Novelties in Microthyriaceae (Microthyriales): Two New Asexual Genera with Three New Species from Freshwater Habitats in Guizhou Province, China

Microthyriaceae is typified by the sexual genus Microthyrium, with eight asexual genera. Three interesting isolates were collected during our investigation of freshwater fungi from the wetlands in Guizhou Province, southwest China. Three new asexual morphs are identified. Phylogenetic analyses using ITS and LSU gene regions revealed the placement of these isolates in Microthyriaceae (Microthyriales, Dothideomycetes). Based on the morphology and phylogenetic evidence, two new asexual genera, Paramirandina and Pseudocorniculariella, and three new species, Pa. aquatica, Pa. cymbiformis, and Ps. guizhouensis, are introduced. Descriptions and illustrations of the new taxa are provided, with a phylogenetic tree of Microthyriales and related taxa.

During a survey of the taxonomy and diversity of freshwater fungi from karst plateau wetlands in Guizhou Province, China [17][18][19][20][21][22], three asexual species were collected and identified based on the morphology and phylogenetic analysis. We therefore introduce two new genera, Paramirandina and Pseudocorniculariella, and three new species, Pa. aquatica, Pa. cymbiformis, and Ps. guizhouensis, with an illustrated account and molecular evidence. An updated backbone tree using ITS and LSU sequences is provided for Microthyriales.

Collection and Examination of Specimens
Specimens of submerged decaying twigs were collected from wetlands in Guizhou Province, China. Samples were taken to the laboratory in zip-lock plastic bags and incubated in plastic boxes lined with moistened sterile filter paper at room temperature for one week. Motic Nikon SMZ-171 (Nikon, Tokyo, Japan) dissecting microscopes were used to observe the fungal colonies and fruiting bodies. Fungal structures were examined and photographed using a Nikon ECLIPSE 80i (Nikon, Tokyo, Japan) compound microscope fitted with a Canon 70D (Canon, Tokyo, Japan) digital camera. Single spore isolations were made onto water agar (WA), and germinated spores were transferred onto potato dextrose agar (PDA) following the method in Luo et al. [23] and Senanayake et al. [24]. Tarosoft Image Frame Work program was used for measurement, and images used for figures were processed with Adobe Photoshop CS6 software. Herbarium specimens were deposited in the herbarium of Guizhou Academic of Agriculture Sciences (GZAAS), Guiyang, China, and herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China. Axenic cultures were deposited in Guizhou Culture Collection (GZCC). Facesoffungi and Index Fungorum numbers were registered as outlined in Jayasiri et al. [25] and Index Fungorum (December 2022) [26].

DNA Extraction, PCR Amplification and Sequencing
Fungal mycelium was scraped using a sterilized scalpel and transferred to a 1.5 mL microcentrifuge tube for genomic DNA extraction. An Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, China) was used to extract DNA following the manufacturer's instructions. DNA amplification was performed by polymerase chain reaction (PCR). ITS, LSU, SSU, tef1-α and rpb2 gene regions were amplified using the primer pairs, ITS5/ITS4 [27], LR0R/LR5 [28,29], NS1/NS4 [27], ef1-983F/ef1-2218R [30], and rpb2-5F/rpb2-7cR [31,32], respectively. The amplification was performed in a 25 µL reaction volume containing 9.5 µL ddH 2 O, 12.5 µL 2 × Taq PCR Master Mix with blue dye (Sangon Biotech, China), 1 µL of DNA template, and 1 µL of each primer (10 µM). The amplification condition for LSU, ITS and tef1-α genes consisted of initial denaturation at 94 • C for 3 min, followed by 40 cycles of 45 s at 94 • C, 50 s at 56 • C and 1 min at 72 • C, and a final extension period of 10 min at 72 • C. The amplification condition for the rpb2 gene consisted of initial denaturation at 95 • C for 5 min, followed by 37 cycles of 15 s at 95 • C, 50 s at 56 • C and 2 min at 72 • C, final extension period of 10 min at 72 • C. Purification and sequencing of PCR products were carried out by Shanghai Sangon Biological Engineering Technology and Services Co., Shanghai, China.

Phylogenetic Analyses
The ex-type and additional strains of Microthyriales species and related orders (Micropeltidales, Natipusillales, Phaeotrichale, Venturiales, and Zeloasperisporiales) were selected in the phylogenetic analyses. Two gene regions, ITS and LSU, were used for the multi-gene analyses. Sequences were optimized manually to allow maximum alignment and maximum sequence similarity. The sequences were aligned using the online multiple alignment program MAFFT v.7 (http://mafft.cbrc.jp/alignment/server/, accessed on 12 January 2023) [33]. The alignments were checked visually and improved manually where necessary.
Maximum likelihood (ML), Bayesian inference (BI), and Maximum parsimony (MP) analyses were employed to assess phylogenetic relationships. ML and BI analyses were performed through the CIPRES Science Gateway V. 3.3 [34]. ML analyses were conducted with RAxML-HPC v. 8.2.12 [35] using a GTRGAMMA approximation with rapid bootstrap analysis followed by 1000 bootstrap replicates. For the BI approach, MrModeltest2 v. 2.3 [36] was used to infer the appropriate substitution model that would best fit the model of DNA evolution for the combined dataset. The GTR+G+I substitution model was selected for ITS and LSU partitions. BI analyses were performed in a likelihood framework implemented in MrBayes 3.2.6 [37]. Six simultaneous Markov chains were run until the average standard deviation of split frequencies was below 0.01, with trees saved every 1000 generations. The first 25% of saved trees, representing the burn-in phase of the analysis, were discarded. The remaining trees were used for calculating the posterior probabilities of recovered branches [38]. MP analyses were conducted with PAUP v. 4.0a167 [39]. A heuristic search was performed with the stepwise-addition option with 1000 random taxon addition replicates and tree bisection and reconnection branch swapping. 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 analysis with 1000 replicates, each with ten replicates of random stepwise addition of taxa [40].
The resulting trees were printed with FigTree v. 1.4.4, and the layout was created in Adobe Illustrator 2019. Sequences generated in this study were deposited in GenBank (Table 1).

Phylogenetic Results
Phylogenetic relationships of three Microthyriales species were assessed in the combined analysis using ITS and LSU gene regions of 54 strains in Microthyriales and related orders Micropeltidales, Natipusillales, Phaeotrichales, Venturiales, and Zeloasperisporiales. The analyzed alignment consisted of combined ITS (1-516 bp) and LSU (517-1321 bp) sequence data, including gaps. Kirschsteiniothelia lignicola (MFLUCC10 0036) served as outgroup taxon. The best scoring RAxML tree is shown in Figure 1. The analyzed ML, MP, and bayesian trees were similar in topology and did not conflict significantly. Paramirandina and Pseudocorniculariella formed two distinct clades and nested within Microthyriaceae.    Saprobic on decaying submerged wood in freshwater habitats. Asexual morph: Colonies on natural substrates effuse, hairy, scattered, yellowish brown to brown, with glistening conidial masses at the apex. Mycelium partly superficial, partly immersed, composed of septate, brown to hyaline, smooth-walled hyphae. Conidiophores macronematous, mononematous, single or in small groups, unbranched, erect, straight or slightly flexuous, cylindrical, smooth-walled, multi-septate, dark brown, becoming pale brown to subhyaline towards the apex, slightly tapering towards the apex. Conidiogenous cells polyblastic, integrated, terminal, cylindrical to lageniform, pale brown to subhyaline, often flexuous at the apex, sometimes elongating percurrently. Conidia holoblastic, solitary or gathered in chains, acropleurogenous, fusiform, cymbiform or narrowly lunate, hyaline, 2-6-septate, smooth-walled, truncate at the base. Sexual morph: undetermined.
Notes: Paramirandina cymbiformis share the similar morphology with Pa. aquatica except for the dimension of conidiophores. However, they are distinct species based on the molecular data.
Attempts to preserve the living culture were unsuccessful since few conidia germinated, with no growth after reaching 1-2 mm diam.. Saprobic on decaying submerged twigs in freshwater habitats. Asexual morph: Conidiomata effuse, sporodochial, synnematous or absent, solitary to gregarious, dark brown to black, stromatic, obpyriform, subcylindrical to subconical, slightly swollen at the base or level of the locule, narrower towards the apex, scattered over the substrate surface, minutely scabrous, reticular. Conidiomatal wall composed of closely interwoven septate hyphae, compacted towards exterior, dark brown to black cells of textura angularis, becoming thin-walled and hyaline toward the inner region. Conidiophores hyaline, cylindrical, branched, developed from the inner layer of the conidiomata, reduced to conidiogenous cells. Conidiogenous cells hyaline, enteroblastic, polyphialidic, subcylindrical or cylindrical to ampulliform, indeterminate, forming conidia at their tips, discrete or integrated, smooth, moderately thick-walled. Conidia solitary, hyaline, smooth, guttulate to granular, septate, slightly constricted at septa, thick-walled, filiform, acerose, tapering towards both ends, slightly curved, base truncate. Sexual morph: Undetermined.
In recent years, the molecular phylogeny of freshwater fungi has been updated several times [48,49,54]. Nevertheless, Microthyriaceae (Microthyriales) was omitted, although a freshwater genus Hamatispora has been reported before then [10]. Recently, four new freshwater genera, Antidactylaria, Isthmomyces, Keqinzhangia, and Pseudocoronospora, were reported [10,11]. In this study, the combined ITS and LSU tree (Figure 1) showed that three new isolates formed two clades in Microthyriaceae. Based on the morphology Culture characteristics: Conidia germinating on PDA medium within 24 h and germ tubes produced from both ends. Colonies growing on PDA medium slow growing, reaching 8-10 mm in three weeks at 25 • C in natural light, circular, with dense, olive-green mycelium in the middle, darker of the inner ring, with sparser, brown mycelium of the outer ring on the surface, in reverse dark brown to black with irregular margin.
In recent years, the molecular phylogeny of freshwater fungi has been updated several times [48,49,54]. Nevertheless, Microthyriaceae (Microthyriales) was omitted, although a freshwater genus Hamatispora has been reported before then [10]. Recently, four new freshwater genera, Antidactylaria, Isthmomyces, Keqinzhangia, and Pseudocoronospora, were reported [10,11]. In this study, the combined ITS and LSU tree ( Figure 1) showed that three new isolates formed two clades in Microthyriaceae. Based on the morphology and molecular evidence, we establish two new asexual genera and three new species, named Paramirandina and Pseudocorniculariella, with Pa. aquatica, Pa. cymbiformis and Ps. guizhouensis. Eight freshwater genera are known in Microthyriaceae, including the two new genera in this study. It is worth noting that both are asexual genera. In the previous studies about freshwater fungi, few aquatic asexual genera were included [55]. The new genus Paramirandina is phylogenetically close to the asexual genus Keqinzhangia (Figure 1). Paramirandina is well distinguishable from Keqinzhangia by relatively long conidiophores (more than 150 µm long vs. prostrate, not differentiated), conidiogenesis (holoblastic vs. holothallic) and the conidial shape (cymbiform or narrowly lunate vs. cylindrical, cylindrical-obclavate, obclavate, bacilliform) [12]. The sequence identity of the LSU gene region between two Paramirandina species and K. aquatica is relatively low (93.29% and 92.88%). Therefore, we introduce Paramirandina as a new genus.
Pseudocorniculariella is phylogenetically close to the asexual genus Isthmomyces (Figure 1). It is distinguished from Isthmomyces by the formation of conidiogenous cells and the morphology of conidia, and was identified as a new genus.
Microthyriaceae has ever been a poorly studied group. However, its taxonomic studies have greatly advanced with molecular data resulting in a rapid increase of genera numbers. Formerly, only seven genera were accepted in the family by Wu et al. [7], nine were accepted by Wijayawardene et al. [8,9], 11 were accepted by Hongsanan et al. [12], and 18 were accepted at present (this study). However, most genera contained fewer species, such as eight monotypic genera, Chaetothyriothecium, Hamatispora, Keqinzhangia, Paramicrothyrium, Pseudomicrothyrium, Pseudocoronospora, Tumidispora, and the new genus Pseudocorniculariella, and four genera with two or three species. Thus, more collections and further molecular evidence are needed to confirm the taxonomy of these genera.
Furthermore, the taxonomy of earlier proposed genera needs to be confirmed by molecular data. Heliocephala is the first hyphomycetous genus described in Microthyriales. There are eight species in the genus, five of which have available molecular DNA data without the type species H. proliferans [56,57]. Gonzalez et al. accepted Heliocephala in Microthyriaceae based on H. variabilis [57]. However, Calabon et al. [54] referred Heliocephala to Microthyriales incertae sedis. Thus, the taxonomy of Heliocephala needs to be reappraised with molecular DNA data of the type species and more collections.

Data Availability Statement:
The datasets generated for this study can be found in the NCBI database.