa, Vanakripa gigaspora, lacks molecular data to clarify the phylogenetic placement. Hence, the congeneric status of Parafuscosporella and Vanakripa is questionable.

Pleurotheciella (family Pleurotheciaceae), typified by Pleurotheciella rivularia, was introduced by Réblová et al. (2012). To date, within the genus Pleurotheciella, only three species, viz., Pleurotheciella erumpens, Pleurotheciella fusiformis, and Pl. rivularia, are known for their sexual morphs, with non-stromatic perithecia, unitunicate asci, and hyaline to subhyaline, 1- or 3–5-septate ascospores, lacking a mucilaginous sheath. The asexual morph of the genus is a dactylaria-like hyphomycete, which is characterized by polyblastic, denticulate conidiogenesis, subhyaline conidiophores, and hyaline, ellipsoidal to ellipsoidal-fusiform, aseptate to multi-septate conidia (Luo et al., 2018). At present, 18 species are accommodated in Pleurotheciella, viz., Pleurotheciella aquatica, Pleurotheciella centenaria, Pleurotheciella dimorphospora, Pl. erumpens, Pl. fusiformis, Pleurotheciella ganzhouensis, Pleurotheciella guttulata, Pleurotheciella irregularis, Pleurotheciella krabiensis, Pleurotheciella lunata, Pleurotheciella nilotica, Pl. rivularia, Pleurotheciella saprophytica, Pleurotheciella submersa, Pleurotheciella sympodia, Pleurotheciella tropica, Pleurotheciella uniseptata, and Pleurotheciella verrucosa, and all accepted species of the genus have been reported for their asexual morphs (Hyde et al., 2018; Luo et al., 2018; Abdel-Aziz et al., 2020; Réblová et al., 2020; Shi et al., 2021; Boonmee et al., 2021; He et al., 2024). The genus has been so far represented from freshwater habitats in China and Thailand, except for Pl. dimorphospora, which is the only species described from terrestrial habitats in China (Boonmee et al., 2021).

In this study, we introduce three new species, Chaetopsina yunnanensis, Parafuscosporella hunanensis, and Pleurotheciella yunnanensis, based on morphology and phylogenetic studies, from freshwater habitats in China. In addition, C. beijingensis is formally synonymized under C. fulva, and the congeneric status of Parafuscosporella and Vanakripa is discussed.

Freshwater fungi exhibit remarkable ecological diversity, playing essential roles in aquatic ecosystems. They act as decomposers, breaking down organic matter like wood and leaf litter, which contributes to nutrient cycling and energy flow. These fungi also form symbiotic relationships with aquatic plants and algae, aiding in nutrient uptake and survival. Some freshwater fungi are pathogens, impacting aquatic plants and animals, while others help control populations within ecosystems. Adapted to both flowing (lotic) and still (lentic) water, they show a variety of morphological and physiological adaptations that allow them to thrive in diverse aquatic environments. Freshwater fungi are also important as bioindicators, providing insights into water quality and ecosystem health. Overall, they are key contributors to ecosystem function, biodiversity, and the balance of freshwater habitats (Gessner et al., 2007; Krauss et al., 2011; Jones et al., 2014; Kuehn, 2016).

Sordariomycetes is the largest class of lignicolous freshwater fungi in Ascomycota, containing approximately 823 species and 298 genera (Luo et al., 2019; Calabon et al., 2022; Shen et al., 2022). In this study, three new freshwater species belonging to the subclasses Hypocreomycetidae and Savoryellomycetidae (Sordariomycetes) are introduced from Hunan and Yunnan provinces, China. The three new species, C. yunnanensis, Pa. hunanensis, and Pl. yunnanensis, are found inhabiting the freshwater habitats, both lotic and lentic. C. yunnanensis was collected in a lake (lentic) and a freshwater stream (lotic), Pa. hunanensis in a freshwater stream (lotic), and Pl. yunnanensis from a lake (lentic). These three species were isolated from submerged decaying branch and wood, indicating their roles as decomposers degrading organic matter in nutrient cycling. It is notable that species of Chaetopsina, Parafuscosporella, and Pleurotheciella are commonly known from aquatic habitats. However, many species such as Chaetopsina guyanensis, C. saulensis, and Pl. dimorphospora have also been found in terrestrial habitats (Lechat and Fournier, 2019; Boonmee et al., 2021). Hence, we speculate the species in genera Chaetopsina and Pleurotheciella tend to inhabit a wide range of environments, including submerged freshwater or wet terrestrial environments. However, all species of Parafuscosporella have so far been found in aquatic environments.

Similar to Tsui et al. (2016), the three new species—C. yunnanensis, Pa. hunanensis, and Pl. yunnanensis—may belong to the group of aquatic–terrestrial hyphomycetes (mitosporic ascomycetes) that are initially found growing on decaying plant material and capable of sporulation underwater. The freshwater fungi in this group such as Canalisporium, Dactylaria, Dictyochaeta, and Sporoschisma, were typically distinguished based on the features of their conidia, conidiophores, and the process of conidiogenesis (Tsui et al., 2016). Since C. yunnanensis, Pa. hunanensis, and Pl. yunnanensis exhibit characteristics similar to those described in aquatic–terrestrial hyphomycetes, they should fit well into this group. Their ability to grow in aquatic environments, along with their possible role in decaying plant material (e.g., branches, leaves, and wood), aligns with the ecological characteristics described for this group of fungi. However, further studies would be required to confirm their precise classification within the group, especially considering conidial morphology and conidiogeneses, as noted by Tsui et al. (2016).

Some freshwater fungi exhibit morphological and ecological specificity (e.g., aquatic spore morphology, attachment structures, growth form adaptation, and spore release strategies), which may display different morphological traits depending on their growth environment, and these traits are often linked to factors such as habitat type, nutrient sources, and interactions with other organisms (Suberkropp, 2011; Naranjo-Ortiz and Gabaldón, 2019). In this study, the three new species, C. yunnanensis, Pa. hunanensis, and Pl. yunnanensis, do not exhibit specific morphological and ecological adaptations to lotic and lentic environments, although detailed studies on their precise adaptations in these habitats are limited. The special morphological characteristics may inconspicuously occur in these three new species, which allow them to adapt to an aquatic environment. However, these three new three species were explored from decaying plant material submerged in freshwater lakes and streams. For instance, most species in Parafuscosporella possess a jelly-like cover on sporodochia or hyaline appendages at the base of conidia. Pa. hunanensis may produce a jelly-like cover; however, this characteristic may disappear during the shifting from the collection site to the laboratory, or this species may generally occur as saprobe in terrestrial and produce some ecological functions that allow its adaptation during its submerged in aquatic environments.

Nonetheless, we can infer potential adaptations based on their general ecological characteristics and roles in aquatic ecosystems. C. yunnanensis, Pa. hunanensis, and Pl. yunnanensis may exhibit morphological traits suited to their respective aquatic environments. In lotic systems, where water flow is continuous, these species may develop stronger attachment structures on their spores or hyphae, which help them remain anchored to substrates such as decaying wood or aquatic plants. In contrast, species in lentic environments, where the water is still, may not require such robust attachment features but may have adaptations for better spore dispersal in stagnant water. Additionally, species in flowing water (lotic systems) may possess conidia with characteristics that allow them to float or travel further, optimizing dispersal in currents. Conversely, in still water (lentic systems), these fungi may produce denser spores or develop morphology that enhances their survival and growth on submerged wood and organic matter, as they would be more reliant on localized nutrient sources (Kuehn, 2016).

The three new species may play crucial roles in nutrient cycling, particularly in wood degradation. In both lotic and lentic environments, they contribute to the breakdown of organic matter such as decomposing wood, which is an important source of nutrients for other organisms in the ecosystem. Their activity in wood degradation releases essential nutrients like nitrogen, carbon, and phosphorus back into the ecosystem, influencing nutrient cycling and supporting a wide range of aquatic life (Gulis et al., 2006, 2008). Moreover, the three new species may interact with other aquatic organisms, including bacteria and invertebrates, through their decomposition activities. The release of small organic molecules during wood degradation can attract microbial communities, facilitating a rich nutrient web. These interactions are crucial for maintaining the health and balance of aquatic ecosystems. The ecological relevance of these fungi in nutrient cycling and wood degradation in lotic and lentic systems is profound. In lotic environments, where constant water movement aids the dispersal of spores, the fungi can contribute to nutrient cycling over large areas, potentially impacting the entire aquatic food web. In lentic environments, they may play a more localized role but still significantly contribute to the recycling of nutrients trapped in stagnant water and organic matter. While studies on these species-specific interactions with aquatic organisms are limited, it is likely that their roles in wood degradation and nutrient cycling are shared across many freshwater fungi.

The species of Chaetopsina are distributed in different climate zones in various countries such as China (C. fulva as C. beijingensis and C. hongkongensis) (Goh and Hyde, 1997; Crous et al., 2014) Egypt (C. aquatica) (Bakhit and Abdel-Aziz, 2021), France (C. guyanensis, C. pnagiana, and C. saulensis) (Lechat and Fournier, 2019, 2020), Thailand (C. penicillata) (Bao et al., 2023), and South Africa (C. gautengina) (Crous et al., 2020). This genus has been reported from both freshwater and terrestrial habitats. For instance, C. penicillata was introduced by Samuels (1985) from terrestrial habitats in Ecuador, Jamaica, and New Zealand. Bao et al. (2023) reported C. penicillata from freshwater habitats in China. Through our study on C. yunnanensis, we noticed the Chaetopsina species can be segregated by their morphological characteristics, such as ascomata dimensions and perithecial wall anatomy, size, shape, and ornamentation of ascospores and conidia of the asexual morph. Furthermore, the morphological traits of our isolates closely resemble those attributed to Chaetopsina. These include red-pigmented erect conidiophores producing unicellular, cylindrical to cylindro-fusoid or ellipsoidal conidia. Phylogenetic analyses of a combined ITS and LSU sequence data indicated that our isolates C. yunnanensis (KUNCC23-12940 and KUNCC23-13014) are grouped with the species of Chaetopsina, forming a distinct lineage that is basal to C. pinicola (CPC 21819). Therefore, considering both the morphological comparisons and the phylogenetic analyses, we proposed the establishment of a new species, C. yunnanensis, in this study.

Meanwhile, we noticed that C. beijingensis has not been formally synonymized under C. fulva so far. While describing a new aquatic species, C. aquatica, from River Nile in Egypt, Bakhit and Abdel-Aziz (2021), quoting Lechat and Fournier (2019), referred that C. beijingensis is a synonym of C. fulva, the type species of the genus. Lechat and Fournier (2019) described two new species of Chaetopsina from Saül (French Guiana) and noted that C. beijingensis is similar to C. fulva, both morphologically and phylogenetically but did not taxonomically synonymize these taxa. In the present study, the phylogenetic analyses coupled with the comparison of their polymorphism demonstrated that C. beijingensis is conspecific with C. fulva, concurring with previous studies (Lechat and Fournier, 2019; Bakhit and Abdel-Aziz, 2021). Hence, C. beijingensis is formally synonymized under C. fulva herein.

Only ITS and LSU sequence data are available for most species of Chaetopsina, of which the ITS gene is currently remarkable as the sufficient phylogenetic marker in delineating the interspecific status of Chaetopsina (Lechat and Fournier, 2019, 2020; Bakhit and Abdel-Aziz, 2021). However, many sufficient phylogenetic markers derived from the protein-coding genes were recommended for resolving a better phylogenetic resolution of the family Nectriaceae where Chaetopsina does belong as well as the class Sordariomycetes [e.g., the ATP citrate lyase (ACL1), α-actin (ACT), β-tubulin (TUB2), calmodulin (CMDA), histone H3 (HIS3), the RNA polymerase II largest subunit (RPB1), the RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1 alpha (TEF1-α)] (Lombard et al., 2015; Perera et al., 2023). Of these, sequences of the protein-coding genes are available for few Chaetopsina species such as C. acutispora, C. fulva (type species), and C. penicillata (Lombard et al., 2015). The limitation on molecular data of Chaetopsina can cause taxonomic ambiguities. We, therefore, recommend utilizing multigene phylogeny for resolving taxonomic ambiguities and also providing a better taxonomic resolution on Chaetopsina, expanding to Nectriaceae as well as the class Sordariomycetes, of which the sufficient phylogenetic markers based on the protein-coding genes of Chaetopsina should be derived.

In our studies on Pa. hunanensis, we noticed that the species of Parafuscosporella are morphologically indistinguishable. Hence, it was found necessary to understand their taxonomic boundaries using multigene phylogenetic markers. Phylogenetic affinities of species in Parafuscosporella have been delineated by ITS, LSU, and SSU phylogenetic markers, of which ITS region is currently recommended as a measurable gene for resolving phylogenetic relationships among species in Parafuscosporella (Boonmee et al., 2021; Boonyuen et al., 2021; Li et al., 2023). Nevertheless, the RPB2 gene has always been utilized for delineating taxa in Fuscosporellaceae (Fuscosporellales) where Parafuscosporella accommodated, with other related orders such as Conioscyphales, Pleurotheciales, and Savoryellales in Savoryellomycetidae (Boonyuen et al., 2016; Yang et al., 2016, 2020; Wang et al., 2023). Unfortunately, the RPB2 gene is only available for Pa. garethii, Pa. pyriformis, and Pa. xishuangbannaensis in the total of the known species. This may cause the insufficient phylogenetic resolution on Parafuscosporella correlated with the closely related genera in the family Fuscosporellaceae, extending to the correlation with taxa among the closely related orders in Savoryellomycetidae. Thus, the RPB2 gene is recommended in further clarifying species levels of Parafuscosporella corresponding with other closely related genera in Fuscosporellaceae.

The confusion between Parafuscosporella and Fuscosporella was also discussed in a previous study (Yang et al., 2016). Fuscosporella is morphologically similar to Parafuscosporella but differs in the structure of conidia, which are produced in culture. Fuscosporella produces multi-celled, filamentous to helicoid conidia, while Parafuscosporella produces globose to obpyriform, uni-septate conidia in culture. The same situation exists for Conioscypha in that the species of Conioscypha are largely indistinguishable in morphology. Li et al. (2024) proposed to use the potential of phylogenetic markers to clarify their phylogenetic relationships. The single gene trees of Conioscypha (ITS, LSU, SSU, and RPB2) and combined sequence datasets (LSU-ITS, LSU-ITS-SSU, and LSU-ITS-RPB2) were previously obtained to compare the reliable phylogenetic markers. The results of these prior analyses demonstrated that analysis of the RPB2 gene could provide a better phylogenetic resolution of Conioscypha. Therefore, the RPB2 gene is recommended as a genetic marker for resolving phylogenetic relationships among species in Conioscypha (Li et al., 2024). Simultaneously, the protein-coding genes such as TEF1-α and RPB2 have been recommended for clarifying the taxonomic ambiguities of various genera in Sordariomycetes due to their high utility in resolving phylogenetic relationships at multiple taxonomic levels. TEF1-α and RPB2 are relatively conserved within Sordariomycetes but also exhibit enough variability to distinguish closely related species or genera. Their nucleotide sequences evolve at rates suitable for both deep and shallow phylogenetic analyses, making them useful for resolving both inter- and intra-generic relationships (Matheny et al., 2007; Lücking et al., 2020, 2021). Therefore, it is concluded that the protein-coding genes, especially the RPB2 gene region, are necessary for resolving the taxonomic ambiguities of some genera in Savoryellomycetidae as well as Sordariomycetes, and hence, TEF1-α and RPB2 should be derived for novel taxa in further study.

It is worth noting that Goh et al. (2023) synonymized Parafuscosporella under Vanakripa due to the morphological features of V. gigaspora, the type of Vanakripa, resembling Parafuscosporella. Based on the principle of nomenclatural priority, all nine Parafuscosporella species were transferred to Vanakripa (Goh et al., 2023). However, V. gigaspora as the type species of Vanakripa lacks molecular data to confirm their phylogenetic placement. Hence, this leads the taxonomic confusion on the phylogenetic placement of Vanakripa. Vanakripa was introduced by Bhat and Kendrick (1993), with V. gigaspora as the type species. The genus was considered as genus incertae sedis in Pezizomycotina (Wijayawardene et al., 2021), while a key to species of the genus was provided by Arias et al. (2008). Vanakripa has a special clavate to vermiform, hyaline, separating cells attached to the conidia (Bhat and Kendrick, 1993). The genus currently accommodated a few species lacking genetic sequences. In Goh et al. (2023), phylogenetic analyses demonstrated that Vanakripa formed separated clades relating to Conioscypha and Parafuscosporella. Two new species that were identified as V. oblonga and V. taiwanensis by Goh et al. (2023) formed a clade with Parafuscosporella in Fuscosporellaceae, whereas V. chiangmaiensis and V. minutiellipsoidea are related to Conioscypha in Conioscyphaceae. Goh et al. (2023) re-circumscribed morphological features of Vanakripa compared to Parafuscosporella. Based on conidial ontogeny resemblance, Goh et al. (2023) demonstrated that Parafuscosporella is congeneric with Vanakripa. Therefore, Goh et al. (2023) transferred all Parafuscosporella to Vanakripa without providing phylogenetic evidence from type studies.

In accordance with the morphological trait, many genera in Fuscosporellaceae and Conioscyphaceae are morphologically somewhat similar; however, these genera can be distinguished based on multigene phylogeny of sufficient genes. Unfortunately, Vanakripa formed polyphyletic clades in Conioscyphales and Fuscosporellales (Goh et al. (2023). Considering the morphology of Vanakripa species, Goh et al. (2023) excluded V. chiangmaiensis and V. minutiellipsoidea (clade in Conioscyphales) and some other described species from Vanakripa (viz., Vanakripa chinensis, Vanakripa ellipsoidea, Vanakripa fasciata, Vanakripa inflata, Vanakripa menglaensis, Vanakripa parva, and Vanakripa rhizophorae) due to their conidial morphology being different from V. gigaspora, the type species of Vanakripa, in producing ellipsoidal or broadly obovoid, one-celled conidia. In contrast, V. oblonga, V. taiwanensis (clade as basal of Parafuscosporella in Fuscosporellales in the present study), and other synonymized Parafuscosporella species were treated as Vanakripa sensu stricto due to the conidial morphology resemblance in producing septate, apiosporous, versicolored conidia, with or without the presence of a hyaline appendage at the conidial base (Goh et al., 2023). With this point of view, it is reasonable to segregate Vanakripa into two morphological groups. However, molecular data of V. gigaspora, the type species of Vanakripa, have not yet been derived, and/or the epitype has not yet been designated, leading to the phylogenetic uncertainty for Vanakripa. Hence, the treatment of Vanakripa in different orders may cause taxonomic confusion in Vanakripa, corresponding with Parafuscosporella. In the present study, we, therefore, tentatively place Parafuscosporella as a distinct genus from Vanakripa to avoid taxonomic confusion until the type strain of V. gigaspora is derived from molecular data and/or the epitype is designated for clarifying its phylogenetic affinity. We also believe that caution of synonymization should be maintained at this stage. However, if future studies could confirm that Parafuscosporella and Vanakripa are congeneric based on morphological and phylogenetic evidence, this would greatly advance the taxonomic discussion of the Fuscosporellaceae. We recognize that any changes in classification should take into account the importance of nomenclatural stability, ensuring that the naming and classification system of the family remains consistent and carefully considered within the scientific community.

It is notable that 17 of the total 18 species of Pleurotheciella were collected from freshwater habitats, and only Pl. dimorphospora is from the terrestrial environment. Furthermore, most species have been found in China and Thailand, as well as in Canada, Egypt, France, and Spain ( Table 4 ). In addition, Pl. dimorphospora is dimorphic (with two types of conidial morphology), which can be well distinguished from other species in Pleurotheciella (Boonmee et al., 2021). Initially, the original authors felt that the two types of conidial morphology in vitro were caused by contamination in the culture. However, through single spore isolation and molecular work on both types of conidia, they confirmed that these two types of conidia are of the same species (Boonmee et al., 2021). It is interesting that our study also meets the same situation with the new species, Pl. yunnanensis, showcasing two-type conidial morphology in vitro.

Although in recent times freshwater fungi have been continuously discovered, compared to soil ecosystems, these fungi still lacked sufficient spatial and temporal resolution, especially from those environments falling in various latitudinal zones, ecosystems (such as the water column and sediments), snowclad mountains, and extreme environments like deep-sea vents (Grossart et al., 2019). Yunnan has emerged as a hotspot for lignicolous freshwater fungal research since 2015, resulting in the discovery of a number of new species and new records in some extremely varied genera such as Acrogenospora, Dictyosporium, Distoseptispora, Pleurotheciella, Sporidesmium, and Sporoschisma (Bao et al., 2020; Wan et al., 2021; Shen et al., 2022). In contrast, the lignicolous freshwater fungi in Hunan Province still remained understudied. Hyde et al. (2016) discussed the impacts of riparian vegetation, water pollution, sampling methods, and global warming on the diversity of lignicolous freshwater fungi. However, the diversity, quantitative abundance, and ecological functions of freshwater fungi, particularly their interactions with other microorganisms, remain largely speculative, unexplored, and overlooked in the current understanding of aquatic ecology and biogeochemistry (Haraldsson et al., 2018; Grossart et al., 2019). The three new species explored in the present study from freshwater lakes and streams in Hunan and Yunnan provinces will add to the species number of lignicolous freshwater fungi in China and upgrade the global species numbers of freshwater fungi.

Additionally, genomic studies have revealed that freshwater fungi contain a vast diversity of secondary metabolite pathways (El-Elimat et al., 2021). However, the genes and gene clusters involved in these metabolic processes, as well as the secondary metabolite products, remain mostly unknown (Chiang et al., 2009; El-Elimat et al., 2021). Over the last 30 years, several freshwater fungi have been subjected to chemical investigations, resulting in the isolation of 283 secondary metabolites of wide chemical diversity and a broad range of biological activities (Wang et al., 2008; Hernández-Carlos and Gamboa-Angulo, 2011; Canto et al., 2022; El-Elimat et al., 2021). El-Elimat et al. (2021) reviewed the secondary metabolites of freshwater fungi and summarized those compounds as mainly belonging to alkaloids, terpenes, polyketides, phenylpropanoids and peptides, and some unclassified secondary metabolites. Dong et al. (2009) isolated two novel naphthalene-containing compounds, colelomycerones A and B, and three known metabolites from the culture broth of an unidentified freshwater fungus YMF 1.01029, and those metabolites showed noticeable antifungal and antibacterial activities. Prabhu et al. (2018) found Greensporone C, a secondary metabolite from freshwater fungi inducing mitochondrial-mediated apoptotic cell death in leukemic cell lines. de Souza et al. (2023) investigated communities of 154 culturable freshwater fungi from Antarctic lakes and the capabilities of all cultured fungi to produce various extracellular enzymes at low temperatures and found that the most widely produced enzymes were proteases and pectinases. These active enzymes produced by freshwater fungi have various applications in biotechnological processes in industries including textile, pharmaceutical, food, detergent, and paper, as well as in bioremediation of environmental pollutants (Raghukumar, 2008; Fathima et al., 2022; de Souza et al., 2023). Unfortunately, the study on secondary metabolites of Chaetopsina, Parafuscosporella, and Pleurotheciella is unexplored. Therefore, further studies on secondary metabolites of freshwater fungi in Chaetopsina, Parafuscosporella, and Pleurotheciella will be most rewarding.

The limitations of the current study are essential for providing a balanced perspective and guiding future research. One of the key challenges encountered in this study was the difficulty in morphological differentiation of some freshwater fungal species, particularly given the high morphological plasticity within certain genera. Additionally, our sampling efforts were constrained by limited geographic coverage and sampling time, which may have affected the comprehensiveness of the data. Furthermore, genetic sequencing efforts were limited by the availability of high-quality DNA samples from all species, which constrained the depth of phylogenetic analyses. These limitations underscore the need for more extensive and targeted genetic studies, such as the use of multigene sequencing or the application of environmental DNA (eDNA) sampling techniques, which could help capture a broader diversity of fungi in various aquatic ecosystems. Future research should prioritize these approaches to better understand the full scope of freshwater fungal biodiversity and address the unresolved taxonomic and ecological questions raised in this study.