﻿Morphological and phylogenetic characterisation of two new soil-borne fungal taxa belonging to Clavicipitaceae (Hypocreales, Ascomycota)

﻿Abstract The fungal taxa belonging to the Clavicipitaceae (Hypocreales, Ascomycota) are widely distributed and include diverse saprophytic, symbiotic and pathogenic species that are associated with soils, insects, plants, fungi and invertebrates. In this study, we identified two new fungal taxa belonging to the family Clavicipitaceae that were isolated from soils collected in China. Morphological characterisation and phylogenetic analyses showed that the two species belong to Pochonia (Pochoniasinensissp. nov.) and a new genus for which we propose Paraneoaraneomycesgen. nov. in Clavicipitaceae.

Morphological and phylogenetic characterisation of two new soil-borne fungal taxa belonging to Clavicipitaceae (Hypocreales, Ascomycota)

Introduction
Fungi are found in a wide array of ecological niches and play key roles as decomposers, mutualists and pathogens (Araújo et al. 2022). Clavicipitaceae (Ascomycota, Hypocreales) is a large fungal family with diverse ecological characteristics and includes saprophytes, symbionts and pathogens that are associated with soils, insects, plants, fungi and other invertebrates (Gams and Zare 2003;Spatafora et al. 2007;Sung et al. 2007a;Steiner et al. 2011;Kepler et al. 2012a). Currently, the family Clavicipitaceae includes 52 genera and more than 500 species (Hyde et al. 2020;Mongkolsamrit et al. 2020aMongkolsamrit et al. , 2021Gao et al. 2021;Chen et al. 2022). Some members of these genera are valuable as biocontrol agents in agriculture and production of antibiotics in the pharmaceutical industry (e.g. cyclosporin, fingolimod, hydroxyfungerins; Uchida et al. (2005); Mapook et al. (2022)). For example, species of Metarhizium are commercially used as biocontrol agents (Kim et al. 2020). Gao et al. (2021) reported two new entomopathogenic species belonging to the genus Parametarhizium (P. hingganense and P. changbaiense) that were isolated from the forest litters in northeast China and exhibited anti-insect activities against three farmland pests (Monolepta hieroglyphica, Callosobruchus chinensis and Rhopalosiphum maidis).
Phylogenetic analyses showed that the Verticillium section Prostrata was heterogenous and Pochonia was recognised as a distinct genus with several species that often form dictyochlamydospores and were parasitic on the nematode cysts and eggs . Pochonia chlamydosporia was the first recognised species of the genus Pochonia. Subsequently, several new taxa have been identified in this genus. Kepler et al. (2012bKepler et al. ( , 2014 showed that the genus Pochonia belonged to Claviciptaceae; Pochonia was polyphyletic and formed two different clades; P. chlamydosporia was the only species assigned to the monophyletic clade in the Pochonia genus, whereas the remaining species were transferred to a new genus, Metapochonia. Currently, Pochonia includes three species (P. globispora, P. boninensis and P. chlamydosporia) and four varieties (P. chlamydosporia var. ellipsospora, P. chlamydosporia var. catenulata, P. chlamydosporia var. spinulospora and P. chlamydosporia var. chlamydosporia). The species of Pochonia are commonly obtained from soil and demonstrate the ability to parasitise plant-parasitic nematodes (Nonaka et al. 2013).
In this study, we report the morphological and phylogenetic characterisation of two new taxa belonging to the family Claviciptaceae that were isolated from the urban soil samples in China.

Fungal isolation and morphology
The soil samples were collected in June 2020 from the Cengong County (27°16'98''N, 108°81'46''E) in Kaili City, Guizhou Province, China. The fungi were isolated using the methods described previously . Colonies on PDA were incubated after 14 days at 25 °C and the cultures were placed to slowly dry at 50 °C to produce the holotypes, which were deposited in the Institute of Fungus Resources, Guizhou University, Guiyang City, Guizhou, China (GZUIFR). All living cultures were stored in a metabolically inactive state (i.e. kept in sterile 30% glycerol in a -80 °C freezer) and were deposited in the GZUIFR.
The phenotype was determined by growing the single isolates in plates containing potato dextrose agar (PDA), malt extract agar (MEA), oatmeal agar (OA) and synthetic low-nutrient agar (SNA) medium. The plates were incubated in the dark at 25 °C for 14 days. The photomicrographs of the fungal structures were obtained using an OLYMPUS BX53 microscope equipped with differential interference contrast (DIC) optics, an OLYMPUS DP73 high-definition colour camera and the cellSens software version 1.18.

DNA extraction, PCR amplification and sequencing
Total DNA was extracted using the 5% chelex-100 solution as described previously ). The small subunit (SSU) rDNA, the internal transcribed spacer (ITS), the large subunit (LSU) rDNA, the second largest subunit of RNA polymerase II (RPB2) and the translation elongation factor EF-1α (EF1A) were PCR amplified and sequenced using primers listed in Table 1. The novel sequences identified in this study were deposited in the GenBank database (Table 2).

Phylogenetic analyses
Lasergene software (version 6.0, DNASTAR) was used to analyse the ambiguous bases of the PCR amplicon sequences. The SSU, ITS, LSU, RPB2 and EF1A sequences were retrieved from the GenBank database, based on previous studies by Mongkolsamrit et al. (2018Mongkolsamrit et al. ( , 2020bMongkolsamrit et al. ( , 2021, Gao et al. (2021), Chen et al. (2022) and others ( Table 2). The sequences for individual loci were aligned using the MAFFT multiple sequence alignment software version 7.037b (Katoh and Standley 2013) and modified manually using the MEGA software version 6.06 (Tamura et al. 2013). The SSU, ITS, LSU, RPB2 and EF1A sequences were then combined using the "Concatenate Sequence" function in the PhyloSuite version 1.2.3 (Xiang et al. 2023). The best-fit substitution model was selected for the Bayesian analysis and the Maximum Likelihood analysis using the corrected Akaike Information Criterion (AICc) in the ModelFinder (Kalyaanamoorthy et al. 2017).
In the present study, the combined loci were analysed using the Bayesian Inference (BI) and the Maximum Likelihood (ML) methods. MrBayes version 3.2 (Ronquist et al. 2012) was used for the BI analysis. The Markov Chain Monte Carlo (MCMC) method was used to perform 10 8 simulations with a sampling frequency of 10 3 generations and a 25% burn-in. ML analysis was performed using the IQ-TREE software version 1.6.11 (Nguyen et al. 2015) and 10 4 bootstrap (BS) tests were performed using the ultrafast algorithm (Minh et al. 2013). The BI and ML analyses were performed in the PhyloSuite platform version 1.2.3 (Xiang et al. 2023).
The phylogenetic trees (Fig. 1) constructed according to the ML and BI analyses were largely congruent and strongly supported in most clades. Most genera were clustered into independent clades (Chen et al. 2022; Fig. 1). Two new isolates, ZY 22.009 and ZY 22.010, belonged to a new species below named Po-  The genus Pochonia was closely related to Rotiferophthora (Fig. 1). This result was in agreement with the previous studies by Kepler et al. (2014) and Chen et al. (2022) Etymology. Based on its close phylogenetic relationship to Neoaraneomyces. Geographical distribution. China. Description. Saprobic in soil. Sexual morph: not observed. Asexual morph: Hyphae hyaline, smooth, branched, septate. Phialides arising from aerial hyphae or hyphae regimental, solitary, straight to flexuous, tapering with enlarged base, smooth, hyaline. Conidia borne on the apex of the phialides or in small globose heads at the apices of the phialides. Conidia cymbiform to reniform, smooth-walled, one-celled, adhering in globose heads or the apex of phialides.
Type species. Paraneoaraneomyces sinensis Zhi. Y. Zhang & Y. F. Han. Notes. Currently, the family Clavicipitaceae includes 52 genera and more than 500 species (Hyde et al. 2020;Mongkolsamrit et al. 2020aMongkolsamrit et al. , 2021Gao et al. 2021;Chen et al. 2022). Of these genera, no SSU, ITS, LSU, RPB2 and EF1A sequences are available for the genera Cavimalum, Epicrea, Helminthascus, Konradia, Loculistroma, Mycomalus and Neocordyceps, while the sequences for the genera Nigrocornus, Pseudomeria and Romanoa are unverified or lacking (https://www.ncbi.nlm.nih.gov/, accessed on 8 May 2023). Therefore, we could not compare the phylogenetic relationships between these genera and Paraneoaraneomyces. In addition, amongst these genera, Cavimalum, Epicrea, Helminthascus, Konradia, Mycomalus and Sphaerocordyceps no asexual morph has been reported (White et al. 2003;Hyde et al. 2020). We, therefore, have not been able to compare the morphological characteristics between these genera and Paraneoaraneomyces. Phylogenetically, Paraneoaraneomyces sinensis represents a well-supported monophyletic lineage in the family Clavicipitaceae and closely related to Neoaraneomyces (Fig. 1). Morphologically, Paraneoaraneomyces can be distinguished from other genera in the family Clavicipitaceae by the cymbiform to reniform conidia adhering to the apex of the phialides or in the form of small globose heads at the apex of the phialides and the phialides were solitary, straight to flexuous and arose from the aerial or regimental hyphae. Geographical distribution. Guizhou Province, China. Description. Culture characteristics (14 days at 25 °C): Colonies on PDA 35-37 mm in diameter, white, slightly raised at centre, fluffy, nearly round, margin regular; reverse: pale yellow. Colonies on MEA 35-37 mm in diameter, white, plicated, flocculent, nearly round, margin regular; reverse: pale yellow. Colonies on SNA 29-31 mm in diameter, white, flat, felty, nearly round, margin regular; reverse: white, compact at centre. Colonies on OA 36-38 mm in diameter, white, felty, early round, margin regular; reverse: white.
Notes. The multi-locus phylogenetic analyses showed that Paraneoaraneomyces sinensis is closely related to Neoaraneomyces araneicola (Fig. 1), but can be distinguished, based on differences in their sequence similarity. The ITS sequence of P. sinensis showed 93.6% similarity, differences in 13 base pairs (bp) and 22 gaps when compared to the 551 bp ITS sequence of N. araneicola DY101711 (Type strain). The LSU sequence of P. sinensis showed 99.3% similarity, differences in 5 bp and without gaps when compared to the 832 bp LSU sequence of N. araneicola DY101711. The RPB2 sequence of P. sinensis showed 83.9% similarity, differences in 158 bp and 8 gaps when compared to the 1,034 bp RPB2 sequence of N. araneicola DY101711. The EF1A sequence of P. sinensis showed 96.2% similarity, differences in 35 bp and without gaps when compared to the 937 bp EF1A sequence of N. araneicola DY101711. Morphologically, the phialides of P. sinensis were solitary, straight to flexuous, arising from the aerial or regimental hyphae compared to the phialides of N. araneicola that were solitary or in groups of two to four and arose from the aerial hyphae (Chen et al. 2022). Furthermore, the conidia of P. sinensis were cymbiform to reniform and adhering to the apex of the phialides or in small globose heads at the apex of the phialides compared with fusiform to ellipsoidal conidia that were arranged as chains in N. araneicola (Chen et al. 2022 Hyphae hyaline, smooth, branched, septate, 0.5-1.5 μm in diameter. Phialides produced from prostrate aerial hyphae, solitary or rarely in whorls of 2-3, slender, tapering towards the tip, 5.5-51.0 × 0.5-1.5 µm (av. 22.0 × 1.0, n = 50). Conidia in small globose heads at the apex of the phialides. Conidia ovoid, sometimes subglobose or ellipsoidal, smooth-walled, one-celled, adhering in globose heads, 3.0-4.5 × 2.0-3.0 µm (av. 3.6 × 2.5, n = 50). Swollen hyphae not observed. Dictyochlamydospores not observed. Crystals absent. Sexual morph undetermined.
Notes. The multi-locus phylogenetic analyses (Fig. 1) and morphological characteristics showed that ZY 22.009 and ZY 22.010 represent a new species of Pochonia. Morphologically, P. sinensis shared similar morphological characters with P. globispora and P. boninensis, but does not produce dictyochlamydospores (Zare and Gams 2007;Nonaka et al. 2013). However, P. sinensis can be easy distinguished from P. globispora and P. boninensis, based on the ovoid conidia and the absence of irregularly swollen hyphae (Zare and Gams 2007;Nonaka et al. 2013).

Discussion
In this study, we proposed a new Pochonia species and a new genus Paraneoaraneomyces within the family Clavicipitaceae. This study has important im- plications for the species diversity, taxonomy and geographic distribution of Clavicipitaceae (Hypocreales).
Fungi are highly abundant eukaryotes (Purvis and Hector 2000) with significant diversity and cosmopolitan distribution and play an essential role in the functions and processes of a wide variety of ecosystems. However, only 150,000 fungal species have been described to date and its plausible that several fungal genera and species are yet to be discovered. Taxonomy is a fundamental discipline of naming, describing and classifying a living organism, plant or fungus and represents the initial step towards understanding its biodiversity, ecological niche and biotechnological utility (Yasanthika et al. 2022). An increasing number of new fungal taxa are constantly being discovered, but mycotaxonomy of a new fungal species is challenging (Aime et al. 2021). Currently, integration of multiple methods is recommended for the taxonomic classification of newly-identified fungal species. Amongst these methods, morphological characteristics and phylogenetic analysis are of primary importance in addition to the ecological habitats, as well as the physiological and biochemical characteristics. The family Clavicipitaceae includes many entomopathogenic fungi, but only a small number of taxa are parasitic and most others show diverse nutritional patterns. Therefore, utmost care is necessary when classifying a new fungal isolate, based on the substrate or a parasitic fungus on an insect host. All the isolates obtained in this study were isolated from soil. Further investigations are necessary to determine if these new fungal isolates were parasitic to insects.
Soil is the largest natural reservoir of microorganisms and is inhabited by a large number of fungi. Taxonomy of soil fungi is an emerging area of research. Currently, only about 800,000 species of soil fungi have been identified worldwide (Senanayake et al. 2022). Majority of studies have focused on the diversity of fungi in the forest, silt, riparian, coastal and contaminated soils (Fracetto et al. 2013;Frac et al. 2018;Satyanarayana et al. 2019), but relatively little is known regarding the fungal taxa in the urban soils. Taxonomic studies of soil fungi use both culture-dependent and non-culture-dependent methods. The culture methods are of great interest because the isolated strains can be used to obtain genetic sequence and morphological data in applied research (Yasanthika et al. 2022). The new fungi described in this study were all isolated from soil and their ecological functions and applications are worthy of further study.