Two new asexual genera and six new asexual species in the family Microthyriaceae (Dothideomycetes, Ascomycota) from China

Abstract The family Microthyriaceae is represented by relatively few mycelial cultures and DNA sequences; as a result, the taxonomy and classification of this group of organisms remain poorly understood. During the investigation of the diversity of aquatic hyphomycetes from southern China, several isolates were collected. These isolates were cultured and sequenced and a BLAST search of its LSU sequences against data in GenBank revealed that the closest related taxa are in the genus Microthyrium. Phylogenetic analyses, based on the combined sequence data from the internal transcribed spacers (ITS) and the large subunit (LSU), revealed that these isolates represent eight new taxa in Microthyriaceae, including two new genera, Antidactylariagen. nov. and Isthmomycesgen. nov. and six new species, Antidactylariaminifimbriatasp. nov., Isthmomycesoxysporussp. nov., I.dissimilissp. nov., I.macrosporussp. nov., Triscelophorusanisopterioideussp. nov. and T.sinensissp. nov. These new taxa are described, illustrated for their morphologies and compared with similar taxa. In addition, two new combinations are proposed in this family.


Introduction
The family Microthyriaceae (Microthyriales, Dothideomycetes) was established by Saccardo (1883), containing foliar epiphytes and saprobes on dead leaves and stems (Wu et al. 2011a). This family is characterised by having superficial, flattened thyriothecia, with cells of the upper wall radiating in a parallel arrangement from the central ostiole opening; the ostiole may or may not be surrounded by setae. Asci are fusiform or obclavate to cylindro-clavate, bitunicate and fissitunicate and ascospores are two-celled, hyaline to brown often with ciliate appendages (Ashton 2009;Wu et al. 2011a;Hyde et al. 2013). Ashton et al. (2009) estimated that there were 54 genera and 278 species in the family. In a subsequent series of papers, Wu et al. (2010Wu et al. ( , 2011aWu et al. ( , b, c 2014 revised Microthyriaceae by examining the generic type species and restricted Microthyriaceae to the species with morphological characteristics similar to Microthyrium Desm. Based on morphological characteristics, 11 genera and about 230 species were listed in this family (Wijayawardene et al. 2014), but in a subsequent outline of Ascomycota, only nine genera were accepted (Wijayawardene et al. 2018a). Recent studies accepted 11 genera in this family (Hongsanan et al. 2020;Wijayawardene et al. 2020).
Microthyriaceae have been poorly studied and there are few DNA sequences in public databases for this group of fungi. In the expanded multigene phylogeny of the Dothideomycetes, Microthyriaceae was not included because of the paucity of DNA sequences (Schoch et al. 2006). In the class-wide phylogenetic assessment of Dothideomycetes, Schoch et al. (2009) included Microthyriaceae, based on Microthyrium microscopicum Desm. (type species of Microthyriaceae). One major contributing reason for the absence of DNA sequences is that few living cultures are available. As a result, researchers might have assumed that many of these species were obligate parasites and could not be cultured (Wu et al. 2011a). Later, Hongsanan et al. (2014) isolated cultures of Chaetothyriothecium elegans Hongsanan & K.D. Hyde and Tumidispora shoreae Hongsanan & K.D. Hyde (Ariyawansa et al. 2015), but failed to observe anamorphs of the two species. Wu et al. (2014) tried to isolate fresh cultures of Microthyrium propagulensis H.X. Wu & K.D. Hyde, but did not observe the germination of ascospores. Based on these situations, asexual genera of Microthyriaceae were recorded only from the literature. Before Wu revised Microthyriaceae, Asterostomula Theiss. and seven other genera were described as asexual morphs (Hyde et al. 2011;Wijayawardene et al. 2012). With the exclusion of many genera from Microthyriaceae (Wu et al. 2010(Wu et al. , 2011a, only Hansfordiella S. Hughes was retained as an asexual genus in Microthyriaceae (Wijayawardene et al. 2018a), but this connection was not confirmed by molecular data because sequences of Hansfordiella were unavailable. Moreover, Hansfordiella was recorded as the asexual state of Trichothyrium Speg., which belongs to Trichothyriaceae (Ashton 2009;Hyde et al. 2011Hyde et al. , 2013Wijayawardene et al. 2012. In the early 1990s, molecular methods, in particular DNA sequence data, provided opportunities for phylogenetic inference and have made a significant impact on the taxonomy and classification of fungi (Shenoy et al. 2007). More importantly, sequence analysis can potentially place an asexual-state taxon within an order or even link it with a teleomorph genus without having to observe the latter (e.g. in Berbee and Taylor 2001). The linkages between asexual and sexual genera have accumulated during implementation of the "One fungus: One name" concept, allowing the asexual genera to be placed in a natural biological framework of fungi ( Wijayawardene et al. 2014( Wijayawardene et al. , 2018aMaharachchikumbura et al. 2015). However, the phylogenetic position of about 1530 genera in Ascomycota still remains incertae sedis (Wijayawardene et al. 2018a).
Aquatic hyphomycetes colonise allochthonous organic matter in fresh waters and are closely involved in the decomposition and conversion of biopolymers in aquatic habitats (Brlocher 1992). They are a polyphyletic group of fungi, mainly consisting of asexual morphs of Ascomycota and Basidiomycota, which have been identified, based on conidium morphology and conidiogenesis (Belliveau and Barlocher 2005). Molecular approaches applied to phylogeny of aquatic hyphomycetes place some genera in a defined class and found multiple origins of aquatic hyphomycetes. Specifically, seven strains (five species) of Tetracladium De Wild. showed close relationships to the Ascomycete orders Onygenales, Erysiphales and Leotiales (Nikolcheva 2002), but subsequently, Baschien (2006) found Tetracladium located in Leotiomycetes, based on combined ITS and 28S analyses. Besides, studies of 31 species of aquatic hyphomycetes placed the majority (74%) within the Leotiomycetes (Belliveau and Barlocher 2005;Campbell et al. 2006). Duarte et al. (2015) constructed an ITS phylogenetic tree for 79 aquatic hyphomycetes, and found Tricladium Ingold and Triscelophorus Ingold are not monophyletic. Of course, with the availability of more and more reference sequences and the establishment of backbone trees of some classes, new aquatic hyphomycetes related to monophyly have been published with confirmed phylogenetic positions (Pratibha et al. 2015;Liu et al. 2016;Su et al. 2016;Qiao et al. 2018a;Wijayawardene et al. 2018a). Although these studies promoted phylogenetic development of aquatic hyphomycetes, the phylogenetic positions of most aquatic hyphomycetes have not been determined at the family level (Wijayawardene et al. 2018a).
In recent years, we have investigated the diversity and phylogeny of aquatic hyphomycetes from southern China which is a hot spot of world biodiversity, such as Yunnan, Sichuan, Guizhou, Guangdong and Hainan Provinces. Many new species collected from these regions have been described (Yang et al. 2011(Yang et al. , 2012Bai et al. 2013;Li et al. 2013Li et al. , 2014Guo et al. 2015Guo et al. , 2019Qiao et al. 2017aQiao et al. , b, 2018bQiao et al. , 2019aQiao et al. , b, c, 2020Peng et al. 2016;Yu et al. 2019;Zheng et al. 2020aZheng et al. , 2021a. In addition, several interesting isolates were collected. These isolates were cultured and sequenced and a BLAST search of its LSU sequences against data in GenBank revealed that the closest related taxa are in the genus Microthyrium. Based on the phylogenetic analysis combined with the internal transcribed spacers (ITS) and the large subunit (LSU) gene sequences and morphological features, two new genera and six new species are proposed within Microthyriaceae. In addition, we also collected Isthmolongispora quadricellularis isolates and describe and illustrate it here.

Collection of samples, fungal isolation and morphological characterisation
Submerged leaves were collected from streams in Guangdong, Hainan Provinces and Tibet region. Samples were preserved in zip-locked plastic bags, labelled and transported to the laboratory at 4 °C. Each leaf was cut into several 3-4 × 4-5 cmsized fragments, then these fragments were incubated on corn meal agar (CMA; 20 g cornmeal, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1 litre distilled water) plates for 5 days at room temperature. Individual conidia were isolated using a sterilised toothpick under a BX51 microscope and cultivated on CMA plates. Morphological characteristics were observed from cultures growing on CMA and potato dextrose agar plates (PDA; 200 g potato, 20 g dextrose, 18 g agar, 1 litre distilled water) after incubation at 25 °C for one week. Microscopic photographs coming from CMA medium were taken with an Olympus BX51 microscope connected to a DP controller digital camera.
The pure cultures and dried cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming (YMF) and the China General Microbiological Culture Collection Center (CGMCC).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fresh mycelia grown on PDA at 25 °C as described by Turner et al. (1997). Fragments of the internal transcribed spacers (ITS) and the large subunit nuclear ribosomal RNA gene (LSU rRNA) were amplified with the following primer pairs: ITS4 and ITS5 for ITS (White et al. 1990) and LROR/LR7 (Vilgalys and Hester 1990), respectively. Each 25 μl PCR reaction volume consisted of 12.5 μl T5 Super PCR Mix (Beijing TsingKe Biotech Co., Ltd., Beijing, China), 1 μl of forward primer (10 μM), 1 μl of reverse primer (10 μM), 1μl DNA template, 5 μl of PCR buffer and 4.5 μl sterile water. The PCR thermal cycle programmes for the amplifications of these three DNA fragments followed those described in Su et al. (2016). PCR products were visualised on 1% agarose gel stained with Goldview (Geneshun Biotech, China) with D2000 DNA ladder (Realtimes Biotech, Beijing, China) and were then purified using a commercial Kit (Bioteke Biotechnology Co., Ltd., Beijing, China). DNA forward and reverse sequencing was performed with a LI-COR 4000L automatic sequencer with the same primers, using a Thermo Sequenase-kit as described by Kindermann et al. (1998). Finally, these new obtained sequences were deposited in the GenBank database at the National Center for Bio-technology Information (NCBI) and the accession numbers are listed in Table 1.

Sequence alignment and phylogenetic analysis
Preliminary searches with newly-generated LSU and ITS gene sequences of these isolates against National Center for Biotechnology Information (NCBI) by the Basic Local Alignment Search Tool (BLAST) determined species closely related to our isolates. Based on this information, sequences of ITS and LSU were downloaded from Microthyriaceae and four sister orders belonging to Dothideomycetes, including 48 strains representing 35 species (Table 1), according to recent studies (Hongsanan et al. 2020;Iturrieta-González et al. 2020). Schismatomma decolorans (Erichsen) Clauzade & Vězda was used as the outgroup taxon. For Microthyriaceae, the phylogenetic analysis was based on the combined ITS and LSU sequences. DNA sequence data of ITS and LSU were aligned using Clustal X 1.83 (Thompson et al. 1997) with the default parameters, then the consensus sequences were manually adjusted and linked through BioEdit v.7.0 (Hall 1999). Manual gap adjustments were carried out to improve the alignment and ambiguously-aligned regions were also excluded. We finally obtained the combined sequence matrix (Fasta file) generated by BioEdit v.7.0, containing 1119 nucleotide positions from two genes and the matrix was uploaded to TreeBASE (www.treebase.org; accession number: S28086). Bayesian Inference (BI) and Maximum Likelihood (ML) were used in this study for phylogenetic analyses. BI analysis was conducted with MrBayes v.3.2.2 (Ronquist et al. 2012) with NEXUS files converted by MEGA6 (Tamura et al. 2013). The Akaike Information Criterion (AIC) implemented in jModelTest 2.0 (Posada 2008) was used to select the best fit models after likelihood score calculations were done. GTR+F+I+G4 was estimated as the best-fit model under the output strategy of AIC. The parameters used were two simultaneous runs of 1,000,000 generations, four Markov chains, sampled every 500 generations. The 50% majority-rule consensus tree and posterior probability values (PP) were calculated after discarding the first 25% of the samples. ML analysis was computed by RAxML (Stamatakis 2006

Phylogenetic analyses
The phylogenic tree, based on a combined sequence of the LSU and ITS, indicated that eight isolates belong to the Microthyriaceae (Fig. 1). After detailed observations of morphological features, these isolates were considered as six new species and one known species. In this tree, five isolates grouped with Isthmolongispora lanceata CBS 622.66 with good support (MLBP/BIPP = 100%/1.0). Combined with morphological differences, we proposed the new genus Isthmomyces to accommodate the three new species, designated as I. dissimilis, I. macrosporus and I. oxysporus and a new combination I. lanceatus. Two isolates, which clustered with Triscelophorus monosporus CBS 440.54 (MLBP/BIPP = 91%/1.0), were considered as two new Triscelophorus species, designated as Triscelophorus anisopteriodeus and T. sinensis. The isolate YMF 1.04578 is phylogenetically close to Isthmolongispora ampulliformis (MLBP/BIPP = 77%/0.96). Considering morphological characters, we proposed a new genus Antidactylaria to accommodate the new species A. minifimbriata and the new combination A. ampulliforma.   (Goh and Hyde 1997;Paulus et al. 2003;Seifert et al. 2011). However, the rhexolytic conidial secession, observed in Antidactylaria, is absent in Dactylaria. Paulus et al. (2003) Tubaki, J. Hattori bot. Lab. 20: 159 (1958) Description. Matsush. 1975 Notes. Antidactylaria ampulliforma was originally isolated by Tubaki from leaves of Cocos nucifera and was described as Diplorhinotrichum species (Tubaki 1958). In 1983, de Hoog and Hennebert included it in the genus Isthmolongispora after examining its morphological character. In this study, A. ampulliforma is phylogenetically close to A. minifimbriata and they are very similar in morphology. Therefore, we assigned it in the newly-established genus Antidactylaria as a new combination. Etymology. Latin, isthmus, Greek (isthmós, "neck") meaning a narrow cellular structure that connects two larger bodies or cells, Greek, myces, referring to fungus.   (Matsushima 1971). The genus is characterised by denticulate, sympodially-extending conidiogenous cells and isthmospore conidia made of two or several cellular structures, which are connected by very narrow isthmuses. In this study, specimens with two and more cellular isthmic-segments were collected, respectively. Phylogenetic analysis inferred from two loci showed that our isolates grouped together with Isthmomyces lanceatus (Isthmolongispora lanceata) in Microthyriaceae. Combining morphological character and phylogenetic analysis, we finally erected the new genus Isthmomyces to accommodate these isolates and I. lanceata. Etymology. Latin, dissimilis, referring to the variation of the conidial shape related to the generic concept of the genus.
Type. China, Hainan Province, Diaoluo Mountain Nature Reserve, on submerged leaves, August 2015, J. Peng. Holotype YMF 1.04604, preserved in a metabolicallyinactive state (deep freezing) in the Conservation and Utilization of Bio-Resources in Yunnan. Ex-type culture CGMCC 3.18826.
Notes. The new species, Isthmomyces dissimilis, varies in conidial shape. Although it has 3-cellular isthmic-segment conidia, its isthmic-segment is not as distinct as Isthmolongispora species. However, the cells of Isthmolongispora are bead-like, while those of I. dissimilis are cylindrical to fusiform.  (Hoog and Hennebert 1983). However, the taxonomic status of this species was Ascomycota incertae sedis. In this study, this is the first report of I. lanceatus isolated from Asia. Morphologically, the conidia of our isolates are larger than the holotype CBS 622.66. Our phylogenetic analysis of combined LSU and ITS sequences reveals that the phylogenetic position of I. lanceatus is in Microthyriaceae and I. lanceatus is close to I. dissimilis in this tree.  4.5-6.7 μm; ii) apical isthmic-segment 0-1-septate, narrow obclavate, sometimes subobspathulate, rounded at the tip, unicellular, guttulate, hyaline or subhyaline, smooth, 21.1-42.0 × 3.3-5.4 μm. Sexual state: unknown.
Type. China, Hainan Province, Diaoluo Mountain Natural Reserve, on submerged leaves, August 2015, J. Peng. Holotype YMF 1.04513, preserved in a metabolicallyinactive state (deep freezing) in the Conservation and Utilization of Bio-Resources in Yunnan. Ex-type culture CGMCC 3.18821.
Notes. Morphologically, Isthmomyces oxysporus resembles Isthmolongispora asymmetrica Aramb. & Cabello in having both tapering isthmic-segment ends, but Is. asymmetrica has asymmetrical conidia, in which the basal isthmic-segment is longer (17-20 μm long) (Arambarri et al. 1987). Besides, I. oxysporusis is somewhat similar to Is. rotundata Matsush. in conidial sizes, but the apical isthmic-segments in Is. rotundatus are rounded at the tip (Matsushima 1987 Notes. Triscelophorus was established by Ingold, with T. monosporus as type species (Ingold 1943). The genus is characterised by macronematous, mononematous, erect, straight or flexuous, sometimes sinuate, septate, unbranched or sparingly branched, hyaline, smooth conidiophores. The conidiogenous cells are monoblastic, sometimes sympodially extended, integrated, hyaline that produce a solitary, acrogenous, septate, staurospore composed of a main axis and 3 or more branches verticillate arranged from the basal cell of the main axis (Ingold 1943;Seifert et al. 2011). Duarte et al. (2015) found that Triscelophorus was polyphyletic, based on ITS analysis, but our phylogenetic analysis, based on two-loci and ITS, showed the genus should be monophyletic. For more details, refer to Discussion.  Etymology. Latin, anisopteriodeus, referring to the resemblance of the conidial body to an adult of Anisoptera sp.
Type. China, Hainan Province, Limu Mountain Nature Reserve, on submerged leaves, April 2015, J. Peng. Holotype YMF 1.04267, preserved in a metabolicallyinactive state (deep freezing) in the Conservation and Utilization of Bio-Resources in Yunnan. Ex-type culture CGMCC 3.18978.
Notes. Isthmolongispora quadricellularia was first described by Matsush. in 1975 from Japan. Subsequently, this species has been isolated from leaves many times in Taiwan. However, no sequences of I. quadricellularia are available in the public database. In this study, it is the first time that I. quadricellularia has been isolated from the aquatic environment. In addition, we also submitted sequence data for this species to the public database (SSU: MT507103-105; LSU: MT507107-110; ITS: OL412746-749).

Discussion
China is considered an important reservoir of Asian biodiversity (Myers et al. 2000); it is estimated that this area harbours an inestimable diversity of fungi. In recent years, more and more new genera and species have been identified and classified for the application of phylogenetic analysis and have led to a significant expansion of species in Dothidomycetes (Zheng et al. 2019(Zheng et al. , 2020b(Zheng et al. , 2021bYang et al. 2021). However, comparatively speaking, aquatic hyphomycetes have been poorly investigated. In recent years, we have been investigating the diversity of aquatic hyphomycetes from southern China. During this process, several interesting isolates have been collected. After studying in detail, two new asexual genera and six new asexual species have been described in Microthyriaceae.
Triscelophorus Ingold was established with T. monosporus Ingold as type species; now, eight species have been accepted in this genus (Ingold 1943;). However, the positions of Triscelophorus in ordinal and familial levels are still unclear. In this study, two isolates which have similarity to Triscelophorus species in morphology were collected. For further study, the two isolates were identified as two new species of Triscelophorus, named as T. anisopteriodeus and T. sinensis. Moreover, phylogenetic analysis of combined LSU and ITS sequences places Triscelophorus in Microthyriaceae (Fig. 1).
Isthmolongispora Matsush. was established in 1971 and, so far, eleven species were accepted in this genus (Matsushima 1971;Wijayawardene et al. 2018bWijayawardene et al. , 2020. In this study, ten isolates have similarity to some Isthmolongispora species. Of these, four isolates were identified as Isthmolongispora quadricellularis, based on morphology. The combined LSU and ITS tree (Fig. 1) showed that the other six isolates formed two clades in Microthyriaceae. Comparing their morphological differences between species of the two clades, we established two new genera Antidactylaria and Isthmomyces. Antidactylaria includes a new species A. minifimbriata and a new combination A. ampulliforma and is phylogenetically close to two asexual species Scolecobasidium tropicum Matsush. and Neoscolecobasidium agapanthi Crous. Isthmomyces includes three new species, I. dissimilis, I. oxysporus and I. macrosporus and a new combination I. lanceatus. Phylogenetically, Isthmomyces is near to the sexual genus Microthyrium and the asexual genus Neoanungitea. Although Ishmomyces is closely related to Microthyrium, their ITS sequence similarity is low, so we cannot determine the connection between them. Based on the two-gene tree, we speculated that Isthmolongispora is polyphyletic. So far, at least 14 genera of aquatic hyphomycetes have shown to be polyphyletic using sequence information from a single or two genes (Nikolcheva 2002;Tsui et al. 2006;Baschien 2006;Campbell et al. 2006;Duarte et al. 2015).
With increasingly widespread use of molecular techniques, multi-genes were concatenated to resolve phylogenetic affiliations and taxonomic placements at family or higher ranks. For example, SSU, LSU, tef1, rpb1 and rpb2 were combined to assess phylogeny (Schoch et al. 2006(Schoch et al. , 2009Wijayawardene et al. 2014). However, sequence data and cultures of many aquatic hyphomycetes were unavailable. By 2013, over 300 aquatic hyphomycete species had been described, based on conidia morphology and conidiogenesis. However, fewer than 50 species had published ITS sequences in the International Nucleotide Sequence Database (Duarte et al. 2013). In addition, most of these species with ITS sequences were considered Ascomycota genera are incertae sedis because of the limitations of ITS as a phylogenetic marker for these organisms.
Molecular phylogeny of freshwater fungi in Dothideomycetes has been studied by Shearer et al. (2009) using SSU and LSU for 84 isolates representing 29 genera. The results showed that the majority of freshwater Dothideomycetes belonged to Pleosporomycetidae, including four clades comprised of only freshwater taxa, while the remaining freshwater taxa were distributed amongst other clades. In the largest phylogenetic assessment of Dothideomycetes up to 2009, members of the class from various ecological niches were included and freshwater taxa were in different clades (Schoch et al. 2009). Unfortunately, like other studies, though representative, these two studies of Dothideomycetes and freshwater ascomycetes had very few aquatic asexual genera. In the paper of Shearer et al. (2009), only 10 asexual genera were included, while in the paper of Schoch et al. (2009), only four asexual genera were included (Monotosporella S. Hughes and Beverwykella Tubaki belonging to Melanommataceae G. Winter, while Helicomyces Link and Helicosporium Nees belonging to Tubeufiaceae). Amongst the accepted genera of Dothideomycetes, only 11 aquatic or aero-aquatic asexual genera have been described as belonging to different families of the subclass Pleosporomycetidae (Wijayawardene et al. 2014). Our study provides the molecular evidence for asexual aquatic fungi.

Conclusions
This study described two new asexual genera and six new asexual species of aquatic hyphomycetes. Our phylogenetic analyses placed several other aquatic genera in the family Microthyriaceae. Though we failed to connect teleomorphs and anamorphs at genus level, our results showed close phylogenetic relationships between aquatic hyphomycetes and Microthyriaceae at the family rank. This study also revealed the importance of obtaining pure cultures of aquatic fungi and multiple gene sequences from them to identify the origins and phylogenetic positions of aquatic hyphomycetes and their relationships with their terrestrial relatives.