Keqinzhangia aquatica gen. et sp. nov. and Pseudocoronospora hainanense gen. et sp. nov., isolated from freshwater in southern China

During an investigation of the diversity of aquatic hyphomycetes from southern China, two interesting isolates were collected. These two isolates were cultured and sequenced, and a BLAST search of their LSU sequences against data in GenBank revealed that the closest related taxa were in the genus Microthyrium. Phylogenetic analyses, based on the combined sequence data from the internal transcribed spacer (ITS) and large nuclear subunit ribosomal DNA (LSU), revealed that our isolates belong to the Microthyriaceae. Combined morphological characters allowed us to describe our isolates as two new genera and species in Microthyriaceae, named as: Keqinzhangia aquatica and Pseudocoronospora hainanense. The full descriptions, illustrations, and a phylogenetic tree showing the position of the two new genera were provided in this paper.


Introduction
Microthyriales was introduced by Arnaud in 1918, with the type family Microthyriaceae. Originally, Microthyriales included two families, Microthyriaceae and Micropeltidaceae, based on their flattened ascomata with a poorly developed base. However, Hongsanan and Hyde (2017) excluded Micropeltidaceae from Microthyriales based on their phylogenetic analyses and morphological characteristics. Moreover, their phylogenetic analyses showed that species of Microthyriales cluster together as a distinct clade within Dothideomycetes with high support. Currently, Microthyriales only contains a single family Microthyriaceae (Hongsanan and Hyde 2017;Wijayawardene et al. 2018Wijayawardene et al. , 2020. Saccardo (1883) established the family Microthyriaceae, with the sexual genus Microthyrium Desm. as the type genus. Theissen (1913) included Microthyriaceae in the order Hemisphaeriales. Subsequently, Arnaud (1918) established a new order Microthyriales to accommodate Microthyriaceae and Microthyriopsidaceae. After that, the family has experienced a complicated taxonomic history, and various genera were included, such as foliar epiphytes or saprobes. Wu et al. (2011) reappraised the Microthyriaceae based on examinations of generic types and provided sequence data of several species. They finally accepted seven sexual genera in the Microthyriaceae. In 2017, Wijayawardene et al. (2018) merged both asexual and sexual genera in the outline of Ascomycota and totally accepted nine genera in the family, including eight sexual genera and one asexual genus. In a recent study, Hongsanan et al. (2020) accepted eleven genera in this family based on morphology and phylogeny, including eight sexual genera and three asexual genera. At the same time, they added the definition of an asexual morph on family level.
China has an enormous fungal diversity, and the southwestern region in this country was assessed as one of the world's 34 biodiversity hotspots (Myers et al. 2000). In recent years, we have been investigating the fungal diversity in China, including soils, submerged leaves, and aquatic plants, and described many new taxa (Qiao et al. 2017(Qiao et al. , 2018aZheng et al. 2019Zheng et al. , 2020aZheng et al. , b, 2021a. During our ongoing studies of freshwater hyphomycetes in the Yunnan and Hainan provinces, two interesting fungi were collected from submerged leaves of unidentified dicotyledonous plants. These two isolates were cultured and sequenced, and a BLAST search of their LSU sequences against data in GenBank revealed that the closest related taxa were in the genus Microthyrium. To further confirm the position of our isolates, phylogenetic analyses with related taxa within Microthyriaceae were carried out based on complete sequences of internal transcribed spacer (ITS) and partial sequences of the nuclear large subunit ribosomal DNA (LSU) genes. Combining morphological characters, we finally described our isolates as two new genera and species in Microthyriaceae, named as: Keqinzhangia aquatica gen. et sp. nov. and Pseudocoronospora hainanense gen. et sp. nov.

Materials and methods
Isolation and morphological study of strains Submerged dicotyledonous leaves were collected from Yunnan and Hainan Provinces. Samples were preserved in zip-lock plastic bags, labeled, and transported to the laboratory. The decomposed leaves were cut into several 2-4 9 2-4 cm sized fragments in the laboratory and then were incubated on corn meal agar (CMA, 20 g cornmeal, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1000 ml distilled water) medium for 10 days at room temperature. Single conidia were isolated with a sterilized needle under an Olympus BX51 microscope and cultivated on. Morphological observations were made from CMA after incubation at 25°C for 1 week, and microscopic photographs were taken on an Olympus BX51 microscope under differential interference contrast model and captured with an Olympus DP 10 digital camera using Olympus DP controller (V.3,1,1208) software. Measurements were based on 30 random conidia and 10 conidiophores.
Pure cultures were 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), the China Center for type Culture Collection (CCTCC), and the China General Microbiological Culture Collection Center (CGMCC).

DNA extraction, PCR amplification, and sequencing
Pure cultures were grown on potato dextrose agar (PDA, 200 g potato, 20 g dextrose, 18 g agar, 1000 ml distilled water) medium for 7 days at 25°C. Actively growing mycelium was scraped off from the surface of the culture and transferred to 2 ml Eppendorf micro-centrifuge tubes. Total genomic DNA was extracted according to the procedures in Turner et al. (1997). Primers used for PCR amplification and sequencing of the nuclear large subunits ribosomal DNA (LSU) and the internal transcribed spacer (ITS) were LROR/LR7 (White et al. 1990) and ITS1/ITS4 (Vilgalys and Hester 1990), respectively. Each 25 lL PCR reaction volume consisted of 12.5 lL T5 Super PCR Mix (containing Taq polymerase, dNTP and Mg2 ? , Beijing TsingKe Biotech Co., Ltd., Beijing, China), 1 lL of forward primer (10 lM), 1 lL of reverse primer (10 lM), 1 lL DNA template, 5 lL of PCR buffer, and 4.5 lL sterile water. PCR reactions were run in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) following the PCR thermal cycle programs described by Qiao et al. (2020). PCR products were purified by using the PCR product purification kit (Biocolor BioScience and Technology Co., Shanghai, China), and forward and reverse sequenced on an ABI 3730 XL DNA sequencer (Applied Biosystems, Foster City, CA, USA) with the same primers, using a Thermo Sequenase Kit as described by Kindermann et al. (1998). These sequences were deposited in the GenBank database at the National Center for Biotechnology Information (NCBI) and the accession numbers are listed in Table 1.

Sequence alignment and phylogenetic analyses
Preliminary BLAST searches with the LSU sequences of our isolates against the GenBank nucleotide database determined the closely related species, it showed that their closest related taxon is in the genus Microthyrium. Based on this information, related sequences at the two marker loci of ITS and LSU, which include 13 representatives belonging to Microthyriaceae, two representatives belonging to Natipusillales, two representatives belonging to Phaeotrichales, three representatives belonging to Venturiales, and three representatives belonging to Zeloasperisporiales, were downloaded according to recent studies (Crous et al. 2019;Gonzalez et al. 2020;Hongsanan et al. 2020). Kirschsteiniothelia lignicola Boonmee & K.D. Hyde was used as the outgroup.
The sequences, together with the newly generated sequences, were manually aligned with ClustalX 1.83 (Thompson et al. 1997). The resulting alignments were subsequently checked and refined using BioEdit version v. 7.0.4.1 (Hall 1999). The two alignments were combined with BioEdit and then converted to a NEXUS file using the programme MEGA6 (Tamura et al. 2013). The resulting combined sequence matrix contained 1,215 nucleotide positions from two loci (855 from LSU, 360 from ITS), and it was uploaded to TreeBASE (www.treebase.org; accession number: S28478).
Bayesian inference (BI) and maximum likelihood (ML) were used in this study for phylogenetic analyses. BI analysis was conducted with MrBayes v3.2.2 (Ronquist et al. 2012) with NEXUS files. 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 bestfit model under the output strategy of AIC. Metropolis-coupled Markov chain Monte Carlo (MCMCMC) searches were run for 1,000,000 generations sampling every 500th generation. Two independent analyses with four chains each (one cold and three heated) were run until stationary distribution was achieved. The initial 25% of the generations of CMC sampling were excluded as burn-in. The refinement of the phylogenetic tree was used for estimating Bayesian inference posterior probability (BIPP) values. ML analysis was computed by RAxML (Stamatakis 2006) with the PHY files generated with ClustalX 1.83, using the GTR-GAMMA model.

Phylogenetic analyses
BLAST analyses with LSU sequences revealed Microthyrium as closely related taxon to our isolates but with relatively low percentage of identities. The combined analysis of the two loci (ITS and LSU), which was analyzed by BI and ML approaches, confirmed the status of our isolates in the family Microthyriaceae. In this tree, YMF 1.04626 and YMF 1.04517 grouped into the Microthyriaceae with good support. YMF 1.04626 was clustered together with the sexual genus Microthyrium with good support (MLBP/BIPP = 92%/1.0), and the clade was close to the asexual genus Neoanungitea Crous. YMF 1.04517 formed an isolated clade, close to Hamatispora L.T.H. Yen, K. Yamag. & K. Ando, Neoanungitea, and Microthyrium with good support (MLBP/BIPP = 80%/0.98). Combined with morphological differences, we described YMF 1.04626 and YMF 1.04517 as two new asexual genera and species in Microthyriaceae, named as Keqinzhangia aquatica and Pseudocoronospora hainanense (Fig. 1).
Etymology: Epithet refers to the collection from stream water.
Notes: In Keqinzhangia aquatica, the fertile hyphae are located at the margin of the colony arise laterally from vegetative hyphae forming aerial mycelium with narrow cylindrical, cylindrical, long cylindrical-obclavate, obclavate, inflated or globose, subulate cellular structures, that include the tip growth. The thallicarthric conidia are formed by random fission at the darker septa of preexisting cells of the fertile hyphae in a similar holothallic mode described by Cole (1986) and Seifert et al. (2011).
Pseudocoronospora  Notes: The genus Coronospora was established by Ellis (1971) with C. dendrocalami M.B. Ellis as the type species, in which after the conidiogenous events the cicatrized loci are produced following sympodial extensions of the polyblastic conidiogenous cells disposed in geniculate conidiophores and the conidia are liberated via schizolytic conidial secession (Seifert et al. 2011;Zhang and Zhang 2004;Ellis 1971), but in Pseudocoronospora hainanense the conidiogenous loci are tiny or conspicuous denticles and the conidial basal cells are fringed after the rhexolytic conidial secession. Matsushima (2001) observed the Coronospora morph in the culture of Ascoronospora Matsush., so he thought that Coronospora is the asexual state of Ascoronospora. Then Kirk et al. (2008) and Wijayawardene et al. (2018)

Discussion
In recent years, more and more molecular data of species in Microthyriaceae has become available. Hongsanan et al. (2020) accepted 11 genera, which include three asexual genera Hamatispora, Neoanungitea, and Pseudopenidiella Crous & Koukol, in Microthyriaceae based on morphological characteristics and sequence analyses of the ITS and LSU barcodes. In this study, our phylogenetic analyses determined two isolates to belong to the Microthyriaceae. Combined with morphological characteristics, we finally described them as two new asexual genera and species in Microthyriaceae, named as Keqinzhangia aquatica and Pseudocoronospora hainanense.
The new genus Keqinzhangia is phylogenetically close to the sexual genus Microthyrium and the asexual genus Neoanungitea. Although we observed cultures for a long time, we did not see any sexual reproductive structures in K. aquatica. Besides, their LSU sequence similarity is relatively low (90%). Therefore, we cannot determine the connection between them. Although Neoanungitea is an asexual genus, Keqinzhangia is obviously different from Neoanungitea in conidiogenesis (holothallic vs. holoblastic) and the shape of conidia (cylindricalobclavate, bacilliform, fusiform vs. fusoid-ellipsoid) (Crous et al. 2019).
Our other new genus, Pseudocoronospora, is phylogenetically close to the asexual genus c Conidia with conidiogenous cells. c, d, e were taken with SEM. Scale bars: (a-e) = 10 lm Hamatispora and Neoanungitea. Hamatispora is a hyphomycetous genus with staurospores that are question-mark-shaped or hook-shaped with 3 arms developing from each cell on the helicoid part (Yen et al. 2018). Therefore, Pseudocoronospora species is easily distinguished from Hamatispora and Neoanungitea by morphology.
Mycrothyriales is a poorly known order. Previously accepted species in this order were based mostly on morphological characters; little molecular data were available. For the past few years, new molecular data are available (Crous et al. 2016(Crous et al. , 2017(Crous et al. , 2019Hongsanan et al. 2020;Wu et al. 2011Wu et al. , 2014. A recent study showed that species of Microthyriales cluster together as a distinct clade within Dothideomycetes with high support based on sequence analyses of LSU and ITS (Hongsanan et al. 2020). This study shows the importance of obtaining pure cultures and gene sequences in order to identify the origins and phylogenetic positions of fungal species.