Keqinzhangia Aquatica Gen. et sp. nov. And Pseudocoronospora Hainanense gen. et sp. nov., Isolated From Freshwater In Southern China

Abstract

During the investigation of the diversity of aquatic hyphomycetes from southern China, two interesting isolates were collected. Then, the two isolates were cultured and sequenced, and a BLAST search of its LSU sequences against data in GenBank revealed that the closest related taxa in the genus Microthyrium. Phylogenetic analyses, based on the combined sequence data from the small and large nuclear subunit ribosomal DNA (SSU and LSU), revealed that our isolates belong to the Microthyriaceae. Combined morphological characters, we finally described 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 type family Microthyriaceae (Arnaud 1918). Originally, Microthyriales included two families, Microthyriaceae and Micropeltidaceae, based on their flattened ascomata with 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 (Wijayawardene et al. 2018; Hongsanan and Hyde Kd 2017), and has 6 genera as family incertae sedis in this order (Wijayawardene et al. 2020).

Saccardo (1883) established the family Microthyriaceae in 1883, with sexual genus Microthyrium Desm. as type genus. In 1913, Theissen included Microthyriaceae in the order Hemisphaeriales (Theissen 1913). Subsequently, Arnaud established a new order Microthyriales to accommodate Microthyriaceae and Microthyriopsidaceae (Arnaud 1918). After that, the family has experienced a complicated taxonomic history, and various genera were included, such as foliar epiphytes or saprobes. Until 2011, 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 outline of Ascomycota and accepted 9 genera in the family, including 8 sexual genera and one asexual genus. In recent study, Hongsanan et al. (Hongsanan et al. 2020) accepted 11 genera in this family based on morphology and phylogeny, including 8 sexual genera and three asexual genera, besides, they added definition of asexual morph in family level.

China has enormous fungal diversity, with the southern region in China 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 in soils, submerged leaves, and aquatic plants, and described many new taxa (Zheng et al. 2019; 2020a,b; 2021a,b; Qiao et al. 2017; 2018a,b; 2019; 2020). During our ongoing studies of freshwater hyphomycetes in Yunnan Province and Hainan Province, two interesting fungi were collected on submerged leaves of an unidentified dicotyledonous plants. The two isolates were cultured and sequenced, and a BLAST search of its LSU sequences against data in GenBank revealed that the closest related taxa 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 nuclear large subunits ribosomal DNA (LSU) genes. Combined morphological characters, we finally described our isolates as two new genera in Microthyriaceae, named as: Keqinzhangia and Pseudocoronospora.

Materials And Methods

Isolation and morphological study of strain

Submerged dicotyledonous leaves were collected from Yunnan Province and Hainan Province. Samples were preserved in zip-lock plastic bags, labelled, and transported to the laboratory. The decomposed leaves were cut into several 2–4 × 2–4 cm sized fragments in the laboratory and then spread onto the surface of corn meal agar (CMA, 20 g cornmeal, 18 g agar, 40 mg streptomycin, 30 mg ampicillin, 1000 ml distilled water) medium for 10 days; single conidium was isolated with a sterilized needle and transferred to CMA plates while viewing with an Olympus BX51 microscope. Morphological observations were made from CMA after incubation at 25°C for one week, and photographs were taken with an Olympus BX51 microscope connected to a DP controller digital camera. Measurement data 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 at 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 µL PCR reaction volume consisted of 12.5 µL T5 Super PCR Mix (containing Taq polymerase, dNTP and Mg2+, 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. 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.

Table 1 Sequence data used in this study. All new sequence data generated in this study are in bold.

Sequence alignment and phylogenetic analysis

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 the genus Microthyrium. Based on this information, related sequences at the two marker loci, 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 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.

These, together with these 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 1215 nucleotide positions from two genes (855 from LSU, 360 from ITS), and the matrix 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 best-fit model under the output strategy of AIC. Me-tropolis-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 MCMC 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. Maximum likelihood bootstrap proportions (MLBP) were computed with 1000 replicates. Trees were visualized in FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/Figtree/, July 2021). Bayesian inference posterior probabilities (BIPP) ≥ 0.95 and maximum likelihood bootstrap proportions (MLBP) ≥ 75% are indicated at nodes.

Results

Phylogenetic analysis

BLAST analyses with LSU sequences revealed that Microthyrium is the closest 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, which close to Hamatispora L.T.H. Yen, K. Yamag. & K. Ando, Neoanungitea, Microthyrium, and YMF 1.04517 with well 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.

Taxonomy

Keqinzhangia Z. F. Yu, M. Qiao & R. F. Castañeda, gen. nov.

Etymology: Name in honors to Prof. Keqing Zhang of the Yunnan University for his contribution to the biological sciences.

MycoBank number: MB 840430

Asexual morph hyphomycetous. Vegetative hyphae cylindrical, branched, microguttulate, septate, hyaline, smooth-walled. Fertile hyphae cylindrical-obclavate, extended inflated subulate to the tip grow, macroguttulate, dark septate, hyaline, smooth-walled. Conidiophores prostrate, not differentiated. Conidiogenous cells holothallic, narrow cylindrical to cylindrical, discrete, indeterminate, forming conidia by random thallic-arthric conidial ontogeny. Conidial secession schizolytic. Conidia thallic-arthric, solitary, polymorphic, cylindrical, cylindrical-obclavate, obclavate, bacilliform, fusiform, sub-oblecythiform or cuneiform, unicellular to septate, hyaline. Chlamydospores globose, terminal, solitary or short catenulate, subhyaline. Sexual state: Unknown.

Type species: Keqinzhangia aquatica Z.F. Yu, M. Qiao & R.F. Castañeda.

Keqinzhangia aquatica Z.F. Yu, M. Qiao & R. F. Castañeda, sp. nov. (Fig. 2–4).

Etymology: Epithet refers to it growing in water.

MycoBank number: MB 840432

Asexual morph hyphomycetous. Colonies flat, growing slowly on CMA, attaining about 2.4 cm diam. after 20 days at 25°C. Pale mouse grey, reverse mouse grey. Mycelium mostly immersed, composed of cylindrical, branched, densely micro-guttulate, septate, subhyaline to hyaline vegetative hyphae and cylindrical-obclavate, extended inflated subulate to the tip grow, macroguttulate, dark septate, hyaline, smooth-walled fertile hyphae. Conidiophores prostrate, undifferentiated. Conidiogenous cells holothallic, narrowly cylindrical, frequently undifferentiated, hyaline, forming conidia by random thallic-arthric disarticulation. Conidia thallic-arthric, solitary, polymorphic, cylindrical-obclavate, long obclavate, cylindrical, bacilliform, fusiform, narrow doliiform, subdolabriform, suboblecythiform or cuneiform, truncate at the ends or truncate at the base and obtuse or rounded at the apex, 0–6(–7)-septate, slightly or strongly constricted at the dark septa, sinuate, macroguttulate, smooth, hyaline, 12–76.5 × 3–6.2 µm, arise after random disarticulation of fertile hyphae at the darker septa. Clamydospores solitary or catenate, broad globose, subglobose to ellipsoidal, terminal, slightly or densely guttulate, smooth, subhyaline, 8–12.6 × 4.1–5.4 µm. Sexual state: Unknown.

Holotype: YMF 1.04262, isolated from leaves of an unidentified dicotyledonous plant submerged in a stream, E’mei National Conservation Area, Sichuan Province, China, 29°35′1′′N, 103°17′3′′E, ca. 1750 m elev., Jun 2014, Zefen Yu, preserved in a metabolically inactive state (deep freezing) in the Conservation and Utilization of Bio‐Resources in Yunnan. Ex-type culture CCTCC AF 2021070.

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 thallic-arthric 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 Z. F. Yu, M. Qiao & R. F. Castañeda, gen. nov.

Etymology: Name refers to it is similar to the genus coronospora in morphology.

MycoBank number: MB 840431

Asexual morph hyphomycetous. Conidiophores macronematous, mononematous, erect, septate, unbranched, brown. Conidiogenous cells polyblastic, denticulate, integrated, sympodial extended, terminal, indeterminate. Conidial secession rhexolytic. Conidia solitary, acropleurogenous obclavate, crowned, with mammiform protuberances arranged near the apex; septate, smooth or verruculose, hyaline, fringed at the base. Sexual state: Unknown.

Type species: Pseudocoronospora hainanense Z.F. Yu, M. Qiao & R.F. Castañeda.

Notes: The genus Coronospora was established by Ellis with C. dendrocalami M. B. Ellis as 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 in culture of Ascoronospora Matsush., so he thought that Coronospora is asexual state of Ascoronospora. Then Asthton et al. (2009) and Wijayawardene et al. (2018) accepted the link between two genera. So far, molecular sequences of two genera were not obtainable, so the connection between two genera was not confirmed by molecular data. However, Ascoronospora was treated as Pleosporales genera incertae sedis (Wijayawardene et al. 2018), which is morphologically different from members of Microthyriaceae.

Pseudocoronospora hainanense Z.F. Yu, M. Qiao & R. F. Castañeda, sp. nov. (Fig. 5,6)

Etymology: Epithet refers to the region Hainan where type strain isolated.

MycoBank number: MB 840433

Asexual morph hyphomycetous. Colonies on CMA attaining 3 cm diam. after 20 days at 25°C, effuse, white to pale flesh, reverse buff. Hyphae thin-walled, septate, hyaline, smooth. Conidiophores macronematous, mononematous, straight or slightly flexuous, somewhat geniculate toward the apex, septate, unbranched, mid brown or pale brown below, pale brown to subhyaline towards the apex, 16.5–49 µm long, 3.5–5.0 µm wide. Conidiogenous cells polyblastic, denticulate, denticle conspicuous, narrowly cylindrical, integrated, sympodial extended, terminal, sometimes intercalary, indeterminate, pale brown to subhyaline. Conidial secession rhexolytic. Conidia solitary, acropleurogenous, obclavate, crowned with 2–3 broadly mammiform protuberances, radially arranged near the rounded to obtuse apex; 2 septate, smooth or slightly verruculose at the basal and central cells, hyaline, 27.2–33 × 3.7–8.0 µm, with a minute basal frill. Sexual state: Unknown.

Holotype: YMF 1.04517, isolated from leaves of an unidentified dicotyledonous plant submerged in a stream, Diaoluoshan National Forest Park, Hainan Province, China, 18°42′11′′N, 109°53′16′′E, ca. 1124 m elev., Apr. 2014, Zefen Yu, preserved in a metabolically inactive state (deep freezing) in the Conservation and Utilization of Bio-Resources in Yunnan. Ex-type culture CGMCC 3.18823.

Discussion

In recent years, more and more molecular data of species in Microthyriaceae were 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 analysis of the ITS and LSU barcodes. In this study, our phylogenetic analysis determined the two isolates 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 was phylogenetically close to the sexual genus Microthyrium and the asexual genus Neoanungitea. Microthyrium is the type genus of Microthyriaceae in Microthyriales (Saccardo 1883). Although we observed cultures for 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. Neoanungitea was introduced by Crous in 2017, with N. eucalypti as type species (Crous et al. 2017). Although Neoanungitea was asexual genus, Keqinzhangia was obviously different from Neoanungitea in morphology.

Our established another new genus Pseudocoronospora was phylogenetically close to the asexual genus 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 was easily distinguished from Hamatispora and Neoanungitea in morphology.

Mycrothyriales is a poorly known order. Previously accepted species in this order was almost based on morphological characters; little molecular data were available. For the past few years, more and more molecular data were available. Recent study showed that species of Microthyriales cluster together as a distinct clade within Dothideomycetes with high support based on sequence analysis of LSU and ITS (Hongsanan et al., 2020). It revealed the importance of obtaining pure cultures and gene sequences in order to identify the origins and phylogenetic positions of fungal species.

Declarations

Acknowledgements

We thank Linlin Fang for her work to this manuscript.

Funding

This work was financed by the National Natural Science Foundation Program of PR China (31770026，31760012).

Conflicts of interest/Competing interests 

The authors declare that have no conflict of interest.

Availability of data and material

The relevant data is uploaded to the public database, and is available.

Code availability

Not applicable

Authors' contributions 

ZY conceived and designed the study. HZ and MQ wrote the manuscript. JG and JP conducted the experiments. R.F.C contributed actively in the identification and the taxonomy of the fungal strains.

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