Taxonomy, phylogeny and evolution of freshwater Hypocreomycetidae (Sordariomycetes)

Hypocreomycetidae is a highly diverse group with species from various habitats. This subclass has been reported as pathogenic, endophytic, parasitic, saprobic, fungicolous, lichenicolous, algicolous, coprophilous and insect fungi from aquatic and terrestrial habitats. In this study, we focused on freshwater fungi of Hypocreomycetidae which resulted 41 fresh collections from China and Thailand. Based on morphological and phylogenetic analyses, we identified 26 species that belong to two orders (Hypocreales and Microascales) and six families (Bionectriaceae, Halosphaeriaceae, Microascaceae, Nectriaceae, Sarocladiaceae and Stachybotryaceae). Ten new species are introduced and 13 new habitats and geographic records are reported. Mariannaea superimposita, Stachybotrys chartarum and S. chlorohalonatus are recollected from freshwater habitats in China. Based on phylogenetic analysis of combined LSU, ITS, SSU, rpb2 and tef1-α sequences data, Emericellopsis is transferred to Hypocreales genera incertae sedis; Pseudoacremonium is transferred to Bionectriaceae; Sedecimiella is placed in Nectriaceae; Nautosphaeria and Tubakiella are excluded from Halosphaeriaceae and placed in Microascales genera incertae sedis; and Faurelina is excluded from Hypocreomycetidae. Varicosporella is placed under Atractium as a synonym of Atractium. In addition, phylogenetic analysis and divergence time estimates showed that Ascocodina, Campylospora, Cornuvesica and Xenodactylariaceae form distinct lineages in Hypocreomycetidae and they evolved in the family/order time frame. Hence, a new order (Xenodactylariales) and three new families (Ascocodinaceae, Campylosporaceae and Cornuvesicaceae) are introduced based on phylogenetic analysis, divergence time estimations and morphological characters. Ancestral character state analysis is performed for different habitats of Hypocreomycetidae including freshwater, marine and terrestrial taxa. The result indicates that marine and freshwater fungi evolved independently from terrestrial ancestors. The results further support those early diverging clades of this subclass, mostly comprising terrestrial taxa and freshwater and marine taxa have been secondarily derived, while the crown clade (Nectriaceae) is represented in all three habitats. The evolution of various morphological adaptations towards their habitual changes are also discussed.


Introduction
Hypocreomycetidae (Sordariomycetes) is an ecologically and morphologically diverse group. It comprises plant or human pathogens, endophytes, parasites, saprobes, fungicolous, lichenicolous, algicolous, coprophilous and insect fungi from various habitats, including freshwater, marine and terrestrial habitats Hyde et al. 2017Hyde et al. , 2020a. The members of Hypocreomycetidae have light-colored perithecia, nonamyloid or amyloid ascal rings, or those which lack apical rings and most taxa lack true paraphyses (Zhang et al. 2006). Hypocreomycetidae was established by Eriksson and Winka (1997) based on morphology and a single gene (SSU) phylogenetic analysis. Eriksson (2006) showed that Hypocreomycetidae formed a monophyletic clade within Sordariomycetes and four orders viz. Coronophorales, Halosphaeriales, Hypocreales and Microascales were accepted in Hypocreomycetidae.
Extended author information available on the last page of the article 1 3 Coronophorales, Halosphaeriales and Hypocreales are monophyletic, while Microascales is paraphyletic. Zhang et al. (2006) updated the phylogenetic tree for Sordariomycetes using LSU, SSU, tef1-α and rpb2 sequence data. Their analysis concurred with Eriksson (2006), except Melanospora, which formed a sister clade with Coronophorales and can be recognized as a distinct order. Thus, a new order Melanosporales was introduced to accommodate Melanospora (Zhang et al. 2006). Subsequently, Tang et al. (2007) accepted five orders in Hypocreomycetidae. In contrast, Hibbett et al. (2007) accepted only four orders in the subclass whereas Halosphaeriales was placed under Microascales based on phylogenetic analysis. Lumbsch and Huhndorf (2010) accepted four orders and 18 families in Hypocreomycetidae. Boonyuen et al. (2011) and Réblová et al. (2011) introduced Savoryellales and Glomerellales in Hypocreomycetidae, respectively. Maharachchikumbura et al. (2015) re-evaluated the classification of Sordariomycetes based on LSU, SSU, tef1-α and rpb2 sequence data. A new order, Falcocladiales, was added to Hypocreomycetidae; thus, seven orders and 30 families were accepted in this subclass . Consequently, Jones et al. (2015) introduced a new order Torpedosporales and Maharachchikumbura et al. (2016) placed Pleurotheciales in Hypocreomycetidae. Yang et al. (2016) introduced Fuscosporellales in the subclass. Hongsanan et al. (2017) and Hyde et al. (2017) showed that Conioscyphales, Fuscosporales, Pleurotheciales and Savoryellales formed a monophyletic clade, sister to Hypocreomycetidae. Therefore, these four orders were transferred to a newly introduced subclass Savoryellomycetidae. This was confirmed by Dayarathne et al. (2019) based on phylogenetic analysis and divergent time estimates. In addition, Parasympodiellales was assigned in Hypocreomycetidae and Melanosporales was placed under Coronophorales Hongsanan et al. 2017). Seven orders Coronophorales, Falcocladiales, Glomerellales, Hypocreales, Parasympodiellales, Torpedosporales and Microascales were accepted in Hypocreomycetidae based on both phylogenetic analysis and divergence time estimates Hyde et al. 2017Hyde et al. , 2020aHernández-Restrepo et al. 2017). The recent treatment of Hypocreomycetidae was provided by Wijayawardene et al. (2022), who accepted eight orders (including Cancellidiales) and 38 families. Xiao et al. (2023) introduced a new family Polycephalomycetaceae in Hypocreales. Eight orders and 39 families are currently accepted in the Hypocreomycetidae (Wijayawardene et al. 2022). However, few orders and families remain polyphyletic; thus, this subclass needs to be revised (Hyde et al. 2020a;Huang et al. 2021).
The evolution study of Hypocreomycetidae is mainly focused on Halosphaeriaceae as this family comprises most marine species with a few species from freshwater and terrestrial habitats (Sakayaroj et al. 2011;Jones et al. 2017Jones et al. , 2019. Several studies have discussed the evolution of morphological characters and the origin of Halosphaeriaceae (Sakayaroj et al. 2011;Jones et al. 2017Jones et al. , 2019. However, there is no specific studies on the evolution of Hypocreomycetidae especially for freshwater fungi. Several studies have recommended the multiple origins of freshwater fungi and the evolution of freshwater ascomycetes from terrestrial habitats (Shearer 1993;Vijaykrishna et al. 2006;Hyde et al. 2021). Belliveau et al. (2005) investigated the evolution of aquatic hyphomycetes based on molecular data and their results demonstrated multiple origins of aquatic hyphomycetes. However, their study did not obtain any firm conclusions concerning their ancestors. It stated that the sexual and asexual morphs have concurrent adaptations to freshwater habitats as several sexual morphs of freshwater hyphomycetes have been reported on tree branches decaying in the water. However, the evolution of aquatic hyphomycetes, either terrestrial asexual or sexual morphs, is undetermined, and this needs further studies based on molecular and morphological data (Webster and Descals 1979;Webster 1992). Vijaykrishna et al. (2006) initially investigated the origin of freshwater ascomycetes based on the molecular clock, and their results stated that freshwater ascomycetes originated from terrestrial fungi with multiple and independent evolution. Freshwater ascomycetes have unique adaptations to survive in freshwater habitats; an example is the freshwater ascomycetes decompose lignocellulose in woody litter, softening the wood, which is thought to be a better adaptation for degrading wood in water-logged conditions. Another example is the unique morphological characters of freshwater ascomycetes, such as the asci with massive apical rings which help eject ascospores into the air and underwater to dispersal in freshwater. The appendages of ascospores can help the species to attached to the substrates in the running water (Ho and Hyde 2000;Hyde and Goh 2003;Vijaykrishna et al. 2006). However, these adaptions have also been found in terrestrial fungi, indicating that freshwater ascomycetes share a common ancestor with terrestrial ascomycetes (Shearer et al. 2009). Recently, Hyde et al. (2021) investigated the evolution of freshwater Diaporthomycetidae based on phylogenetic analysis and divergence time estimates; their result indicated that freshwater Diaporthomycetidae have evolved from terrestrial fungi and has evolved on several occasions.
Studies concluded that the fungi originated from aquatic habitats and then migrated to terrestrial habitats (Vijaykrishna et al. 2006;Beakes and Sekimoto 2009;Beakes et al. 2012;Jones et al. 2014). Vijaykrishna et al. (2006) made a conclusion based on the previous studies (e.g. Shearer 1993;Wong et al. 1998;Hyde and Wong 2000;Cai et al. 2003) that fungi may occur as pathogens, saprobes or endophytes on plants, then become adapted to the aquatic environment, when these plants invaded water. Some studies stated that fungi originated from the sea and then migrated to terrestrial habitats (Beakes and Sekimoto 2009;Jones et al. 2011Jones et al. , 2014. There is still controversial, and it would be interesting if further studies could focus on this. Hypocreomycetidae is widely distributed worldwide and has been reported in freshwater, terrestrial and marine environments. One-hundred and fifty-six Hypocreomycetidae species have been reported from freshwater habitats and are distributed in five orders viz. Coronophorales, Glomerellales, Hypocreales, Microascales and Torpedosporales (Calabon et al. , 2022. Thus, studying the evolution of Hypocreomycetidae is important for understanding the possible transition and evolution of aquatic and terrestrial ascomycetes. In this study, we aim to (1) investigate freshwater fungi in Hypocreomycetidae with fresh collections based on morphological and multi-gene phylogenetic analyses; (2) establish the divergence time of orders and families in Hypocreomycetidae based on molecular clock analyses and (3) explore the evolution of Hypocreomycetidae based on ancestral state analysis.

Isolation and morphological examination
Samples (submerged woods) were collected from freshwater habitats (lakes and streams) in China and Thailand. The samples were brought to the laboratory in plastic bags. Sample incubation, observation and morphological studies were done following the methods outlined by Luo et al. (2018). Fungal species were isolated using single spore isolation following the method described in Senanayake et al. (2020). Germinating ascospores and conidia were transferred to fresh potato dextrose agar (PDA) media and incubated at room temperature for 2-4 weeks, and cultures were grown for 1-2 months. The cultures obtained from Thailand are deposited at the Mae Fah Luang University Culture Collection (MFLUCC) and the cultures obtained from China are deposited at the Kunming Institute of Botany Culture Collection (KUNCC). Herbarium specimens from China and Thailand were prepared following the methods provided by Luo et al. (2018), and the herbariums were deposited at Mae Fah Luang University (MFLU) and Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (Herb. KUN-HKAS) respectively. Facesoffungi and Index Fungorum numbers are registered as outlined in Jayasiri et al. (2015) and Index Fungorum (2023). The descriptions of the species are added to GMS database (Chaiwan et al. 2021).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fungal mycelium or directly from the ascomatal tissue thalli of fungi as outlined by Wanasinghe et al. (2018). EZ gene™ Fungal gDNA Kit (GD2416) was used to extract genomic DNA following the manufacturer's instructions. The primers are summarized in Table 1. The amplification reactions were performed in 25 μL of PCR mixtures containing 12.5 μL of 2 × Power Taq PCR Master Mix (a premix and ready-to-use solution, including 0.1 Units/μL Taq DNA Polymerase, 500 μm dNTP Mixture each (dATP, dCTP, dGTP, dTTP), 20 mM Tris-HCl pH 8.3, 100 Mm KCl, 3 mM MgCl 2 , stabilizer and enhancer), 1 μL of each primer, 1 μL DNA template and 9.5 μL nuclease-free water. PCR amplification was confirmed on 1% agarose electrophoresis gels stained with ethidium bromide. Purification and sequencing of PCR products were carried out using the above-mentioned PCR primers at Kunming Tsingke Biological Engineering Technology and Services Co., Ltd. (Kunming, P.R. China).

Sequence alignment and phylogenetic analyses
Sequences featuring a high degree of similarity were determined from a BLAST search for each gene to identify the closest matches with taxa in Sordariomycetes. The sequences were assembled using BioEdit and aligned with MAFFT v.7 online program (http:// mafft. cbrc. jp/ align ment/ server/) (Katoh and Standleym 2013;Katoh et al. 2019) and final improvements were made when necessary, using BioEdit v7.2.3 (Hall 1999).
Maximum likelihood (ML) analysis was performed using RAxML-HPC v.8 (Stamatakis 2006;Stamatakis et al. 2008) on the XSEDE Teragrid of the CIPRES Science Gateway online flatform (Miller et al. 2010) 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 GTRGAMMA substitution model. MP bootstrap analyses were performed with PAUP v4.0b10 (Swofford 2002). The analysis was performed using the heuristic search option with 1000 random taxa addition and tree bisection and reconnection (TBR) as the branch-swapping algorithm. All characters were unordered and of equal weight, and gaps were treated as missing data. Maxtrees were unlimited, branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. Clade stability was assessed using a bootstrap (BT) analysis with 1000 replicates, each with 10 replicates of the random stepwise addition of taxa (Hillis and Bull 1993).
Bayesian analysis was performed by MrBayes v3.1.2 (Ronquist et al. 2012). The model of evolution was estimated by MrModeltest 2.2 (Nylander 2004). Posterior probabilities (Rannala and Yang 1996) were performed by Markov Chain Monte Carlo Sampling (MCMC) in MrBayes v. 3.1.2. Six simultaneous Markov Chains were run for 1 billion generations, and trees were sampled every 1000 generation (resulting in 100,000 trees). The first 20,000 trees representing the burn-in phase of the analyses were discarded and the remaining 80,000 (post-burning) trees used for calculating posterior probabilities (PP) in the majority rule consensus tree.
Several recent studies have discussed the criteria to define a species (Boekhout et al. 2021;Chethana et al. 2021;Maharachchikumbura et al. 2021;Voigt et al. 2021). Maharachchikumbura et al. (2021) detailed the integrative approaches including morphological species concept (MSC), biological species concept (BSC), and Evolutionary/phylogenetic species concept (PSC) with chemotaxonomy and divergence time estimation for species delimitation in Ascomycota. In this study, the new species and families were established based on morphological characters, phylogenetic analysis and divergence time estimates. In addition, we also performed base pair comparison for the newly described taxa following Jeewon and Hyde (2016).

Calibration, divergence time and evolutionary rate estimations
This study used one secondary and a fossil calibration for the divergence time estimates. Paleoophiocordyceps coccophagus, a fossil of Hypocreales, which is similar to the asexual morph of Hirsutella and Hymenostilbe (Sung et al. 2007) that are synonyms of Ophiocordyceps (Ophiocordycipitaceae, Hypocreales; Quandt et al. 2014) was used to calibrate the Ophiocordyceps crown, using an exponential distribution (offset = 100, mean = 27.5, with 97.5% CI of 200 MYA) (Sung et al. 2008;Samarakoon et al. 2016). The crown age of Sordariomycetes with a normal distribution (mean = 250, SD = 40, with 97.5% CI = 338 MYA) was selected as a secondary calibration point .
Divergence time estimates were carried out by BEAST v 1.8.4 (Drummond et al. 2012). Aligned sequence data were partitioned separately for LSU, ITS, SSU, rpb2 and tef1-α dataset, and loaded to prepare an XML file constructed in BEAUti v1.8.0. The substitution models were selected based on jModeltest2.1.1; GTR + I + G for ITS ACC CTC AGT GTA GTG ACC CTT GGC Glass and Donaldson (1995) and LSU, TIM3 + I + G for SSU, TrN + I + G for tef1-α and TIM2 + I + G for rpb2. However, the models TIM2, TIM3 and TrN were unavailable in BEAUti 1.8.4; thus, TN93 was selected by setting the ''All Equal'' for the base frequencies.
An uncorrelated relaxed clock model (Drummond and Rambaut 2007) with a lognormal distribution of rates for each gene estimate was used for the analyses. We used a Yule tree prior, which assumes a constant speciation rate per lineage, and a randomly generated starting tree ). The analysis was run for 300 million generations and parameters were sampled every 30,000 generations. The effective sample sizes were checked in Tracer v.

Ancestral character state analyses
Ancestral character state analysis (Thiyagaraja et al. 2020(Thiyagaraja et al. , 2021 was carried out to reconstruct the evolutionary relationship of habitual changes in Hypocrealmycetidae. The following states were established: freshwater, marine and terrestrial fungi. The platform Reconstruct Ancestral State in Phylogenies (RASP 3.2.1) was used to construct ancestral character analysis, using the Bayesian Binary MCMC based on the divergence time estimate tree (Yu et al. 2015(Yu et al. , 2019. This approach was performed and visualized in RASP 3.2.1 using settings: 10 chains, a sampling frequency of 100, a temperature of 0.1, state frequencies fixed (JC), and among-site rate variation equal. The trees were edited using Microsoft PowerPoint 2013 and converted to jpeg files using Adobe Photoshop CS6 Extended 10.0 (Adobe Systems. U.S.A.).

Phylogenetic analysis
The multi-gene dataset (LSU, ITS, SSU, rpb2 and tef1-α) was used to reveal the relationships of orders and families in Hypocreomycetidae (Fig. 1) The statistical analyses resulted in largely the same topology with high support for most branches in the ML and BI analyses and with similar overall topologies of order and family level relationships in agreement with previous work based on ML and BI analyses Hyde et al. 2020a). Therefore, the best scoring RAxML tree is shown in Fig. 1

Molecular clock analysis
In recent years, divergence time estimates have been used in fungal taxonomic studies, especially in ranking higher taxa. Hyde et al. (2017) and Liu et al. (2017a) have recommended the range to recognize subclasses, orders, and families in the classes Sordariomycetes and Dothideomycetes. In Sordariomycetes, the stem ages of orders and families are recommended within 150-250 MYA and 50-150 MYA, respectively. According to the divergence time estimates (Fig. 2), the crown and stem ages of Sordariomycetes are 302 MYA and 323 MYA (Fig. 2), which are quite similar to Hyde et al. (2020a). The crown and stem ages of Hypocreomycetidae are 290 MYA and 302 MYA, respectively. The stem ages of all the orders in this subclass are listed in Table 2, including a new order Xenodactylariales. The phylogenetic analysis and divergence time estimates support all orders.
The stem ages of the orders are compared with previous studies (Hyde et al. , 2020a. In our study, the stem ages of Falcocladiales, Glomerellales, Hypocreales and Microascales are similar to the previous studies (Table 2). However, the divergence time of Coronophorales and Torpedosporales are older than Hyde et al. (2017Hyde et al. ( , 2020a (Table. 1). The different fossils, models and substitution rate variation can cause different results of divergence times (Beimforde et al. 2014). Our study uses one fossil data (Paleoophiocordyceps coccophagus and a secondary calibration (crown age of Sordariomycetes). In contrast, Hyde et al. (2017) used one fossil datum (Paleoophiocordyceps coccophagus) and two secondary calibrations (the divergence time of Sordariomycetes and Leotiomycetes and crown age of Sordariomycetes) and Hyde et al.

Fig. 1
Phylogenetic tree based on RAxML analyses of combined LSU, SSU, ITS, tef1-α and rpb2 sequence data. Maximum likelihood bootstrap ≥ 75% (MLBS) and Bayesian posterior probabilities ≥ 0.95 (PP) are indicated at the nodes. The freshwater species are in red and newly obtained strains are in bold. The tree is rooted with Aureobasidium microstictum, Dothidea insculpta, D. sambuci and Pseudoseptoria collariana (2020a) used two secondary calibrations (crown ages of Sordariomycetes and Dothideomycetes). In addition, Hyde et al. (2017) mentioned that the taxon sampling and number of base pair differences between fungal groups could affect the crown node age, and the crown node age can become older by including more taxa in the data set. Our analysis is focused on the subclass Hypocreomycetidae and the taxa were mostly selected from Hypocreomycetidae. In comparison, Hyde et al. (2017Hyde et al. ( , 2020a focused on the class Sordariomycetes. Thus, the divergence time of Coronophorales and Torpedosporales were different from that of Hyde et al. (2017Hyde et al. ( , 2020a.

Ancestral character analysis
Savoryellomycetidae, Diaporthomycetidae, Sordariomycetidae, Lulworthiomycetidae and Xylariomycetidae show a terrestrial ancestor. Hypocreomycetidae also shares terrestrial as the common ancestor, whereas marine and freshwater taxa have independently evolved. Within the Hypocreales clade, the Stachybotryaceae clade comprised mostly freshwater taxa and clustered together with mostly terrestrial Incertae sedis species with terrestrial ancestor. Gliomastix represents marine, terrestrial and newly sequenced freshwater fungi which were recovered in the Bionectriaceae clade with several terrestrial and marine fungal genera. Clonostachys and its sexual genus Bionectria mainly comprised terrestrial fungi except for two newly sequenced species which were reported from freshwater habitats. All these genera represent Bionectriaceae which formed a sister clade to the mostly marine fungal genera Emericellopsis with a marine ancestor which had high support. Calcarisporiaceae, Clavicipitaceae, Cocoonihabitaceae, Cordycipitaceae, Flammocladiellaceae, Hypocreaceae, Niessliaceae, Ophiocordycipitaceae and Polycephalomycetaceae show terrestrial ancestor and marine and freshwater taxa show independent evolution. The Myrotheciomycetaceae clade mostly comprises terrestrial fungi with terrestrial ancestors. Cai et al. (2010) reported the first freshwater Mariannaea species and expected a large discovery of freshwater taxa (Cai et al. 2010;Hu et al. 2017). Mariannaea humicola and Mariannaea punicea were recorded from soil (Lombard et al. 2015;Hu et al. 2017), while all other species, including our newly sequenced strains reported from freshwater habitats. Thelonectria are mostly saprobes in terrestrial habitats (Salgado-Salazar et al. 2016), while T. discophora (= Nectria discophora) was reported from aquatic (Shearer and Webster 1991) and terrestrial habitats (Zeng and Zhuang 2013). Our newly sequenced strains clustered together with terrestrial species of Thelonectria with freshwater ancestor and the node was not statistically supported. Throughout the Nectriaceae clade, several genera such as Acremonium, Atractium, Fusicolla, Rodentomyces and Sedecimiella comprised freshwater, marine or terrestrial species, while several clades represent all three habitats such as Chaetopsina, Cosmospora, Neocosmospora and Volutella. The basal clade of Nectriaceae are mostly marine habitats which also shows terrestrial ancestor, and the freshwater and terrestrial habitats show independent evolution. The Ophiocordycipitaceae clade mainly comprised terrestrial fungi and several Tolypocladium species reported from marine habitats which together formed a clade with exclusively terrestrial Niessliaceae clade. Clavicipitaceae to Hypocreaceae clades comprised marine and terrestrial taxa with the ancestor of a terrestrial habitat. Halosphaeriaceae, one of the most prominent marine ascomycetous families, assigned within Microascales has a marine ancestor (Raghukumar 2017), which was also confirmed in our study.

Sordariomycetes, Hypocreomycetidae
The recent treatment of Hypocreomycetidae was provided by Hyde et al. (2020a) and Wijayawardene et al. (2022), and are followed in the present study. We provided an updated phylogenetic tree for Hypocreomycetidae which includes 41 families and seven orders. Based on phylogenetic analysis and divergence time estimates, a new order (Xenodactylariales) and three new families (Ascocodinaceae, Campylosporaceae, and Cornuvesicaceae) are introduced. Furthermore, the placements of several orders, families and genera are discussed and revised based on phylogenetic analysis. Emericellopsis and Pseudoacremonium are transferred to Hypocreales genera incertae sedis and Bionectriaceae, respectively; Sedecimiella is placed in Nectriaceae; Nautosphaeria and Tubakiella are excluded from Halosphaeriaceae and placed in Microascales genera incertae sedis; Varicosporella is placed under Atractium with synonymy of A. aquatica instead of V. aquatica. Faurelina is excluded from Hypocreomycetidae.
In this study, 41 fresh collections were made from freshwater habitats. Based on phylogenetic analysis and morphological characters, ten new species are introduced, and 13 new habitats and geographic records and 3 new collections are reported. Detailed descriptions and illustrations are provided.
In our phylogenetic analysis, Glomerellales formed a monophyletic clade basal to Hypocreomycetidae; Plectosphaerellaceae is basal to Glomerellales; Australiascaceae, Glomerellaceae and Malaysiascaceae clustered together as a monophyletic clade and sister to Reticulascaceae ( Fig. 1) with the stem ages of Australiascaceae (48 MYA), Malaysiascaceae (35 MYA), and Glomerellaceae (35 MYA) which fall within the genus status (Fig. 2). Hyde et al. (2021) mentioned the revision of Malaysiascaceae as the stem age of this family accord within genus level and our analysis also agree with Hyde et al. (2020a). Thus, the status of these three families may need further study. In addition, the genus Ascocodinaea formed a distinct lineage within Glomerellales (Fig. 1), and the divergence time estimates showed that the stem age of Ascocodinaea (96.8 MYA, Fig. 2 Fungicolous. Sterile setae and conidiophores abundant, arising from the host surface among perithecia. Sexual morph: Perithecia forming directly on the hymenial surface, gregarious, superficial to semi-immersed, gray to black, translucent brown in 3% KOH, ovoidal, with an acute apex, collapsing deeply by lateral pinching when dry, with stiff, erect, acute, unbranched, septate, black setae arising as modified cells of the surface of the upper half of the perithecium, thick-walled. Perithecial wall translucent brown by transmitted light, thin-walled cells of textura epidermoidea at the surface. perithecial apex formed of cells enlarged and arranged in files. Ostiolar canal periphysate; periphyses continuous with the paraphyses. Paraphyses abundant among, and overreaching, mature asci, infrequently branched, septate, slightly enlarged at the tip. Asci cylindrical, 8-spored; apex with a thin ring pierced by a pore. Ascospores uniseriate with overlapping ends, ellipsoidal to fusiform, slightly curved, multi-septate, central cells translucent brown and end cells hyaline, smooth-walled. Asexual morph: Conidiophores macronematous, mononematous, stiff, erect, unbranched, black, morphologically indistinguishable from the sterile setae, each bearing a single, terminal, integrated conidiogenous cell. Conidiogenous cells monophialidic, enteroblastic, proliferating percurrently or sympodially; tip not flared, with slight periclinal thickening at the conidiogenous locus. Conidia broadly ellipsoidal, cylindrical, or inequilateral, often slightly curved, 0-1-septate, hyaline, lacking a visible basal abscission scar, smooth-walled, held in a drop of hyaline slime at the tip of each conidiophore (Samuels et al. 1997).
Notes: Ascocodinaea was introduced by Samuels et al. (1997) to accommodate two fungicolous species, Ascocodinaea polyporicola and A. stereicola (type). Previously, the placement of Ascocodinaea was not well-resolved. Ascocodinaea was originally placed in Lasiosphaeriaceae (Sordariales), based on similar morphological characters with Lasiosphaeriaceae, such as the dark, setose, pseudoparenchymatic ascomata and dematiaceous phialidic asexual morphs (Samuels et al. 1997). Réblová et al. (1999) transferred Ascocodinaea to Chaetosphaeriaceae based on characters of asci, ascospores and perithecial and the dictyochaeta-like asexual morph. Huhndorf et al. (2004) provided a phylogenetic analysis for Sordariales based on LSU sequence data and the result showed that the placement of Ascocodinaea is far from Lasiosphaeriaceae and had close affinities to Glomerella (Collectotrichum). Thus, Ascocodinaea was excluded from Lasiosphaeriaceae and treated as genus incertae sedis in Hypocreomycetidae Wijayawardene et al. 2012). Maharachchikumbura et al. (2015) placed Ascocodinaea in Glomerellales genera incertae sedis, which was accepted by later studies Hyde et al. 2020a).
Only LSU sequence is available for A. stereicola; in our LSU phylogenetic analysis Ascocodinaea stereicola clustered as a distinct clade and sister to Plectosphaerellacea. In our multi-locus phylogenetic analysis, Ascocodinaea stereicola formed a distinct lineage within Glomerellales (Fig. 1). Currently, only A. polyporicola and A. stereicola are accepted to the genus, and sequence data of A. polyporicola were not available in the GenBank. However, A. polyporicola differs from A. stereicola in having larger ascospores, conidia and conidiophores, finer and much more intricately branched paraphyses (Samuels et al. 1997).
Morphologically, Ascocodinaea differs from Australiascaceae in having uniseriate ascospores with translucent brown central cells and hyaline end cells and broadly ellipsoidal, cylindrical, or inequilateral, 0-1-septate conidia. While, ascospores of Australiascaceae are biseriate, hyaline and conidia are ellipsoid to cylindrical-ellipsoidal, septate, aggregated in slime or in chains. Ascocodinaea can be easily distinguished from Glomerellaceae by the uniseriate, multi-septate, ascospores with translucent brown central cells and hyaline end cells. However, ascospores of Glomerellaceae are uni-to biseriate, hyaline and aseptate, in addition, conidia of Glomerellaceae sometimes have a filiform appendage, the base is rounded to truncate, sometimes with a prominent hilum, a character were not found in Ascocodinaea Hyde et al. 2020a). Ascocodinaea differs from Reticulascaceae in having unbranched conidiophores and broadly ellipsoidal or inequilateral, 0-1-septate conidia, whereases, conidiophores of Reticulascaceae are branched or unbranched and conidia are pyriform to cylindrical, 1-or multi-septate. In addition, conidiogenous cells of Ascocodinaea have proliferating percurrent growth; this was not observed in Reticulascaceae (Réblová et al. 2011). Malaysiascaceae differs from Australiascaceae in having bi-seriate, hyaline ascospores that become 1-septate and pale brown after discharge. Ascocodinaea is different from Plectosphaerellaceae in having asci with a thin ring pierced by a pore and uniseriate with overlapping ends ascospores with translucent brown central cells and hyaline end cells. However, the asci of Plectosphaerellaceae lack an apical ring, and ascospores are irregularly arranged, hyaline or pale brown Giraldo and Crous 2019).
Divergence time estimates showed that the stem age of Ascocodinaea is around 96.8 MYA which falls within the family range ). Therefore, a new family Ascocodinaceae is introduced to accommodate Ascocodinaea based on morphological and phylogenetic analyses and divergence time estimates.
The most recent treatment of Hypocreales was provided by Hyde et al. (2020a) and Wijayawardene et al. (2022). Hyde et al. (2020a) listed 14 families under Hypocreales, while, Wijayawardene et al. (2022) accepted 15 families in the order where Cylindriaceae was additionally added. Earlier, Hyde et al. (2020a) placed Cylindriaceae in Xylariomycetidae. This was later confirmed by the analysis of Samarakoon et al. (2022). Hence, Cylindriaceae should be excluded from Hypocreales and placed in Xylariomycetidae. Xiao et al. (2023) recently introduced a new family Polycephalomycetaceae to Hypocreales. Perera et al. (2023) provided an updated phylogenetic analysis of combined gene analysis of ITS, LSU, rpb2, tef1-α and tub2 for Hypocreales and accepted 17 families including three new families (Ijuhyaceae, Stromatonectriaceae and Xanthonectriaceae). Based on our phylogenetic analysis, the placements of a few genera, such as, Emericellopsis, Pseudoacremonium and Sedecimiella need further revisions. In our study, Pseudoacremonium and Sedecimiella are transferred to Bionectriaceae and Nectriaceae, respectively. Emericellopsis was excluded from Myrotheciomycetaceae and placed in Hypocreales genera incertae sedis. In our fresh collections, 39 freshwater strains were placed in Bionectriaceae, Nectriaceae, Sarocladiaceae and Stachybotryaceae within Hypocreales.
Bionectriaceae was introduced by Rossman et al. (1999) to accommodate 26 genera. The classification of Bionectriaceae has been revised and refined by several studies based on phylogenetic analyses (Rossman et al. 2001;Maharachchikumbura et al. 2015Maharachchikumbura et al. , 2016Wijayawardene et al. 2018). The recent treatment of Bionectriaceae was provided by Wijayawardene et al. (2022) with the acceptance of 47 genera. In our phylogenetic analyses, Bionectriaceae clustered with Tilachlidiaceae. Tilachlidiaceae was introduced by Lombard et al. (2015) and three genera Psychronectria, 1 3 Septofusidium and Tilachlidium, are accepted in the family. Our phylogenetic analysis suggests that Tilachlidiaceae may need to be revised and placed under Bionectriaceae (Fig. 1). In addition, our phylogenetic analysis showed that two Septofusidium species (S. berolinense and S. herbarum) and Pseudoacremonium sacchari clustered within Bionectriaceae (Fig. 1). Septofusidium was previously placed within Tilachlidiaceae (Lombard et al. 2015;Hyde et al. 2020a), recently, Perera et al. (2023) transferred it to Bionectriaceae based on phylogenetic analysis. However, the taxonomy of Septofusidium needs further studies, as Septofusidium is polyphyletic (Perera et al. 2023) and the type species lacks sequence data. Pseudoacremonium was placed in Hypocreales genera incertae sedis (Crous et al. 2014;Hyde et al. 2020a). Based on our phylogenetic analysis, Pseudoacremonium is transferred to Bionectriaceae.
In addition, our three fresh collections made from freshwater habitats and the phylogenetic analysis placed them in Clonostachys and Gliomastix within Bionectriaceae. The three fresh collections are identified as Clonostachys rosea, Gliomastix masseei and a new species Clonostachys aquatica based on phylogenetic analysis and morphological characters. Clonostachys rosea, and Gliomastix masseei were reported from freshwater habitats for the first time.
Clonostachys has a worldwide distribution and is commonly found in tropical and subtropical regions (Schroers 2001;Domsch et al. 2007). Species in the genus are saprobes, endophytes, plant pathogens and mycoparasites from various habitats including soil (Schroers 2001;Toledo et al. 2006;Zhang et al. 2008;Moreira et al. 2016). Clonostachys was linked to Bionectria by Rossman et al. (2013). Based on One Fungus-One Name concept, Rossman et al. (2013) synonymized Bionectria under Clonostachys by giving priority to older and frequently used name Clonostachys. The asexual morph of Clonostachys is characterized by penicillate, frequently sporodochial and, in many cases, dimorphic conidiophores (Schroers 2001). The sexual morph of Clonostachys is characterized by solitary to gregarious, subglobose or globose to ovoid, white, yellow, pale orange, tan, or brown perithecia with KOH-and LA-perithecial walls and narrowly clavate to clavate asci containing eight ascospores (Schroers 2001). There are more than 100 records of Clonostachys listed in Index Fungorum (2023), of which 65 species are commonly accepted (Rossman 2014;Lombard et al. 2015;Dao et al. 2016;Prasher and Chauhan 2017;Lechat and Fournier 2018, 2020aZeng and Zhuang 2022). In this study, a new species Clonostachys aquatica is introduced with detailed description and illustration and C. rosea was collected from freshwater habitat for the first time. Etymology: Refer r ing the fungus was collected from aquatic habitat.
Culture characteristics: Conidia germinating on PDA within 12 h. Colonies growing on PDA reaching 3 cm in 14 days at room temperature, surface effused, mycelium sparse, white.
GenBank numbers: ITS = OP876724, LSU = OP875077. Notes: In our phylogenetic analysis, Clonostachys aquatica clustered sister to C. rossmaniae with 94%ML/98%MP/1.00PP support (Fig. 5). Morphologically, Clonostachys aquatica is similar to C. rossmaniae in having penicillate, branched hyaline conidiophores and one-celled, ellipsoidal to ovoidal, hyaline and similar size of conidia. However, C. aquatica is different from C. rossmaniae in having subulate to subcylindrical phialides which are swollen at the apex, while, phialides of C. rossmaniae are almost cylindrical, terminate, flask-shaped and the apex are not swollen (Schroers 2001). We also compared the base pair differences of ITS nucleotides between C. aquatica and C. rossmaniae and found 1.75% differences. Therefore, Clonostachys aquatica is introduced as a new species based on both morphological and phylogenetic analyses, as recommended by Chethana et al. (2021).
Culture characteristics: Conidia germinating on PDA within 12 h. Colonies growing on PDA reaching 3.5 cm in 7 days at room temperature, surface effused, smooth, margin entire, initially white, becoming pale yellowish orange, reverse yellowish, orange at center.
In our phylogenetic analysis, the new collection KUNCC 22-12453 clustered with Clonostachys rosea (Fig. 5). Our new isolate fits well with the description of C. rosea (Schroers et al. 1999). Therefore, we identified our new isolate as C. rosea, which has been reported from freshwater habitats for the first time.
Culture characteristics: Conidia germinating on PDA within 24 h. Colonies growing on PDA reaching 5.0 cm in 7 days at room temperature, circular, with velvety to cotton, dense, greyish aerial mycelium, initially white, with light grey immersed hyphae, forming dark, grey, concentric rings.

Fig. 8 Aquanectria penicillioides (KUN-HKAS125807)
a-c Appearance of ascomata on the host. d Section through ascoma. e, f Section of peridium in 3% KOH (arrowed in e and f, turning red dark to purple in 3% KOH). g section of peridium. h Paraphyses. i-l Asci. m Ascal apical ring. n-t Ascospores. Scale bars: d-f = 50 μm, g = 30 μm, h-l = 20 μm, g, m-r = 10 μm Aquanectria species have been reported from China, Colombia, Ecuador, French Guiana, Singapore, the UK, and the USA (Lombard et al. 2015;Huang et al. 2018;Gordillo and Decock 2019). This study describes a new geographical and habitat record Aquanectria penicillioides based on morphological characteristics and phylogenetic evidence.
Aquanectria penicillioides is characterized by perithecial, superficial, ovate to subglobose, brown-orange to orangered ascomata, with a papillate ostiolar region, cylindrical to clavate, 8-spored asci and ellipsoid to fusiform, hyaline, 1-septate ascospores. The new isolate fits well with the original description of A. penicillioides (Ranzon 1956). In our phylogenetic analysis, the new isolate KUN-HKAS 125807 clustered with A. penicillioides (CBS 257.54) with 100%ML /100%MP /1PP support (Fig. 9). Thus, we identified the new isolate as A. penicillioides. This species has been reported from freshwater habitats in the USA (Ingold 1942;Ranzoni 1956;Lombard et al. 2015). In this study, the new isolate was collected from freshwater habitat in China, it is a new record for China.
Notes: Atractium was established with A. stilbaster as the type species by Link (1809). Link (1825) reinterpreted the generic concept of Atractium to include the pale or colourful synnematous taxa with slimy conidial masses, usually with falcate, septate conidia. The genus was previously listed as a synonym of Fusarium (Wollenweber and Reinking 1935). However, Atractium has synnematous conidiophores which is different from Fusarium. Hence, Gräfenhan et al. (2011) proposed them as two distinct genera. In addition, the epitype of A. stilbaste (the type of Atractium) was designated by Gräfenhan et al. (2011), and they accepted three species (A. crissum, A. holubovae and A. stilbaster) in Atractium. Currently, 26 names of Atractium are listed in the Index Fungorum (2023), of which only three species are accepted in the genus while the placement of the other 23 species remains uncertain . Atractium species are commonly associated with water  and species in the genus have been found from Canada, Germany, and the Philippines (Seifert 1985;Seifert et al. 1995;Sivichai et al. 2002;Fryar et al. 2004).
Atractium is only known by its asexual morphs and is characterized by synnematous conidiophores branching once or twice monochasial, 2-level verticillate, monoverticillate or irregularly biverticillate; monophialidic, hyaline, subulate conidiogenous cells and septate, clavate, obovoid or gently curved, rarely ellipsoidal, conidia with a rounded apical cell, and somewhat conical basal cell, lacking a differentiated foot, some species produce chlamydospores . In this study, a sexual species Varicosporella aquatica is transferred to Atractium and a new species Atractium fusiformis is introduced based on morphological and phylogenetic analyses. The morphological description of sexual morph of this genus is also provided.
Atractium aquatica (Lechat & (2015) with a single species V. aquatica. Phylogenetic analysis of Lechat and Fournier (2015) showed that V. aquatica grouped within Nectriaceae in a basal branch distant from extant genera. However, they did not include all the genera of Nectriaceae in their phylogenetic analysis. In our phylogenetic analysis, V. aquatica clustered with Atractium species within Nectriaceae (Fig. 11). Thus, we transferred V. aquatica to Atractium, and synonymized it under A. aquatica based on phylogenetic analysis.
Culture characteristics: Ascospore germinated on PDA media within 12 h. Colony reached 2.5-3 cm at room temperature for one week, circular, flat, with fluffy, dense, white mycelium, edge entire, reverse pale yellowish.
Material Notes: In this study, we introduce a new sexual species Atractium fusiformis in Atractium. Morphologically, A. fusiformis fits well with the generic concepts of Nectriaceae in having orange, orange-red, perithecial, globose to subglobose, pyriform ascomata, unitunicate, cylindrical or ellipsoidal asci with apical ring and uniseriate, hyaline to yellow, fusiform or ellipsoidal, septate ascospores (Hyde et al. 2020a). In the phylogenetic analysis, four newly obtained strains of Atractium fusiformis (KUNCC 22-12522, KUNCC 22-12523, KUNCC 22-12521 and KUNCC 22-12452) clustered together and sister to A. aquatica with low support ( Fig. 11). However, A. fusiformis can be distinguished from A. aquatica in having ellipsoid with rounded ends ascospores and much larger asci and ascospores (Lechat and Fournier 2015). Thus, we introduce our new isolate as a new species based on both phylogeny and morphology.
Cosmospora Rabenh., Hedwigia 2: 59 (1862) Cosmospora was established by Rabenhorst (1862) with C. coccinea as the type. The generic concept of Cosmospora was previously relatively broad, encompassing a great deal of asexual morph variability and the sexual morph usually with a small, orange or reddish KOH + and thin-walled perithecia, cylindrical asci with or without an apical ring, and 8-spored, uniseriate, 1-septate ascospores. Gräfenhan et al. (2011) strictly refined the generic concept of Cosmospora to include only the species growing on polypores and xylariaceous fungi and having acremonium-like or verticillium-like asexual states. They accepted eight species in Cosmospora, 12 species were subsequently introduced to the genus. Currently, 20 species are accepted in the genus Herrera et al. 2015;Zeng and Zhuang 2016;Luo et al. 2019;Lechat and Fournier 2021).
Species in Cosmospora are characterized by superficial, solitary to gregarious, orange-red to bright red, pyriform perithecia, cylindrical to narrowly clavate asci and ellipsoidal, multi-septate, verrucose or tuberculate, yellow-brown ascospores; ellipsoidal, oblong, clavate or allantoid and aseptate conidia Herrera et al. 2015;Zeng and Zhuang 2016). This study introduces a new species Cosmospora cylindricospora based on phylogenetic and morphological analyses.
Culture characteristics: Colonies on PDA reaching 4-4.5 cm diam. after three weeks at room temperature. Colony medium dense, circular, flattened to raised, surface slightly rough with hyphal tufts, edge entire, velvety to fluffy; from above, white to white yellowish at the margin, light green to yellowish green at the centre; from below, radiating outwards colony, white to cream at the margin, dark green at the middle, dark yellowish green at the centre.
Notes: Cosmospora cylindricospora matches the generic concept of Cosmospora in having hyaline and aseptate conidia. However, Cosmospora cylindricospora is distinct from other species of the genus by its cylindrical, straight or slightly curved and longer conidia. Cosmospora cylindricospora shares similar morphological characteristics such as simple, unbranched conidiophores and hyaline, unicellular, smooth conidia with C. khandalensis and C. lavitskiae. However, C. cylindricospora differs from C. khandalensis and C. lavitskiae in having polyblastic conidiogenous cells and cylindrical, straight or slightly curved conidia with rounded apex and truncated base. Whereas conidiogenous cells of C. khandalensis and C. lavitskiae are monophialidic and conidia are ovoid to ellipsoidal or reniform (Sukapure and Thirumalachar 1966;Zhdanova 1966). In addition, our phylogenetic analysis showed that C. cylindricospora formed a distinct lineage within the genus (Fig. 13). Thus, Cosmospora cylindricospora is introduced as a new species and it is the second Cosmospora species reported from freshwater habitats in China.
Chaetopsina, a hyphomycetous genus, was introduced by Rambelli (1956) with C. fulva as the type species. Since then, many numbers of hyphomycetes species have been  Sutton and Hodges 1976;1979;Morgan-Jones 1979;Crane and Schoknecht 1982;Kirk 1985;Samuels 1985;Wingfield 1987;Holubova-Jechova 1990;Merli 1992;Zucconi and Rambelli 1993). Samuels (1985) described four sexual species of Nectria sensu lato having Chaetopsina asexual morphs. These species were later placed in a newly introduced genus Chaetopsinectria by Luo and Zhuang (2010a). Rossman et al. (2016) recommended the generic name Chaetopsina instead of Chaetopsinectria based on its priority, widespread use, and a greater number of names and this was accepted by later studies Fournier 2019, 2020b).
The sexual morph of Chaetopsina is characterized by perithecial, solitary, superficial, non-stromatic, obpyriform, red, becoming dark red in KOH ascomata with an acute apex, 8-spored, clavate asci with a simple apex or an apical ring and ellipsoid to fusiform, 1-septate, hyaline, smooth to striate ascospores. The asexual morphs are characterized by setiform, tapering towards acutely rounded apex, base bulbous, mostly flexuous, yellow-brown, turning red-brown in KOH, fertile in mid region, unbranched, verruculose conidiophores and the fertile region consisting of irregularly branched dense aggregated conidiogenous cells, conidiogenous cells are hyaline, mono-to polyphialidic, ampulliform to lageniform and conidia are subcylindrical, aseptate hyaline, smooth, with bluntly rounded ends and base rarely with flattened hilum (Rambelli 1956;Luo and Zhuang 2010a).
Currently, 27 species are included in Chaetopsina of which only 12 species have sequence data in GenBank. Chaetopsina species are widespread in tropical and neotropical areas and have been found on leaves, bark, dead palm leaves or ascomycetous stromata (Sutton and Hodges 1976;Rambelli and Lunghini 1976;Crane and Schoknecht 1982;Kirk 1985;Wingfield 1987;Merli 1992;Zucconi and Rambelli 1993;Fournier 2019, 2020b). In this study, Chaetopsina penicillate was collected from freshwater habitats in China, and it is the first species of this genus reported from freshwater habitats. Samuels, Mycotaxon 22(1): 24 (1985).
So far, only one sexual species (G. pseudotenuis) has been reported in Gliocladiopsis. Gliocladiopsis pseudotenuis was iniality introduced by Schoch et al. (2000) as Glionectria tenuis and it was incorrectly linked to the asexual morph of Gliocladiopsis tenuis. However, Phylogenetic analysis and morphological observations showed that it is a distinct species wihtin Gliocladiopsis. Therefore, Lombard and Crous (2012) provided a new name Gliocladiopsis pseudotenuis for Glionectria tenuis. The sexual morph of Gliocladiopsis is characterized by superficial, obovoid to broadly obpyriform ascomata that are turning red-brown in 3% KOH + with a dark red stromatic base, unitunicate, 8-spored, cylindrical, sessile asci with flattened apex and a refractive apical apparatus and uniseriate, hyaline, ellipsoidal, smooth, 1-septate ascospores that are becoming brown and verruculose with age; The asexual morphs are characterized by hyaline, penicillate conidiophores, which consist of a simple-septate Bootstrap support values for RAxML (blue) and MP (red) greater than 70% and Bayesian posterior probabilities (black) greater than 0.95 are given at each node stipe bearing 2-4 successive whorls of branches subtending whorls of phialides and hyaline, cylindrical, 0-1-septate conidia accumulating in whitish to pale yellowish mucoid drops (Lombard and Crous 2012;Gordillo and Decock 2019).
Culture characteristics: Colonies on PDA reaching 3.5-4.0 cm diameter after 3 weeks at room temperature; colony from above, dense, circular, with edge entire, fluffy to floccose, with white tufts and black droplets, dark brown to black at the margin; reverse reddish-brown to dark brown.
GenBank numbers: ITS = OP876701, tub2 = OQ025194, his3 = OQ064512, tef1-α = OQ064522. Bootstrap support values for RAxML (blue) and MP (red) greater than 70% and Bayesian posterior probabilities (black) greater than 0.95 are given at each node Notes: In the phylogenetic analysis, the new isolate KUNCC 22-12663 clustered with the ex-type of G. tenuis (IMI 68205) and four extant strains of G. tenuis with 100% ML/ 100% MP/ 1 PP support (Fig. 17). Morphologically, the new isolate fits well with the original description of G. tenuis (Crous and Wingfield 1993), thus, we identified the new isolate as G. tenuis based on phylogenetic analysis and morphological characters.
Gliocladiopsis tenuis has been reported from terrestrial habitats in India, Indonesia, South-East Asia, Thailand and Vietnam (Crous and Wingfield 1993;Crous and Peerally 1996;Perera et al. 2020). In this study, the new collection was isolated from freshwater habitats for the first time and introduced as a new geographical record for China.
GenBank numbers: ITS = OP876732, LSU = OP875086. Notes: Mariannaea dimorpha was introduced by Zeng and Zhuang (2014) with its sexual and asexual morphs. It was isolated on rotten bark from China. Phylogenetic analysis showed that our new isolate (KUNCC 22-12458) clustered with the holotype of M. dimorpha (Fig. 19). There are no differences in ITS region between these two species, we therefore identified our species as M. dimorpha. Our new isolate was collected from China and first reported from freshwater habitat.
GenBank numbers: ITS = OP876704, LSU = OP875060. Notes: Mariannaea suae is introduced here based on phylogenetic analysis and its distinct morphology. Mariannaea suae is unique in the genus by its conidiophores which bear short branches in the upper part with a terminal whorl of more than ten phialides, branching one level verticillate. While, conidiophores of other species less than ten phialides in the terminal whorl, and usually branching 1-3 levels verticillate (Cai et al. 2010;Gräfenhan et al. 2011;Zeng and Zhuang 2014;Lombard et al. 2015;Hu et al. 2017;Yang et al. 2021). In our phylogenetic analysis, Mariannaea suae formed a distinct lineage within Mariannaea (Fig. 19). Therefore, we introduce our new isolate as a new species based on phylogenetic and morphological analyses.
Mariannaea superimposita has been reported from China, Japan and Venezuela (Matsushima 1975;Luo et al. 2019). In this study, the new isolate was recollected from freshwater habitats in China.
Currently, 129 records of Neocosmospora are listed in Index Fungorum (2023). However, only N. haematococca has been reported from freshwater habitats. In this study, two species were collected from freshwater habitats. Based on multi-locus phylogenetic analysis and morphological characters, the species were identified as N. aquatica sp. nov. and N. brevis. Etymology: Epithet refers to the collection from an aquatic habitat.
Notes: Neocosmospora brevis was described by Sandoval-Denis et al. (2019), which was collected from soil-water polluted with diethyleneglycerol and ethylenglycerol, Citrus sinensis and a human eye in Belgium, Italy and the USA (Sandoval-Denis et al. 2019). In this study, a new isolate (KUNCC 22-12461) was identified as Neocosmospora brevis based on both phylogenetic and morphological analysis which was collected on submerged decaying wood from freshwater habitats in China. It is a new record for China.
In our phylogenetic analysis, the new isolate clustered with Neocosmospora brevis with significant support (Fig. 24). The nucleoide comparision of ITS and tef1-α between our new isolate (KUNCC 22-12461) and Neocosmospora brevis (CBS 130326) revealed 1 bp and 3 bp differences, respectively. Our new isolate is morphologically similar to the holotype of N. brevis in having cylindrical, unbranched or rarely branched, septate conidiophores, phialidic conidiogenous cells and oval, ellipsoidal straight or slightly curved, hyaline, smooth-and thin-walled microconidia, the macroconidia were not observed in the new isolate. However, our new isolate (KUNCC 22-12461) has longer conidiophores than the holotype (100-140 vs. 36.5-59 μm) and aseptate conidia, while the holotype has 0-1(-2)-septate, which may due to their different hosts and habitats. The holotype of N. brevis was collected from soil-water with diethyleneglycerol and ethylenglycerol (Sandoval-Denis et al. 2019). However, our new isolate is a saprobe collected on submerged wood from freshwater habitats.
Neonectria Wollenw., Annls mycol. 15(1/2): 52 (1917). Neonectria, is a cosmopolitan genus commonly distributed in tropical and temperate regions. The species occur as saprobes, pathogens and some species are isolated as soil inhabitants (Brayford 1993;Chaverri et al. 2011). Species of Neonectria sensu lato are characterised by subglobose to broadly obpyriform, smooth to roughened, red perithecia that are becoming dark red in 3% KOH, and with an acute to constricted apex that is sometimes knobby, the perithecial wall is ca. 50 μm thick and generally composed of two regions, sometimes with an outer region that forms textura epidermoidea, that may or may not be covered with another region of cells; and the ascospores are hyaline, generally bicellular, rarely multi-cellular, and smooth or finely ornamented (Rossman et al. 1999;Chaverri et al. 2011).
Neonectria was linked to the asexual genus Cylindrocarpon. Neonectria and Cylindrocarpon was informally classified into several groups based on the combined morphology of asexual morph and sexual morph (Neonectria into five informal groups and Cylindrocarpon into four groups, Booth 1959Booth , 1966Brayford and Samuels 1993;Samuels and Brayford 1994). Phylogenetic analysis revealed that Neonectria and Cylindrocarpon are phylogenetically congeneric (Mantiri et al. 2001;Brayford et al. 2004;Seifert et al. 2003). Halleen et al. (2004) introduced a new asexual morph genus Campylocarpon to accommodate C. fasciculare, which is the first formal segregation from Cylindrocarpon. A taxonomic revision of Neonectria sensu lato was provided by Chaverri et al. (2011) based on multi-locus phylogenetic analysis, morphological characters and ecological data. Their phylogenetic analysis showed that five distinct highly supported clades that correspond to some extent with the informal Neonectria and Cylindrocarpon groups. Hence, three genera llyonectria, Rugonectria and Thelonectria were introduced to accommodate three Neonectria sensu lato informal groups. Currently, 55 Neonectria species epithets are listed in the Index Fungorum (2023). However, only N. lugdunensis has been reported from freshwater habitats. This study introduces a new freshwater species Neonectria aquatica based on phylogenetic analysis and morphological characters. Etymology: Referring to the aquatic habitat of this fungus.
Paracremonium is an acremonium-like genus, was established by Lombard et al. (2015) with P. inflatum as the type species. Eight species were further introduced in the genus (Lynch et al.2016;Crous et al. , 2021aZhang et al. 2017Zhang et al. , 2021aAl-Bedak et al. 2019;Ming et al. 2021). Paracremonium is only known by the aseual morphs and characterized by hyaline, septate, branched hyphae which sometimes forming sterile coils from which conidiophores arise (Lombard et al. 2015). Species of Paracremonium are widely distributed and have been reported from Canada, California, China, India, Netherlands and Egypt (Lynch et al. 2016;Crous et al. , 2021aAl-Bedak et al. 2019;Zhang et al. 2021a;Ming et al. 2021). In this study, a new geographic record Paracremonium binnewijzendii is described.
Culture characteristics: Colonies on PDA, 1.5-2 cm diam after one week at room temperature, margin regular, smooth surface, entire edge, cottony or woolly, whitish, well-defined edges with no pigmentation of the agar, reverse brown to dark yellow ate centre, pale yellow at the edge. Notes: In our phylogenetic analysis, our new isolate clustered with two strains of Paracremonium binnewijzendii with 100% ML/ 100% MP/ 1.00 PP support (Fig. 29). Paracremonium binnewijzendii was described by , and is characterized by subcylindrical, hyaline, smooth, phialides with an inconspicuous collarette conidiogenous cells and aseptate, ellipsoidal, smooth conidia. Morphologically, our species is almost identical to the holotype of P. binnewijzendii, except the conidiophores of our new isolate that bear branches on conidiophores with phialides in verticils, branching 1-2 levels verticillate. Their morphological differences are probably due to the different growing environments and hosts. The holotype of P. binnewijzendii was collected in soil from stream embankment in the Netherlands. While, our new isolated was collected on submerged decaying wood from freshwater habitats in China, and it is a new record for China. Sedecimiella was introduced with a single species S. taiwanensis by Pang et al. (2010). Sedecimiella taiwanensis is a mangrove-based marine fungus collected from Taiwan, China. Sedecimiella taiwanensis is placed in Hypocreales genera incertae sedis (Pang et al. 2010;Hyde et al. 2020a) and was characterized by orange to dark brown, pyriform with globose to subglobose venter, coriaceous, ostiolate ascomata, two-layered peridium, 16-spored, unitunicate, cylindrical asci with short pedicellate lacking an apical pore and globose, one-celled, hyaline ascospores. In our phylogenetic analysis, Sedecimiella taiwanensis clustered with two Acremonium species (A. minutisporum and A. vitellinum) within Nectriaceae (Fig. 1). We therefore place S. taiwanensis in Nectriaceae. However, the relationship between S. taiwanensis and the two species of Acremonium (A. minutisporum and A. vitellinum) needs to be resolved.
Culture characteristics: Ascospore germinated on PDA within 24 h. Colonies growing on MEA, reaching 2-2.5 cm in one week at room temperature. Mycelium superficial, circular, with entire margin, flat, smooth, from above white, from below dark yellowish at centre, white at the edge.
GenBank numbers: ITS = OP876736, LSU = OP875057. Notes: In the phylogenetic analysis, two new isolates Thelonectria aquatica and T. cylindricospora clustered as sister taxa (Fig. 31). The nucleotide comparison between these species revealed 15 bp (2.7%) differences in the ITS gene region. We therefore identified them as two distinct species in Thelonectria as recommended by Jeewon and Hyde (2016).
Phylogenetic analysis showed that our three new isolates clustered within V. ciliata clade. Morphologically, our new isolates fit well with the description of V. ciliata . Therefore, we identified the three new isolates as V. ciliata, which is the first time of this species was collected from freshwater habitats.
Sarocladiaceae was recently established by Crous et al. (2018). The family comprises two genera, Parasarocladium and Sarocladium, placed in Hypocreales (Crous et al. 2018;Hyde et al. 2020a). Phylogenetic analysis of Crous et al. (2018) showed that the family is sister to Bionectriaceae. While, Hyde et al. (2020a) showed that Sarocladiaceae is close to Flammocladiellaceae. In our phylogenetic analysis, Sarocladiaceae formed a sister clade to Myrotheciomycetaceae with 86% ML/ 1.00 PP support values (Fig. 1). In this study, Sarocladium kiliense was collected from freshwater habitats for the first time in China.
Sarocladium W. Gams & D. Hawksw., Kavaka 3: 57 (1976. Sarocladium was established with S. oryzae as the type by Gams and Hawksworth (1975). The genus is characterized by cylindrical, phialidic conidiogenous cells and the phialides arise solitarily from undifferentiated hyphae or conidiophores that are sparsely to densely branched, with ellipsoidal conidia formed in false heads (Summerbell et al. 2011;Ou et al. 2020). Summerbell et al. (2011) reviewed  Colonies on substrate. c-d Squashed conidioma with setae. e-j Conidiophores, conidiogenous cells and developing conidia. k Conidia. Scale bars: c-d = 60 μm, e-g, k = 20 μm, h-j = 10 μm the taxonomy of Sarocladium and included the species that belong to the A. bacillisporum and A. strictum clades and accepted eight species in the genus. Since then, several new species have been introduced to the genus (Yeh and Kirschner 2014;Giraldo et al. 2015;Liu et al. 2017b;Crous et al. 2018;Ou et al. 2020). Currently, 30 species are included in the genus (Index Fungroum 2023).
Sarocladium kiliense is ubiquitous, commonly found from soil, also isolated from man, cattle and maize (Sukapure and Thirumalachar 1965). In this study our new isolate was collected on submerged wood from freshwater habitats in China. Morphologically, the conidiogenous cells and conidia of our new isolate are identical to S. kiliense. We compared the base pairs differences for the ITS gene region which revealed 2 bp differences. We therefore identified our new isolate as S. kiliense, and it is a new record for China.
Stachybotryaceae L. Lombard & Crous, Persoonia 32: 283 (2014).  Hyde et al. (2020a), and accepted 39 genera. Species in Stachybotryaceae are characterized by asexual morphs with mononematous to sporodochial to synnematous conidiomata, usually with phialidic conidiogenous cells that produce 0-1-septate conidia in dark green dry chains or slimy masses. Only three species of Stachybotryaceae (Koorchalomella salmonispora, Stachybotrys chartarum and S. chlorohalonata) have been reported from freshwater habitats. In this study, two Memnoniella species viz. Memnoniella echinate and M. oenanthes were collected from freshwater habitats for the first time. Stachybotrys chartarum and S. chlorohalonata were recollected from freshwater habitats in China.
Memnoniella was introduced by Von Höhnel (1924) based on M. aterrima. It is characterized by macronematous, mononematous, unbranched conidiophores, phialidic conidiogenous cells with conspicuous collarettes, and unicellular, aseptate, smooth to verrucose conidia arranged in dry chains or slimy masses. The morphology of Memnoniella and Stachybotrys are similar and several studies have treated them as congeneric. (Jong and Davies 1976;Castlebury et al. 2004;Wang et al. 2015). However, Lombard et al. (2016) showed that Memnoniella species forming a well-supported clade distant to the Stachybotrys clade, hence, Memnoniella was resurrected as a distinct genus in Stachybotryaceae which was accepted by later studies Hyde et al. 2020a;Mapook et al. 2020;Samarakoon et al. 2021). Currently, 24 records of Memnoniella are listed in Index Fungorum (2023), of which 10 species were transferred to other genera viz. Brevistachys and Stachybotrys. In this study, we introduce two new habitat and geographic records of Memnoniella.
Culture characteristics: Colonies on PDA attaining 5 cm diam., within three weeks at room temperature, white in the beginning and brown to black with age, greyish white at middle, grey to brown at edge, circular, entire edge with raised on media surface, velvety.
Notes: During our investigation of freshwater fungi from China, a stachybotrys-like taxon was collected and identified as Memnoniella echinata based on morphology and phylogeny. Our collection fits well with M. echinata in having macronematous, mononematous, erect conidiophores,  (KUN-HKAS 125795). a-c Colonies on substrate. d-h Conidiophores and conidia. i Conidiogenous cells and conidia. j-m Conidia. n Surface view of culture on PDA. o Reverse view of culture on PDA. Scale bars: d-f = 30 μm, h, k-m = 20 μm, i, j = 10 μm monophialidic, discrete, determinate, terminal conidiogenous cells and globose to subglobose, olivaceous brown to dark brown, verrucose conidia Tennakoon et al. 2021). Phylogenetic analysis showed that the new collection clustered with four strains of M. echinata (Fig. 37). Memnoniella echinata has a worldwide distribution, commonly found in soil (Jarvis et al. 1998;Lombard et al. 2016;Tennakoon et al. 2021). Our collection was from submerged wood in freshwater habitats in China for the first time.
Culture characters: Colonies on PDA slow growing, reaching 2.5-3.0 cm diam. after 2 weeks at room temperature, white to cream, or pale yellowish at the margins, cream at the centre, distinguished from the margin by white embossed hyphae with grey tufts in the centre; slightly radiating; reverse white cream at the margin, yellowish at the centre.
Notes: Memnoniella oenanthes was reported as Stachybotrys oenanthes on dead stems of Oenanthe crocata by Ellis (1971). Lombard et al. (2016) re-evaluated the taxonomy of Stachybotriaceae based on phylogenetic analysis and morphological characters. The phylogenetic analysis showed that S. oenanthes clustered within Memnoniella. They, therefore, transferred S. oenanthes to Memnoniella as M. oenanthes.
Memnoniella oenanthes is characterized by erect, simple, 1-2 septate, solitary, smooth conidiophores, monophialidic, cylindrical or obovoid conidiogenous cells and reniform or ellipsoidal, smooth or verrucose conidia (Eills 1971(Eills , 1976Wang et al. 2015). Our collection fits well with the description of M. oenanthes except the size of conidiophores and conidiogenous cells. The conidiophores of our collection are much longer (212-302 vs. 120-180 μm) and conidiogenous cells are smaller (8.5-12.5 × 2-4 vs.12-21 × 4-7 μm)  (Ellis 1971(Ellis , 1976. Phylogenetic analysis showed that our new collection clustered with M. echinate with good support (Fig. 37). Thus, we identified our new collection as M. echinates based on phylogeny and morphology. Memnoniella echinate has been reported as saprobes on the stems of Euphorbia tirukalli and Oenanthe crocata from Guernsey and India. In this study, our new isolate was collected from freshwater habitats in China and it is a new record for China.
Stachybotrys Corda, Icon. fung. (Prague) 1: 21 (1837). The asexual genus Stachybotrys is ubiquitous distributed worldwide. Species in the genus are common in soil, plant litter (hay, straw, cereal grains, and decaying plant debris) and air and a few species have been found from damp paper, cotton, linen, cellulose-based building materials waterdamaged indoor buildings, and air ducts from both aquatic and terrestrial habitats (Izabel et al. 2010;Lombard et al. 2016;Hyde et al. 2020a). Stachybotrys was established by Corda (1837) with S. atrus as the type. The genus was revised by Lombard et al. (2016), their phylogenetic analysis showed that Stachybotrys s.l. is poloyphyletic and it can be segregated into ten genera, which is also supported by morphological observations. Hence, Lombard et al. (2016) refined the generic concept of Stachybotrys in a strict sense as conidiophores branching at the basal septum and the formation of thick-walled conidia sometimes bearing ornamentations.
There are 88 records of Stachybotrys on Species Fungorum of which 33 species have DNA sequence data in GenBank. Recently, Samarakoon et al. (2021) introduced a new species S. musae on Banana. Currently, 88 species are included in the genus, of which, only Stachybotrys chartarum and S. chlorohalonatus have been reported from freshwater habitats. In this study, we reported S. chartarum and S. chlorohalonatus from freshwater habitats again.
Culture characteristics: Colonies on PDA reaching 3 cm diam, after 14 days at room temperature, white at first, irregular, raised, undulate, rough, after maturity, smooth at the margin, white from above, reverse cream to yellow at the margin, dark yellowish brown at centre. Notes: Stachybotrys chlorohalonata was introduced by Andersen et al. (2003) and isolated from cardboard on gypsum board. Stachybotrys chlorohalonata likely inhabits wet cellulose-containing materials such as fabric, hay, seaweed, grain, paper and soil and is found in Belgium, Denmark, Finland, Iraq, New Guinea, Spain and the USA. This study collected four fresh collections from freshwater habitats in China. Phylogenetic analysis showed that the four new isolates grouped with S. chlorohalonata (Fig. 37). With identical morphology to each, they were identified as S. chlorohalonata.
Emericellopsis was described by Van Beyma (1940) with E. terricola as the type species. Since then, several species have been introduced worldwide in various habitats such as agricultural and forest soils, peat, rhizomes, prairies and freshwater-, estuarine-and marine-mud sediments (Stolk 1955;Gilman 1957;Thirumalachar 1960, 1962;Backus and Orpurt 1961;Davidson and Christensen 1971;Zuccaro et al. 2004;Grum-Grzhimaylo et al. 2013). The placement of Emericellopsis has been revised several times by different authors. Emericellopsis was initially placed in Eurotiales (Van Beyma 1940). Phylogenetic analysis of Emericellopsis was firstly provided by Glenn et al. (1996) and placed in Hypocreales. Ogawa et al. (1997) set Emericellopsis in Hypocreaceae based on SSU and LSU sequence data. Subsequently, the genus was transferred to Bionectriaceae (Rossman et al. 1999(Rossman et al. , 2001Grum-Grzhimaylo et al. 2013). Recently, Crous et al. (2018) transferred Emericellopsis to Myrotheciomycetaceae based on phylogenetic analysis. However, they did not include the type species of Emericellopsis (E. terricola) in their phylogenetic analysis.
Notes: Cornuvesica was introduced by Viljoen et al. (2000) to accommodate Ceratocystiopsis falcata, originally placed in Ceratocystis (Wright and Cain 1961). A 1 3 phylogenetic analysis based on SSU sequence data showed that Cornuvesica clustered as a sister clade to Ceratocystis within Microascales (Hausner and Reid 2004). Therefore, Cornuvesica was placed in Ceratocystidaceae based on phylogenetic analysis (Réblová et al. 2011;de Beer et al. 2013ade Beer et al. , 2014. Marincowitz et al. (2015) introduced three additional species in the genus. They reconstructed the phylogenetic analysis for Cornuvesica based on SSU and LSU sequence data and the result showed that Cornuvesica formed a monophyletic clade sister to Ceratocystidaceae within Microascales. Marincowitz et al. (2015) further placed Cornuvesica in Microascales genera incertae sedis. The placement of the genus was accepted by Hyde et al. (2020a) and Wijayawardene et al. (2020).
In our phylogenetic analysis, Cornuvesica formed a monophyletic clade grouping with Ceratocystidaceae, Graphiaceae, Gondwanamycetaceae and two strains of Sporendocladia bactrospora (Microascales genera incertae sedis) (Fig. 1). Morphologically, Cornuvesica is known by both sexual and asexual morphs. Cornuvesica is different from Ceratocystidaceae in having falcate, straight or slightly curved, 1-septate, subhyaline ascospores. While, ascospores of Ceratocystidaceae are aseptate, hyaline and varied in shape, hat-shaped, ellipsoidal or elongate to slightly curved, with rounded ends, oblong, cylindrical or narrowly fusiform to spindle-shaped. Graphiaceae is only known by the asexual morphs. It is different from Cornuvesica in having macronematous, synnematous conidiophores, conidiogenous cells in whorls of two to six, with annellidic extensions and cylindrical to obovoid conidia often with a distinct basal frill. While Cornuvesica has two types of conidiophores which are mononematous, conidiogenous cells of Cornuvesica are phialidic, collarette in distinct, discrete or integrated, intercalary or terminal and conidia are cylindrical to obovoid, often with a distinct basal frill. Gondwanamycetaceae is different from Cornuvesica in having hyaline, aseptate, fusiform to lunate or falcate or allantoid ascospores without sheath, mono-verticillate or penicillate conidiophores and cylindrical to allantoid, slimy conidia that are not in chains. Whereas, Cornuvesica has falcate or allantoid, 1-septate ascospores surrounded by hyaline sheath with both ends attenuated and Cornuvesica has two types of conidiophores which are mononematous, unbranched or branched and conidia are cylindrical to obovoid, often with a distinct basal frill.
In addition, Cornuvesica is phylogenetically distinct from Ceratocystidaceae, Graphiaceae, Gondwanamycetaceae (Fig. 1). The stem age of Cornuvesicaceae (209.88 MYA) falls within the family range . Therefore, a new family Cornuvesicaceae is introduced to accommodate Cornuvesica based on morphology, phylogeny and divergence time estimates.
Halosphaeriaceae E. Müll. & Arx ex Kohlm., Can. J. Bot. 50: 1951(1972. Halosphaeriaceae (Microascales, Hypocrealmycetidae) comprises 64 genera (Pang 2002;Jones et al. 2009Jones et al. , 2015Jones et al. , 2017Jones et al. , 2019Maharachchikumbura et al. 2015;Wijayawardene et al. , 2018Wijayawardene et al. , 2022Hyde et al. 2020a). Species in the family are commonly found in marine habitats and few species are from freshwater and terrestrial habitats (Jones et al. 2009Hyde et al. 2020a). In our phylogenetic analysis, Halosphaeriaceae is polyphyletic and the genera Nautosphaeria and Tubakiella clustered out of Halosphaeriaceae (Fig. 1)  Ascosacculus was introduced by Campbell et al. (2003) to accommodate two Halosarpheia species (A. aquaticus and A. heteroguttulatus) with A. aquaticus as the type species. The genus is known only by sexual moprs and it is characterized by immersed or superficial, globose to subglobose ascomata with long neck, 8-spored, thin-walled, early deliquescent asci, which lacking an apical pore and apical apparatus and fusiform to cylindrical, hyaline, 1-septate ascospores filled with many small guttules and having a hamate appendage at each apex that unfurls to form long, threadlike, sticky appendages (Campbell et al. 2003).
Ascosacculus fusiformis differs from the morphological concepts of the genus, such as having cylindrical, pedicellate asci with an apical ring, and uniseriate, fusiform, 3-septate conidia. Asci of other Ascosacculus species are obovoid to broadly cylindrical lacking an apical ring and ascospores are bi-seriate and uniseptate. Thus, we doubt the reliability of the sequence data of A. fusiformis. Currently, only three species are introduced in the genus and with the few taxa sampled, a clear understanding of the characters of Ascosacculus cannot be provided. Hence, further fresh collections are required to better understand this genus.
Microascaceae was introduced by Luttrell (1951) to accommodate Microascus which was originally placed in Ophiostomataceae (Nannfeldt 1932) or Eurotiaceae (Emmonas and Dodge 1931;Moreau and Moreau 1953;Doguet 1957). Malloch (1970) Hyde et al. (2020a), and 23 genera accepted. Species in this family have a worldwide distribution and most species are saprobes in soil, dung or on decaying plant materials Sandoval-Denis et al. 2016a, b;Hyde et al. 2020a), while a few species are opportunistic pathogens of humans Sandoval-Denis et al. 2013, 2016aLackner et al. 2014). In our phylogenetic analysis, Microascaceae clustered basal to Microascales, and the stem age of Microascaceae is around 153 MYA, which falls within the family range .
Notes: Parascedosporium was established by Gilgado et al. (2007) with P. tectonae as the type. Parascedosporium is a polymorphic genus with two asexual morphs, one is characterized by solitary conidiophores with sympodial conidia emerging from denticulate conidiogenous cells. The second one showed the features typical of Graphium wich has annellidic conidiogenous cells (Gilgado et al. 2007;Lackner and de Hoog 2011). Lackner and de Hoog (2011) examined the ex-type culture (CBS 127.84) of Parascedosporium tectona and their phylogenetic analysis based on ITS sequence data showed that P. putredinis clustered with P. tectonae and there were maximally two base pairs differences within the clade. Therefore, they considered P. tectona as a synonym of P. putredinis. Later, de Beer et al. (2013b) examined the holotype of Parascedosporium tectona (IMI 95673d) and accepted the treatment of Lackner and de Hoog (2011). Zhang et al. (2021b) introduced another species P. sanyaense which was initially placed in Scedosporium. Zhang et al. (2021b) showed that Scedosporium sanyaense clustered with P. putredinis. They therefore transferred Scedosporium sanyaense to Parascedosporium as P. sanyaense. There are three Parascedosporium species listed in the Index Fungorum (2023). In this study, P. putredinis was collected from freshwater habitats in China.
Culture characteristics: Conidia germinated within 24 h on PDA, colonies grow rapidly on PDA at room temperature, reaching around 4 cm diam., after three weeks. Colonies on medium appear circular to irregular, medium dense, flat or effuse, with fimbriate edge, colonies from above and below Bootstrap support values for RAxML (blue) and MP (red) greater than 70% and Bayesian posterior probabilities (black) greater than 0.95 are given at each node white to greyish, white at middle, cream to pale grey at edge; was collected on leaves of unidentified vine in Thailand. Phylogenetic analysis of Crous et al. (2018) showed that X. thailandica formed a distinct lineage within Hypocreales and close to Ophiocordycipitaceae. However, Crous et al. (2018) placed the family in Myrmecridiales, Diaporthomycetidae (Sordariomycetes). Hyde et al. (2020a) showed that Xenodactylariaceae grouped within Hypocreomycetidae and close to Torpedosporales. Recently, a phylogenetic analysis of Hyde et al. (2021) found that Xenodactylariaceae did not cluster with members of Diaporthomycetidae and suggested excluding this family from Diaporthomycetidae.
In our phylogenetic analysis, Xenodactylariaceae grouped within Hypocreomycetidae as a distinct lineage close to Torpedosporales ( Fig. 1) which is consistent with the analysis of Hyde et al. (2020a). Xenodactylariaceae has subcylindrical, hyaline, septate conidia that are in branched chains. These characters are quite different from Torpedosporales; Conidia of Torpedosporales are solitary and helicoid e.g. Juncigenaceae has single, brown, helicoid conidia and conidia of Torpedosporaceae are solitary, irregularly helicoid and muriform (Hyde et al. 2020a). In addition, falls within the order range ). Thus, we transfer the family to Hypocreomycetidae in a newly introduced order Xenodactylariales based on the phylogenetic analysis and divergence time estimates.
Hypocreomycetidae families incertae sedis. Saprobic on submerged leaves or endophytic in plants. Sexual morph: Undetermined. Asexual morph: Colonies hyaline to pale brown hyphae including variously shaped inflated cells. Conidiophores lateral or rarely terminal or intercalary, cylindrical or somewhat nodose, mostly simple or rarely sparsely branched. Conidiogenous cells integrated, typically proliferating sympodially. Conidia tetraradiate, hyaline, composed of two parts, deltoid and allantoid, both with two diverging branches at the ends; deltoid triangular to pyramidal, basal cells with ends rounded; apical cells of both parts rounded; branches aseptate. (Fiuza and Gusmão 2013;Marvanová and Laichmanová 2014).
Placement of Campylospora was unclear since it was established. Hyde et al. (2020a) and Wijayawardene (2022) place Campylospora in Hypocreomycetidae genera incertae sedis. In our phylogenetic analysis, Campylospora formed a monophyletic clade sister to Falcocladiaceae (the only family in Falcocladiales) within Hypocreomycetidae (Fig. 1). Our phylogenetic analysis suggests it can be introduced as a new family. In addition, the divergence time estimates showed that the stem age of Campylospora is 193.9 MYA which accords with the family level suggested by Hyde et al. (2017). Morphology of Campylospora is distinct in the subclass in having tetraradiate conidia, which are composed two parts of deltoid and allantoid, both with two diverging branches at the ends (Fiuza and Gusmão 2013;Marvanová and Laichmanová 2014). Hence, a new family Campylosporaceae is introduced to accommodate Campylospora based on phylogenetic analysis and divergence time estimates.
Faurelina was described by Locquin-Linard (1975) for several cleistothecial ascomycetes. The placement of Faurelina is controversial and has been changed many times by different authors (Locquin-Linard 1975;Parguey-Leduc and Locquin-Linard 1976;von Arx 1978;Cannon and Kirk 2007;Tang et al. 2007;Réblová et al. 2011;Maharachchikumbura et al. 2015;Hyde et al. 2020a). The last treatment of Faurelina was provided by Wijayawardene et al. (2022), and placed in Chadefaudiellaceae (Microascales). Tang et al. (2007) firstly provided sequence data for a single strain of Faurelina indica (CBS 126.78) and this strain was similar to the type species of Ceratocystis (C. fimbriata). However, Réblová et al. (2011) doubted the reliability of this strain. They studied and sequenced two strains of F. indica including the ex-type strain (CBS 126.78), and confirmed that the strain of Tang et al. (2007) was based on different fungus. In addition, phylogenetic analysis of Réblová et al. (2011) showed that the two strains of F. indica have relationship with Didymellaceae (Pleosporales, Dothideomycetes). Therefore, we exclude Faurelina from Chadefaudiellaceae (Microascales). However, Hyde et al. (2020a) and Wijayawardene et al. (2022) placed Faurelina in Chadefaudiellaceae (Microascales). Our study is in agreement with Réblová et al. (2011) excluded Faurelina from Chadefaudiellaceae (Microascales). In our preliminary phylogenetic analysis, F. fimigena and F. indica clustered out of the Sordariomycetes (thus Faurelina was excluded from our phylogenetic analysis). The blast result showed that these two species have higher similarity to Dothideomycetes rather than Sordariomycetes. Our result is consistent with Réblová et al. (2011). Hence, we suggest to exclude Faurelina from Chadefaudiellaceae (Microascales). However, the placement of Faurelina need further studies based on phylogenetic analysis and morphological characteristics.

Evolution of Hypocreomycetidae
The evolution of fungi is still a matter of debate , and transitions from aquatic to terrestrial habitats have been commonly postulated (Hibbett and Binder 2001). The basal clade of the kingdom Fungi is Chytrids, also known as lower fungi, and are mainly found in the freshwater habitats (Raghukumar 2017). They developed uniflagellate zoospores, which helps to disperse in water (Raghukumar 2017). The unicellular group of fungi are thought to have changed their lifestyles several times and developed into multicellular organisms in the terrestrial environment, resulting in Ascomycota and Basidiomycota (Raghukumar 2017). The initial colonizer of terrestrial environment faced harsh physical environment thus, they underwent macroevolutionary jump (Selosse and Le Tacon 1998). The initial terrestrial colonization was formed by the association of fungus and phototrophs (Selosse and Le Tacon 1998). In some lineages, mutualism with fungi was an ancestral feature (Selosse and Le Tacon 1998). The colonization of fungi on land is thought to have started with the establishment of arbuscular mycorrhiza-like symbioses between fungi belonging to Glomeromycota and plant roots (Redecker et al. 2000;Raghukumar 2017). The fungi facilitated nutrient intake of these earlier plants (Bidartondo et al. 2011). Later studies revealed that the earliest mycorrhizal symbiosis was formed between Mucoromycota with land plants or dual Mucoromycotina-Glomeromycotina partnerships with land plants (Strullu-Derrien et al. 2014;Rimington et al. 2015;Feijen et al. 2018). Terrestrial fungi may be originated directly from marine or freshwater (Little 1990;Berbee et al. 2017). However, the transition from marine to freshwater is difficult, based on its limited frequency in phylogenies (del Campo and Ruiz-Trillo 2013; Berbee et al. 2017).
Marine Ascomycota have independently derived from terrestrial and freshwater ascomycetes (Spatafora et al. 1998). They clustered as sister clades to terrestrial or freshwater species and several genera of Ascomycota contain terrestrial and freshwater species together with marine species (Raghukumar 2017). Primary marine Ascomycota species are assumed to be derived from the marine environment, and secondary marine species are assumed to have a terrestrial ancestor (Spatafora et al. 1998;Raghukumar 2017). The phylogenetic analyses conducted by Vijaykrishna et al. (2006) showed that freshwater taxa have derived from terrestrial habitats several times independently. The freshwater taxa could be moved from terrestrial when the associated plants invaded water or ran off rainwater and sediments (Vijaykrishna et al. 2006). Evidently, some freshwater Ascomycota mainly occur on bamboo and in many countries, bamboo grows near river banks (Vijaykrishna et al. 2006). Freshwater Ascomycota shows various adaptations to aquatic environments, such as enhanced mechanisms for dispersal and subsequent attachment in freshwater (Vijaykrishna et al. 2006) and various environments (Ruisi et al. 2007). The freshwater Sordariomycetes reported in this study mainly occur on decayed wood in submerged lakes, rivers and streams. Vijaykrishna et al. (2006) also stated the potential of aquatic fungi to degrade submerged substrates which help the survival in water-logged conditions. Goh and Hyde (1996) proposed four artificial groups of hyphomycetes based on occurrences, namely 1) ingoldian fungi, 2) aero-aquatic hyphomycetes, 3) terrestrial-aquatic hyphomycetes and 4) submerged-aquatic hyphomycetes. Aero-aquatic hyphomycetes are mainly reported from ponds, ditches, or slow-running streams. They are characterized primarily by conspicuous conidiophores which occur in submerged leaves or woody substrates under semi-anaerobic conditions. Our new collections of Clonostachys rosea, Gliomastix masseei and Memnoniella echinate show visible larger conidiophores and are reported from submerged decaying wood near the lakes.
Despite being one of the largest classes, freshwater Sordariomycetes account for more than 60% of total freshwater ascomycetes (Shearer and Raja 2013). Samarakoon et al. (2016) and Hongsanan et al. (2017) studied several subclasses of unitunicate fungi based on divergence time estimate analysis. Hyde et al. (2021) recently reviewed the evolution of freshwater Diaporthomycetidae based on molecular clock analyses. In addition, evolutionary studies have been mostly conducted for Halosphaeriaceae and related orders such as Koralionastetales and Lulworthiales (Raghukumar 2017). In the study of Spatafora et al. (1998), marine Halosphaeriales (Microascales) species showed the independent evolution from terrestrial environment based on phylogenetic analyses. Further species of Halosphaeriaceae clustered as sister clades to terrestrial or freshwater species, and several genera of Ascomycota contain terrestrial and freshwater species together with marine species (Raghukumar 2017). As for accommodating freshwater, marine and terrestrial fungi, studying the evolution of Hypocreomycetidae are important for understanding the possible transition and evolution of aquatic and terrestrial ascomycetes.
In this study, we tried to address the possible evolutionary transitions of aquatic Hypocreomycetidae with broad taxon sampling. The result supports the transition of aquatic Hypocreomycetidae from terrestrial habitat to freshwater and marine habitats. Our finding concurred with the study of Vijaykrishna et al. (2006) who stated the evolution of freshwater ascomycetes form terrestrial habitats based on molecular phylogeny. Several molecular studies also stated the close association of several aquatic ascomycetes with terrestrial relatives (Liew et al. 2002). Shearer (1993) noted the evolution of aquatic hyphomycetes from terrestrial plantassociated or litter-associated fungi (Shearer 1993;Selosse et al. 2008), which was later supported in other ascomycetous fungi (Baschien et al. 2013). In the present study, the aquatic hyphomycetes, Clonostachys rosea, Gliomastix masseei and Memnoniella echinate were first reported from freshwater habitats. In contrast, previously they have been reported only in terrestrial habitats. However, we could not find a firm conclusion of the origin for these aquatic hyphomycetes. The early diverging clades of Hypocreomycetidae mostly comprise terrestrial fungi, while the marine and freshwater species show secondary independent evolution, represented by the order Glomerellales (Figs. 1, 2). The crown clade is represented by Nectriaceae (Hypocreales), which moderately comprises all three habitats with terrestrial ancestors. The following clades are represented by Coronophorales and Falcocladiales, which entirely include terrestrial fungi. The adjacent clade represents Torpedosporales which constitutes all three habitats, whereas a single species from the terrestrial habitat represents the next clade Xenodactylariales. Halosphaeriaceae (Microascales) clade mainly represents marine fungi, and only a few species show terrestrial and freshwater habitats that were secondarily derived. The representative families of Microascales, including Ceratocystidaceae, Cornuvesicaceae, Gondwanamycetaceae, Graphiaceae and Microascaceae, entirely accommodate terrestrial fungi, while Triadelphiaceae comprises a few freshwater fungi. The specific morphological adaptations observed among the representative species in various habitats were discussed under morphological adaptions of freshwater Hypocreomycetidae. However, no clear pattern in habitual transitions were observed within several lineages, and the absence of molecular data for several genera of the subclass renders the definitive conclusion. The details studies on fungal adaptation strategies, sexual and asexual connections, and their evolutionary changes can provide more information about their origin.

Divergence time estimates of Hypocreomycetidae
The divergence time estimates revealed that Hypocreomycetidae evolved during the early Permian (251-290 MYA) (Hyde et al. , 2020aDayarathne et al. 2019). In this study, the estimated crown age of Hypocreomycetidae (290 MYA) is similar to previous studies and falls within the early Permian period (Hyde et al. , 2020aDayarathne et al. 2019). However, previous studies used different methods to calibrate the evolution tree. Hyde et al. (2017) used fossil data (Paleoophiocordyceps coccophagus) and two secondary calibrations (the divergence time of Sordariomycetes and Leotiomycetes and crown age of Sordariomycetes). Dayarathne et al. (2019) used Paleoophiocordyceps coccophagus fossil data, and crown age of Sordariomycetes as secondary calibration and Hyde et al. (2020a) used two secondary calibrations (crown ages of Sordariomycetes and Dothideomycetes). In this study, we used Paleoophiocordyceps coccophagus fossil data and the crown age of Sordariomycetes as secondary calibration. The result showed that the stem and the crown age of Hypocreomycetidae are around 290 and 302 MYA, respectively. The crown ages of several orders in Hypocreomycetidae viz. Coronophorales (220 MYA), Glomerellales (218 MYA), Hypocreales (222 MYA), Microascales (249 MYA), and Torpedosporales (175 MYA) lie within the Jurassic period (251.9-201.3 MAY). The Jurassic period had a warm climate, which promoted the flourishing of gymnosperms and true ferns, and formed vast forests covering the world, which providing various hosts for fungi. In addition, during the mass extinction event, the death of animals or insect also provided nutrients for fungi to survive and diversify during this period.
Studies suggested that basal fungi originated from aquatic habitats and fungal territorialization occurred during the Cambrian, over 500 million years ago (MYA) (Taylor and Osborn 1996;Brundrett 2002;Wijayawardene et al. 2018). Ascomycota and Basidiomycota are divergent at a similar time with the fungal territorialization and evolved from terrestrial ancestors (Vijaykrishna et al. 2006;James et al. 2006;Liu et al. 2006;Lucking et al. 2009;Gueidan et al. 2011;Hyde et al. 2021). Based on the molecular clock and ancestral state analysis, freshwater taxa in Hypocreomycetidae have evolved from terrestrial ancestors (Fig. 2). They have divergent much later than the terrestrial taxa (Fig. 2).
The Hypocreomycetidae may evolved from terrestrial to freshwater habitats in two pathways: 1) fungi may occur initially as pathogens, endophytes or saprobes on plants and they develop adaptation for freshwater when plants invaded freshwater habitats, 2) freshwater fungi may on the branches, stems or leaves in riparian vegetation and these substrates fallen into streams, the fungi colonized and adapted to the freshwater habitats (Shearer 1993;Hyde et al. 2021). Most freshwater Hypocreomycetidae have been reported on submerged wood or leaves, supporting Sherear (1993). The evolution of morphological characters in freshwater fungi along the molecular study can provide more information for evolutionary strategies.

Morphological adaptions of freshwater Hypocreomycetidae
Fungi show morphological adaptation when they transit from land to aquatic habitats. Sherer (1993) proposed several morphological adaptations of freshwater Ascomycota such as ascospores with appendages or sheaths, which help the ascospores attaching the substrate and remain connected as the water moves (Aniptodera, Ceriospora, Ceriosporopsis and Halosarpheia) (Shearer and Crane 1980;Shearer 1993). Filiform-like ascospores become sigmoid shape in the water which expands the area of the orthogonal projection, while the long filamentary shape enhances entanglement with the matrix (Webster and Davey 1984;Webster 1987). Few genera also developed deliquescent asci, which facilitate the dispersal of ascospores. Massive apical rings of asci also help the strong ejection of ascospores (Hyde and Goh 2003). The asexual freshwater species show branched, tetraradiate, sigmoid, helicosporous, and multicellular air trapping conidia which facilitate the dispersal and attachment of conidia to substrata shapes (Sherer 1993). However, it is hard to conclude that these characteristics have been developed as either pre-adaptation or convergent evolution since these characteristics are also found in terrestrial species (Vijaykrishna et al. 2006).
In our phylogenetic analysis, freshwater Hypocreomycetidae are mainly distributed in Campylosporaceae, Halosphaeriaceae, Nectriaceae and Reticulascaceae and therefore, our discussion is mainly focused to these families.
The asexual genus Campylospora (Campylosporaceae) entirely composed of freshwater species. The taxa are characterized by tetraradiate and hyaline conidia composed of two parts, deltoid and allantoid, and both show diverging branches at the ends. The presence of tetraradiate with diverging branches is a typical character of freshwater fungi, which helps the conidia to attach to the substrate and dispersal (Read et al. 1992;Vijaykrishna et al. 2006).
Halosphaeriaceae is a well-studied family with most marine species and few species from freshwater and terrestrial habitats (Pang 2002;Jones et al. 2009Jones et al. , 2015Jones et al. , 2017Jones et al. , 2019Maharachchikumbura et al. 2015;Wijayawardene et al. , 2018. Molecular data showed that Halosphaeriaceae has a marine ancestor (Fig. 2), which developed morphological adaptations to marine habitats. Based on morphological characters, Vijaykrishna et al. (2006) divided Halosphaeriaceae into two groups: (1) species with early deliquescing asci and appendaged ascospores and (2) species with persistent asci, often with an apical apparatus and mostly with ascospores with polar filamentous unfurling appendages. However, deliquescing asci may not be a good adaptation as deliquescing asci are rarely found in freshwater species . The second group are common in freshwater and marine habitats. Persistent asci with an apical apparatus may have the ability to facilitate strong ejection of ascospores and ascospores with polar filamentous unfurling appendages, which is important for dispersal and subsequent attachment in freshwater habitats (Goh and Hyde 1996). Jones (2006) assumed the ancestral ascospore lacked appendages or spore wall ornamentation. Sakayaroj et al. (2011) suggested that the appendages were lost several times during the evolution based on the phylogenetic analysis. However, ascospores of few species lack appendages when they release from the ascus, yet when the ascospores mounted in seawater, which produce apical appendages, but not in freshwater (Savoryella appendiculata, Halosarpheia aquatica, Thalespora appendiculata) (Jones and Hyde 1992;Hyde 1992;Jones 2006). Our phylogenetic analysis showed that freshwater fungi clustered with marine taxa in four clades (Fig. 1). Most freshwater Halosphaeriaceae species have ascospores with appendages, and only a few species lack appendages (e.g. Nais inornata). Thus, we assume that the appendages of these species may be lost during their evolution, as proposed by Sakayaroj et al. (2011).
Nectriaceae is an ecologically diverse group with species that have been found as endophytes, saprobes and pathogens of plants, and some are entomogenous, a few species are human pathogens (Hyde et al. 2020a). Nectriaceae comprises the mostly freshwater taxa of Hypocreomycetidae (Luo et al. 2019). Several freshwater species have also been reported from terrestrial habitats (e.g., Aquanectria penicillioides, Chaetopsina penicillata, Gliomastix masseei, Neocosmospora brevis and Volutella ciliata). Only a few species (e.g. Atractium aquatica and Mariannaea aquaticola) are restricted to freshwater habitats (Luo et al. 2019;Calabon et al. 2022). However, there are no special morphological adaptations have been reported in the freshwater Nectriaceae species except for a few which show apical rings (e.g., A. aquatica, Atractium fusiformis and Thelonectria aquatica), that help the ascospores to release into the water (Vijaykrishna et al. 2006).
Reticulascaceae comprises four genera, two genera (Cylindrotrichum; Cylindrotrichum aquaticum, C. gorii, C. submersum and Kylindria; K. aquatica and K. chinensis) include freshwater species but also encompass terrestrial species (Luo et al. 2019). Shearer (1993) mentioned that freshwater fungi adapt to aquatic habitats by their asexual morphs (e.g. hyphomycetes). Cylindrotrichum and Kylindria were reported in their asexual morphs Luo et al. 2019). No definite morphological adaptations were observed among the species except Kylindria aquatica, which has conidia with a slimy mucilaginous coating around it ). This indicates the independent evolution of morphological adaption from freshwater to terrestrial habitats.

Diversity of freshwater Hypocreomycetidae
Fungi are ubiquitous and have been found in all habitats. The number of fungi has been estimated between 2.2 and 3.8 million; however, only about 150,600 species of fungi and fungus-like taxa have been so far described (Hyde et al. 2020c;Phukhamsakda et al. 2022;Wijayawardene et al. 2020). Freshwater fungi as a unique ecological group of highly diverse fungi; since Shearer (1993) reported 288 ascomycetes species found in watery settings, and this number has now expanded to 3077 (Calabon et al. 2022). However, many species are yet to be discovered Calabon et al. 2022).
Hypocreomycetidae are an ecologically diverse group with species found in various environments. Hu et al. (2013) reported 57 Hypocreomycetidae species from freshwater habitats in China, and Luo et al. (2019) documented 76 species. Bao et al. (2021) provided a checklist of freshwater fungi in Yunnan Province during 2015-2020 and listed 21 freshwater Hypocreomycetidae species, and most species were collected from submerged decaying wood. In the latest study of Calabon et al. (2022), 156 Hypocreomycetidae species have been reported from freshwater habitats, with most documented from Lotic habitats (river and stream) (Calabon et al. 2022). In this study, 26 Hypocreomycetidae species have been reported from lentic freshwater habitats (lakes). This indicates that freshwater Hypocreomycetidae is highly diverse not only in lotic habitats but also in lentic habitats.

Update of Hypocreomycetidae based on the current study
In this section, we provide an updated classification of Hypocreomycetidae based on current study and recent publications (Hyde et al. 2020a;Huang et al. 2021;Wijayawardene et al. 2022;Xiao et al. 2023;Perera et al. 2023