Discovery of Three New Mucor Species Associated with Cricket Insects in Korea

Species in the genus Mucor have a worldwide distribution and are isolated from various substrata and hosts, including soil, dung, freshwater, and fruits. However, their diversity from insects is still much too little explored. The aim of this study was to characterize three new species of Mucor: Mucor grylli sp. nov., M. hyangburmii sp. nov., and M. kunryangriensis sp. nov., discovered in Kunryang-ri, Cheongyang in the Chungnam Province of Korea, during an investigation of Mucorales from cricket insects. The new species are described using morphological characters and molecular data including ITS and LSU rDNA regions. Mucor grylli is characterized by the highly variable shape of its columellae, which are subglobose to oblong, obovoid, strawberry-shaped, and sometimes slightly or strongly constricted in the center. Mucor hyangburmii is characterized by the production of azygospores and growth at 40 °C. Mucor kunryangriensis is characterized by the variable shape of its columellae, which are elongated-conical, obovoid, cylindrical ellipsoid, cylindrical, and production of abundant yeast-like cells on PDA, MEA, and SMA media. Based on the sequence analysis of two genetic markers, our phylogenic assessment strongly supported M. grylli, M. hyangburmii, and M. kunryangriensis as new species. Detailed descriptions, illustrations, and phylogenetic trees are provided.

The purpose of this study was to expand the present knowledge of the fungal diversity found in poorly studied substrates or unexplored areas. Herein, we describe and illustrate three new species of Mucor isolated from insects in Korea.

Sampling and Isolation
Cricket insect (Gryllus sp.) samples were collected from Kunryang-ri, Cheongyang, Chungnam Province, Korea, between April 2020 and October 2021. The insects were collected in polyethylene bags, stored at ambient temperature, and transported to the laboratory. Fungal isolation from the insect samples was conducted following our previous methods [28]. Briefly, the samples were transferred to clean Petri dishes. The insect bodies were then broken up into small pieces and placed on PDA. The plates were then incubated at 25 • C for 2-5 days. Then, hyphal tips were transferred to fresh PDA. All isolates were purified by single spore isolation as previously described [28].
Ex-type living cultures were deposited at Environmental Microbiology Laboratory Fungarium, Chonnam National University (CNUFC), Gwangju, Korea and the Culture Collection of National Institute of Biological Resources (NIBR), Incheon, Korea. Dried cultures were deposited in the Herbarium Chonnam National University, Gwangju, Korea.

DNA Extraction, PCR, Cloning, and Sequencing
Total genomic DNA was extracted from fresh fungal mycelia that were grown on cellophane at 25 • C after 4 days using the Solg TM Genomic DNA Preparation Kit (Solgent Co. Ltd., Daejeon, Korea) according to the manufacture's protocol, and then stored at −20 • C. Two regions were amplified, including the internal transcribed spacer (ITS) region using primers ITS1 and ITS4 [29], and the large subunit rDNA region using primers LR0R and LR5 [30]. The PCR products were purified with the Accuprep PCR Purification Kit (Bioneer Corp., Daejeon, Korea) and sequenced at Macrogen (Daejeon, South Korea). Direct sequencing of the ITS PCR product failed; thus, we performed the cloning. PCR products after gel purification were ligated into the pGEM-T Easy Vector (Promega, Madison, WI, USA), following the manufacturer's instructions. The ligation mixture was transformed into Escherichia coli DH5α by heat shock. The positive white colonies were grown in Luria broth (LB) media containing 100 µg of ampicillin per milliliter. The plasmids were purified using the Plasmid Purification Mini Kit (Nucleogen, Si-heung, South Korea). Then, purified plasmids of three clones were sequenced using the primers M13F forward (5 -GTAAAACGACGGCCAGT-3 ) and M13R reverse (5 -GCGGATAACAATTTCACACAGG-3 ).

Phylogenetic Analyses
DNA sequences were checked and were assembled by Seqman Pro 7.1.0 in Lasergene package (DNASTAR, Madison, WI, USA). All newly generated sequences were submitted to GenBank database under the accession numbers provided in Table 1.  Sequence data of closely related Mucor spp. were selected from data previously published by Walther et al. [10], Li et al. [26], Wanasinghe et al. [11], Lima et al. [27], and Hurdeal et al. [5], and downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/ (accessed on 10 March 2022) [31] for molecular phylogenetic analyses (Table 1). Sequences of datasets ITS (44 taxa) and LSU (45 taxa) were aligned using MAFFT (http: //mafft.cbrc.jp/alignment/server (accessed on 12 March 2022) with the algorithm L-INS-I [32], and the alignment was checked in MEGA7 [33]. Aligned sequences were automatically trimmed using trimAl with the gappyout method [34]. Data were converted from fasta format to nexus and phylip formats using the online tool Alignment Transformation Environment (https://sing.ei.uvigo.es/ALTER/ (accessed on 12 March 2022). Phylogenetic reconstructions by maximum likelihood (ML) and Bayesian inference (BI) were carried out using PhyML 3.0 [35], and MrBayes 3.2.2 [36], respectively. The most appropriate model was obtained using the software jModelTest v.2.1.10 [37,38]. We performed the ML analysis using 1000 bootstrap replicates under the best substitution model for the ITS (TPM2uf+I+G) and LSU (TIM3+I+G). BI analyses were performed using 5 million Markov chain Monte Carlo (MCMC) generations and the best substitution model HKY+G and HKY+I+G for ITS and LSU, respectively. The sample frequency was set to 100, the first 25% of trees were removed as burn-in, and the remaining trees were used to calculate the posterior probabilities. The resulting trees were viewed using FigTree v.1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 12 March 2022). The alignment files generated for phylogenetic analyses are provided in the Supplemental Materials Files S1 and S2.

Molecular Phylogenetic Analysis
To understand the evolutionary relationship between isolated strains, sequences of ITS and LSU were used for the phylogenetic analysis (Figures 1 and 2). The ITS and LSU phylogenetic analyses revealed that isolate CNUFC CY22 grouped with M. pernambucoensis, having strong statistical support in the ITS tree (ML/BI = 99/1) (Figure 1). However, this relationship was supported with a moderate bootstrap percentage (86%), but a low posterior probability value (<0.90) in the LSU tree ( Figure 2). Isolate CNUFC CY223 was closely related to M. zachae in the ITS tree with high statistical support (ML/BI = 99/0.99) (Figure 1) but formed an independent clade with high statistical support (ML/BI = 99/0.99) in the LSU tree ( Figure 2). In the ITS analysis (Figure 1

Taxonomy
Based on our phylogenies and morphological data, three new species of Mucor from cricket insects in Korea were described and illustrated here.

Discussion
This study reports on new Mucor species isolated from Gryllus insects collected from Kunryang-ri, Cheongyang, located in Chungnam Province, Korea. Three new Mucor species belonging to the M. amphibiorum group [10]  respectively. However, M. grylli differs from these species by production of strawberryshaped columellae, sometimes slightly or strongly constricted in the center. Wagner et al. [4] reported the presence of strawberry-shaped columellae in M. variicolumellatus, but M. grylli produces larger sporangiospores and includes granules at the end. Moreover, M. grylli can grow at 37 • C, while M. variicolumellatus cannot [4]. Treschew [39] has mentioned that M. odoratus grew slowly at 40 • C, whereas M. grylli was not able to grow at 40 • C. Mucor ucrainicus differs from M. grylli in producing larger sporangia (up to 175 µm diam.) and smaller sporangiospores (4.4-8.1 × 2.7-4.7 µm) [40].
It is also noteworthy that M. grylli, M. hyangburmii, and M. kunryangriensis are the first species in the M. amphibiorum group collected on insects. Interestingly, the three new species can grow optimally at near-human-body temperature, which needs attention as a potential cause of diseases.
Mucor species are dimorphic fungi and exhibit either hyphal or yeast growth depending upon the conditions such as cultivation time, temperature, presence or absence of oxygen, and carbon and nitrogen sources [47,48]. Several studies reveal that M. indicus in different morphologies (filamentous and yeast-like forms) can produce ethanol with relatively high yields and productivity [49,50]. Interestingly, M. kunryangriensis also produces a yeast-like form, necessitating further studies.
Three new species described here were recovered from samples collected at Kunryangri, Cheongyang, located in Chungnam Province, Korea, an area recognized as a biodiversity hotspot and known as the "Alps of Chungnam" with significant mucoralean species richness [13,28]. With further investigations, we expect to discover additional unreported species in this genus. New species could be a source of novel drugs and other useful compounds.

Data Availability Statement:
The sequencing data were submitted to GenBank.