Phylogenetic analyses of new aquatic hyphomycetes provide molecular evidence for Microthyriaceae (Dothideomycetes, As- comycota) anamorph

The fungal 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. Here, based on DNA sequences at four gene fragments (nuLSU rDNA, nuSSU rDNA, TEF1 and RPB2) in our analyses of aquatic hyphomycetes from southern China, we identify and report four new genera (Antidactylaria, Isthmomyces, Keqinzhangia, Pseudocoronospora) and thirteen new species (Antidactylaria minifimbriata, Pseudocoronospora hainanensis, Isthmomyces oxysporus, I. dissimilis, I. macrosporus, I. relanceatus, Keqinzhangia aquatica, Triscelophorus anakonajensis, T. anisopterioides, T. guizhouensis, T. mugecuoensis, T. multibrachiatus, T. neoseptatus; new combinations Isthmomyces asymmetrica, I. basitruncata, I. geniculata, I. lanceata, I. minima, I. rotundata) belonging to Microthyriaceae. Our results provided the first molecular evidence of asexual morph of this family and strengthened the phylogenetic placement of the family in class Dothideomycetes. The addition of these new taxa made Microthyriaceae the largest family comprising freshwater asexual genera in Pleosporomycetidae. In addition, we confirmed the monophyly of the genus Triscelophorus, the paraphyly of the genus Isthmolongispora, and revised 6 new combinations in Isthmolongispora. ITS barcoding of 13 species were also provided to help identify aquatic hyphomycetes in the future. Our results suggest that the asexual genera and sexual genera identified so far within this family have completely different ecological niches.


Introduction
The family Microthyriaceae (Microthyriales, Dothideomycetes) was established by Saccardo [1], containing foliar epiphytes and saprobes on dead leaves and stems [2]. This family is characterized by having superficial, flattened thyriothecia, with cells of the upper wall radiating in a parallel arrangement from the central opening; the opening may or may not be surrounded by setae. Asci are fusiform or obclavate to cylindroclavate, bitunicate, fissitunicate, and ascospores are two-celled, hyaline to brown often with ciliate appendages [2][3][4]. Asthton et al. [3] estimated that there were 54 genera and 278 species in the family. In a subsequent series of papers, Wu et al. [2,[5][6][7][8] 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 proposed [9], but in a subsequent outline of Ascomycota, only 9 genera were listed in this family [10].
Microthyriaceae have been poorly studied, there are very 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 sequence [11]. In the class-wide phylogenetic assessment of Dothideomycetes, Schoch et al. [12] included Microthyriaceae based on one strain of Microthyrium microscopicum Desm. (the type species of Microthyriaceae). So far, among the accepted 9 genera of the family, sequences of only five species (out of more than 200 species) are available from public databases, representing Chaetothyriothecium Hongsannan & K.D. Hyde, Microthyrium Desm., Palawania Syd. & P. Syd., and Tumidispora Hongsannan & K.D. Hyde. 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 [2]. Later, Hongsannan et al. [13] isolated cultures of Chaetothyriothecium elegans Hongsanan & K.D. Hyde and Tumidispora shoreae Hongsanan & K.D. Hyde [14], but failed to observe anamorphs of the two species. Wu et al. [7] tried to isolate fresh cultures from Microthyrium propagulensis H.X. Wu & K.D. Hyde, but did not observe the germination of ascospores. Based on these situation, asexual genera of Microthyriaceae were recorded only from the literature. Before Wu revised Microthyriaceae, Asterostomula Theiss. and seven other genera were listed as asexual [15,16]. With the exclusion of many genera from Microthyriaceae [2,5,6,8], only Hansfordiella S. Hughes was retained as an asexual genus in Microthyriaceae [10], but this connection was not confirmed by molecular data because sequences of Hansfordiella were unavailable. Moreover, Hansfordiella was recorded as asexual state of Trichothyrium Speg., which belongs to Trichothyriaceae [3,4,[15][16][17]. So strictly speaking, no asexual genus has been reported within the modern circumscription of Microthyriaceae.
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 [18]. 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 [19]). The linkages between asexual and sexual genera have been accumulated during implementation of the "One fungus: One name" concept, allowing the asexual genera to be placed in a natural biological framework of fungi [9,10,20]. However, the phylogenetic position of about 1530 genera in Ascomycota still remain incertae sedis [10].
Aquatic hyphomycetes colonize allochthonous organic matter in fresh waters and are closely involved in the decomposition and conversion of biopolymers in aquatic habitats [21]. 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 [22]. Molecular approaches applied to phylogeny of aquatic hyphomycetes place some genera in a defined class and found multiple origins of aquatic hyphomycetes. Specifically, 7 strains (5 species) of Tetracladium De Wild. showed close relationships to the Ascomycete orders Onygenales, Erysiphales and Leotiales [23], but subsequently, Tetracladium was placed in Leotiomycetes based on combined ITS and 28S analyses [24]. Studies of 31 species of aquatic hyphomycetes placed the majority (74 %) within the Leotiomycetes [22,25]. Duarte et al. [26] constructed and 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 have been published with confirmed phylogenetic positions [10,[27][28][29][30]. Although these studies promoted phylogenetic development of aquatic hyphomycetes, the phylogenetic positions of most aquatic hyphomycetes have not been determined at the family level [10].
In recent years, we have studied the diversity and phylogeny of aquatic hyphomycetes from southern China in Yunnan, Sichuan, Guizhou and Hainan Provinces, a hot spot of world biodiversity. Previously we have reported some new species from these regions [29,[31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48]. During this process, we found some isolates similar to those in Microthyriaceae. After studying in detail, we described and illustrated these new taxa, placed them in Microthyriaceae based on analyses of four gene regions: nuclear large subunit (nuLSU rDNA), nuclear small subunit (nuSSU rDNA), translational elongation factor 1α (TEF1), and RNA polymerase II subunit 2 (RPB2), and discussed difference between each new taxon and its most similar genera or species. In addition, the internal transcribed spacers including the 5.8s subunit rDNA (ITS) were provided for each of the new species as DNA barcodes.

Collection of fresh samples, fungal isolation and characterization
Submerged dicotyledonous leaves were collected from streams in Yunnan, Guizhou, Sichuan, Hainan Provinces and Tibet. Samples were preserved in zip-locked plastic bags, labeled and transported to the laboratory. Each rotted leaf was cut into several 3-4 × 4-5 cm sized fragments, which were incubated on CMA (20 g cornmeal, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1000 ml distilled water) for 5 days at room temperature. Conidia were isolated using a sterilized toothpick under a BX51 microscope and cultivated on CMA plates. Morphological characteristics were observed from cultures growing on CMA after incubation at 25°C for a week. Pure cultures have been deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming, Yunnan, P.R. China (YMF, formerly Key Laboratory of Industrial Microbiology and Fermentation Technology of Yunnan). Ex-holotype living cultures were deposited in the China General Microbiological Culture Collection Center (CGMCC).

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from fresh mycelia grown on potato dextrose agar (PDA) at 25°C as described by Turner et al. [49]. The air-dried precipitate was dissolved in 50 µl of sterilized distilled water and stored at -20°C until use for amplification reactions. The primer pairs NS1/NS4, LROR/LR7 [50], EF1-728F [51] and TEF1LLErev [52], and tRPB2-5F and tRPB2-7R [53] were, respectively, used for the amplification of the small subunit nuclear ribosomal RNA gene (SSU rRNA), the large subunit nuclear ribosomal RNA gene (LSU rRNA), translation elongation factor 1 alpha gene (TEF1), and the second largest subunit of the DNA-directed RNA polymerase II (RPB2). The PCR thermal cycle programs for the amplifications of these three DNA fragments followed those described in Su et al. [30]. PCR products were visualized 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., China). DNA forward and reverse sequencing was performed with a LI-COR 4000L automatic sequencer, using a Thermo Sequenase-kit as described by Kindermann et al. [54].

Sequence alignment and phylogenetic analysis
Preliminary BLAST searches with the LSU gene sequences of the new isolates against GenBank nucleotide databases determined species closely related to our isolates. Based on this information, sequences at the four marker loci were downloaded from Microthyriaceae and five sister families belonging to Dothideomycetes, including 44 strains representing 30 species (Supplementary Table 1). Schismatomma decolorans (Erichsen) Clauzade & Vězda was used as outgroup. For phylogenetic analyses of Isthmolongispora, LSU sequences of 32 strains representing 16 species of 7 genera, belonging to three families Hyaloscyphaceae, Mollisiaceae and Loramycetaceae were downloaded following BLAST searching of LSU. Endocronartium harknessii (J.P. Moore) Y. Hirats. belonging to Cronartiaceae was used as outgroup (Supplementary Table 2).
For Microthyriaceae the sequences of these representative strains were combined with the ones from our own cultures (Table 1). Four alignment files were generated, one for each gene, and there were then converted to NEXUS files with ClustalX 1.83 [55]. The four aligned were then concatenated with BioEdit 7.1.9.0 [56]. All characters were weighted equally and gaps were treated as missing characters. Maximum likelihood (ML) analysis was computed by RAxML [57] with the PHY files generated with ClustalX 1.83 [55], using the GTR-GAMMA model. Maximum likelihood bootstrap proportions (MLBP) were computed with 1000 replicates. Bayesian inference (BI) analysis was conducted with MrBayes v3.2.2 [58]. The Akaike information criterion (AIC) implemented in jModelTest 2.0 [59] was used to select the best fit models after likelihood score calculations were done. The base tree for likelihood calculations was ML-optimized. HKY+I+G was estimated as the best-fit model under the output strategy of AIC, Metropolis-coupled Markov chain Monte Carlo (MCMCMC) searches were run for 2,000,000 generations, sampling every 1000th generation. Two independent analyses with four chains each (one cold and three heated) were run until the average standard deviation of the split frequencies dropped below 0.01. The initial 25% of the generations of MCMC sampling were discarded as burn-in. The refinement of the phylogenetic tree was used for estimating Bayesian inference posterior probability (BIPP) values. The Tree was viewed in FigureTree v1.4. The values of maximum likelihood bootstrap proportions (MLBP) greater than 70% and Bayesian inference posterior probabilities (BIPP) greater than 90% at the nodes are shown along branches. For Isthmolongispora and Leotiomycetes, only Bayesian inference analysis based on LSU was used.

Phylogenetic analyses
In the phylogenetic analyses of Microthyriaceae, several major clades were found, consistent with results of earlier multi-gene phylogenetic analyses [4,9]. Our 13 species along with four known species of Microthyriaceae formed a clade with 98 % Maximum Likelihood Bootstrap (MLB) and 100% Bayesian Posterior Probability supports (BPP) (Figure 1), which is still as a sister clade of Phaeotirchaceae as previously indicated. Our analyses revealed four distinct new clades which we describe as four new genera Antidactylaria, Isthmomyces, Keqinzhangia, Pseudocoronospora. In addition, Triscelophorus was found to form a distinct clade with 95 % MLB support and 100% BPP, while both the MLB and BPP supports of Isthmomyces were 100%. Of the four new genera, only Isthmomyces was closely related to the known genus Microthyrium, and these two genera formed a sister clade with a high support value.
In the phylogenetic analyses of Isthmolongispora and members of Leotiomycetes, three main clades were present, representing three families, Hyaloscyphaceae, Mollisiaceae and Loramycetaceae. Four strains of I. quadricellularia fell into the clade of Hyaloscyphaceae, as the closest sister clade to Hyaloscypha Boud ( Figure 2).  Notes: Microthyriaceae have been poorly studied, therefore, there are very few DNA sequences in public databases for this group of fungi. Our results provided the first molecular evidence of asexual morph of this family. We erected four new genera (Antidactylaria, Isthmomyces, Keqinzhangia, Pseudocoronospora) and recognized thirteen new species in Microthyriaceae based on DNA sequences at four gene fragments. In addition, six new combinations are proposed for Isthmolongispora species composed of two cellular isthmic segments. Notes: The genus Dactylaria Sacc., typified with D. purpurella (Sacc.) Sacc., is characterized by unbranched, septate, hyaline or pigmented conidiophores and denticulate, integrated, mostly terminal, sympodially extending conidiogenous cells and cylindrical, fusiform, filiform, ellipsoid, clavate, obclavate, unicellular or septate, hyaline or pale pigmented conidia that are liberated after schizolytic secession [60][61][62]. The rhexolytic conidial secession in Antidactylaria separates it from Dactylaria morphologically as conidiogenous event and an important criterion for generic delimitation, discussed by Paulus et al. [61] and supported by the molecular phylogeny analysis obtained from Antidactylaria minifimbriata. Etymology. Latin, mini-meaning very small, minutely, + Latin, fimbriata-, referring to edged, delicately toothed, fringe or frill that remained on the conidial base after rhexolytic secession.

Discussion
Molecular phylogeny of freshwater fungi in Dothideomycetes has been studied by Shearer et al. [73] 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 among other clades. In the largest phylogenetic assessment of Dothideomycetes by 2009, members of the class from various ecological niches were included, and freshwater were in different clades [12]. 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. [73], only 10 asexual genera were included, while in the paper of Schoch et al. [12], only four asexual genera were included (Monotosporella S. Hughes and Beverwykella Tubaki belonging to Melanom-mataceae G. Winter, while Helicomyces Link and Helicosporium Nees belonging to Tubeufiaceae). Among 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 [9]. By our addition of new aquatic hyphomycetes to Microthyriaceae makes this family the largest of Pleosporomycetidae comprising aquatic asexual genera.
With increasingly widespread use of molecular techniques, multigenes were concatenated to resolve phylogenetic affiliations and taxonomic placements at family or higher ranks. For example, nucSSU, nucLSU rDNA, TEF1, RPB1 and RPB2, were combined to assess phylogeny [9,11,12]. 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 [74]. In addition, most of these species with ITS sequences were considered Ascomycota genera incertae sedis because of the limitations of ITS as a phylogenetic marker for these organisms. Duarte et al. [26] found that Triscelophorus spp. were polyphyly based on ITS analysis, but did not determine its phylogenetic position. We carried out phylogenetic analysis based on all ITS of Triscelophorus spp. available from GenBank and generated from our Triscelophorus strains, found four strains of T. cf. acuminatus (GenBank accession number: KF730836 -838) and one unnamed Triscelophorus strain (KF730841) formed a clade, but other 6 unnamed Triscelophorus strains (Genbank accessible number: KF730842 -847) fell into another clade, we suspected these six strains might be misidentified at genus level. Based on our four-loci phylogenetic analysis, in our opinion, Triscelophorus should be monophylogenetic. At present, the majority of ascomycetous aquatic hyphomycetes were only placed at the class level [75][76][77]. In order to bring more aquatic hyphomycetes into lower taxonomic levels, it is necessary to obtain sequences from more genes.
The most obvious result achieved on the phylogeny of aquatic hyphomycetes is that the multiple origins of aquatic hyphomycetes was found [22]. So far, at least 14 genera have shown to be polyphyletic using sequence information from a single or two genes [23][24][25][26]78]. Likewise, polyphyly of Isthmolongispora was found for the first time here. Although there are 9 ITS sequences, one sequence is from I. lanceate, and another one from I. ampulliformis (Tubaki) de Hoog & Hennebert, while other 7 sequences were from unidentified species. Three LSU sequences were available from GenBank. However, Isthmolongispora was listed as Ascomycota genera incertae sedis [17]. These results show it is necessary to sequences more loci for confirm polyphyly or monophyly.
Difficulty in conidia development may be the reason why the asexual states of Microthyriaceae have not been found. According to previous studies, cultures were isolated but no sporulation [13,14]. According to our experience from studying aquatic hyphomycetes and their relevant relationships in Microthyriaceae, in our opinion, it is necessary to induce conidia using various methods. Aquatic hyphomycetes often grow slowly, and do not produce generative structures easily. Based on our experiment, conidia can be induced after preserving the isolates at a low temperature of 4°C, sometimes water is needed. Anyhow, development of conidia takes about two weeks or longer.
Morphologically, five asexual genera of Microthyriaceae do not have any characteristics in common. Genetically there are large differences between each other, including between species within the same genus. For example, ITS of Isthmomyces lanceatus has 98.02 similarity with that of I. relanceatus, but only 59.76% similarity between I. lanceatus and I. macroporus. Within Triscelophorus, the largest ITS similarity of 95.83% is between T. anakonajensis and T. mugecuoensis, while the lowest ITS similarity of 73.96% is between T. anakonajensis and T. mugecuoensis. The larger genetic difference suggests that there are likely many more un-identified taxa in this genus and/or that the evolution of ITS in Microthyriaceae is very fast.
In this study, all taxa were described based on their asexual characteristics. Although we observed cultures for long time on CMA, we did not see any sexual reproductive structures. According to our phylogenetic analyses, only Ishmomyces is closely related to the genus Microthyrium, but their ITS sequence similarity is low, so we can't determine the connection between them.
In conclusion, this study described four new genera and 13 new species of aquatic hyphomycetes. Our phylogenetic analyses placed several other aquatic genera in 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 in order to identify the origins and phylogenetic positions of aquatic hyphomycetes and their relationships with their terrestrial relatives.
Author Contributions: KZ, and ZY conceived and designed the study. MQ, JG and HZ wrote the manuscript. JG and JP conducted the experiments. R.F.C contributed actively in the identification and the taxonomy of the fungal strains. ZY and JX revised the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This work was financed by the National Natural Science Foundation Program of PR China (31770026， 31760012).