Discovery of Novel Backusella (Backusellaceae, Mucorales) Isolated from Invertebrates and Toads in Cheongyang, Korea

Three novel fungal species, Backusella chlamydospora sp. nov., B. koreana sp. nov., and B. thermophila sp. nov., as well as two new records, B. oblongielliptica and B. oblongispora, were found in Cheongyang, Korea, during an investigation of fungal species from invertebrates and toads. All species are described here using morphological characters and sequence data from internal transcribed spacer sequences of ribosomal DNA and large subunit of the ribosomal DNA. Backusella chlamydospora is different from other Backusella species by producing chlamydospores. Backusella koreana can be distinguished from other Backusella species by producing abundant yeast-like cells. Backusella thermophila is characterized by a variable (subglobose to oblong, applanate to oval, conical and ellipsoidal to pyriform) columellae and grows well at 37 °C. Multigene phylogenetic analyses of the combined ITS and LSU rDNA sequences data generated from maximum likelihood and MrBayes analyses indicate that B. chlamydospora, B. koreana, and B. thermophila form distinct lineages in the family Backusellaceae. Detailed descriptions, illustrations, phylogenetic tree, and taxonomic key to the Backusella species present in Korea are provided.

To isolate the fungal strains from Bufo gargarizans: the skin was swabbed with sterilized cotton swabs and then streaked on potato dextrose agar (PDA: Difco Laboratories, Detroit, MI, USA) with antibiotics (streptomycin sulfate 100 mg/L and penicillin 100 mg/L). The plates were then placed in the dark at 25 • C for 3-7 days. Bufo gargarizans were quickly released to the original site of collection after isolation of fungi.
To isolate the fungal strains from Theuronema hilgendorfi hilgendorfi, Porcellio scaber, Timomenus komarovi, Gryllus bimaculatus, and Scolopendra morsitans: samples were removed from the bags or Falcon tubes and transferred to clean Petri dishes. Bodies were then broken up into small pieces and placed on PDA. The plates were then placed in the dark at 25 • C and checked under a stereomicroscope every day after the second day of incubation. Then, hyphal tips were transferred to fresh PDA. Single-spore cultures were established as described by Choi et al. [14]. Briefly, spores from a single sporangium after 2-4 days incubation were transferred to sterilized water using capillary tubes and then vortexed to obtain a homogeneous spore suspension. Spore suspensions were diluted several times (1:10) and 50 µL of each dilution was spread on PDA medium. The plates were then incubated at 25 • C to allow for spore germination. Colonies from single spores were transferred to a new PDA plate and used for DNA extraction. Ex-type living cultures were deposited at Chonnam National University Fungal Collection, Gwangju, South Korea (CNUFC), Gwangju, Korea. Dried cultures were deposited in the Herbarium Chonnam National University, Gwangju, Korea. To isolate the fungal strains from Bufo gargarizans: the skin was swabbed with sterilized cotton swabs and then streaked on potato dextrose agar (PDA: Difco Laboratories, Detroit, MI, USA) with antibiotics (streptomycin sulfate 100 mg/L and penicillin 100 mg/L). The plates were then placed in the dark at 25 °C for 3-7 days. Bufo gargarizans were quickly released to the original site of collection after isolation of fungi.
To isolate the fungal strains from Theuronema hilgendorfi hilgendorfi, Porcellio scaber, Timomenus komarovi, Gryllus bimaculatus, and Scolopendra morsitans: samples were removed from the bags or Falcon tubes and transferred to clean Petri dishes. Bodies were

Morphological Studies
Pure cultures of Backusella spp. were grown on synthetic mucor agar (SMA: 40 g dextrose, 2 g asparagine, 0.5 g KH 2 PO 4 , 0.25 g MgSO 4 ·7H 2 O, 0.5 mg thiamine hydrochloride, and 15 g agar in 1 L of deionized water), PDA, and malt extract agar (MEA: 40 g malt extract, 4 g yeast extract, and 15 g agar in 1 L of deionized water). Plates were incubated at 25 • C in the dark for 5 to 14 days. Cultures grown on SMA at 25 • C were photographed, and the dimensions of sporangia, sporangiola, columellae, sporangiospores, and chlamydospores were measured. Fragments of mycelia were removed from each culture and placed onto microscope slides with 60% lactic acid. A differential interference contrast microscope (Olympus BX51, Olympus, Tokyo, Japan) was used to generate digital images. For temperature studies, the isolates were grown in triplicates on SMA and were incubated at 10, 20, 25, 30, 37, or 40 • C. Colony growth was measured every 24 h for 7 days.

DNA Extraction, PCR, Cloning, and Sequencing
Backusella spp. were cultured on cellophane over PDA at 25 • C for 3 days. The mycelial mass was collected by scraping from the surface of cellophane and was placed in 1.5 mL sterile Eppendorf tubes. Genomic DNA was then extracted using the SolgTM Genomic DNA Preparation Kit (Solgent Co. Ltd., Daejeon, Korea) according to the manufacturer's protocol. The purified DNA was stored at −20 • C for later use. Two regions were amplified: the internal transcribed spacer (ITS) region, using the primers V9G/ITS 4 [15,16] and ITS1/ITS4 [16]; and the large subunit (LSU) rRNA gene region, using the primers LROR [17] and LR7 [18]. These target regions, the ITS, and the LSU rRNA gene, were amplified by PCR in 20 µL reaction mixtures containing 2 µL of genomic DNA, 1.5 µL of each primer (5 pM), 14 µL of sterile water, and 1 µL of PCR premix (Bioneer, Daejeon, Korea). Reaction mixtures were pre-heated at 95 • C for 4 min, and PCR was performed as follows: 30 cycles of 45 s at 95 • C, 30 s at 52 • C, and 45 s at 72 • C, with a final extension at 72 • C for 7 min, followed by cooling at 4 • C. PCR products were visualized in 1% (w/v) agarose gel electrophoresis. The PCR products were then purified using an Accuprep PCR Purification Kit (Bioneer) and sequenced on the ABI PRISM 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using the same primers employed for PCR. In some cases, it is necessary to clone the ITS sequences before sequencing. PCR products were cloned using the pGEM-T Easy Vector System cloning kit (Promega, Madison, WI, USA), following the manufacturer's instructions. These clones were sequenced using the primers M13F forward (5 -GTAAAACGACGGCCAGT-3 ) and M13R-pUC reverse (5 -CAGGAAACAGCTATGAC-3 ).

Sequence Alignment and Phylogenetic Analyses
Each generated sequence was checked for the presence of ambiguous bases and was assembled using Lasergene SeqMan program from DNASTAR, Inc. (Madison, WI, USA). Edited sequences were blasted against the NCBI GenBank nucleotide database (https://blast.ncbi.nlm.nih.gov/Blast.cgi; 8 September 2020) to search for closest relatives. Sequences of all the accepted Backusella species according to the last update of the Index Fungorum database were retrieved from GenBank. Sequences were aligned using MAFFT (http://mafft.cbrc.jp/alignment/server; 15 January 2021) with the algorithm L-INS-I. Aligned sequences were automatically trimmed using trimAl [19] with the gappyout method. Data were converted from a fasta format to nexus and phylip formats using the online tool Alignment Transformation Environment (https://sing.ei.uvigo.es/ALTER/; 15 January 2021) [20]. Phylogenetic reconstructions by maximum likelihood (ML) and Bayesian inference (BI) were carried out using PhyML 3.0 [21] and MrBayes 3.2.2 [22], respectively. We performed the ML analysis using 1000 bootstrap replicates. BI analyses were performed using three million Markov chain Monte Carlo (MCMC) generations. The sample frequency was set to 100, and the first 25% of trees were removed as burn-in. The best substitution models for each data partition were estimated using jModelTest v.2.1.10 according to the Akaike criterion [23,24]. The newly obtained sequences were deposited in the GenBank database under the accession numbers provided in Table 1.

Phylogenetic Analysis
The phylogenetic relationship of the new Backusella species with accepted species was determined by analysis of concatenated sequence datasets of two loci (ITS and LSU). The combined ITS and LSU sequence dataset consisted of 49 taxa, and the aligned dataset was comprised of 1120 characters including gaps (ITS: 1-530 and LSU: 531-1120). The BI and ML analyses of this dataset were based on the GTR + I + G model. The trees generated from Bayesian analyses showed the similar topologies (data not shown here). Therefore, we only present the tree obtained from ML analysis ( Figure 2).  The two new species, CNUFC PS1 and CNUFC CM05, are close to B. locustae, B. morwellensis, B. tarrabulga, B. australiensis, B. westeae, and B. luteola, and they form a separate lineage based on multi-loci gene phylogeny. The new species CNUFC CS02 is close to B. constricta, B. grandis, and B. variabilis, and it forms a separate lineage, while strains CNUFC IL02 and CNUFC TKB11 group together with reported species B. oblongielliptica and B. oblongispora, respectively, in a monophyletic clade (Figure 2).

Taxonomy
Our analysis revealed the presence of three new species and two new records in genus Backusella. These species are described below.

Discussion
Cheongyang, located in Chungnam Province, South Korea, covers a total area of 479.7 square kilometers, about 65.8% of which is forest, 7.3% field, and 14.7% paddy field. It is also known for the spicy gochu peppers that are produced in the region and for the fruit of the Chinese matrimony vine, or gugija, which make Cheongyang a favorable region for the growth of fungi. This area is more recognized as a hotspot for biodiversity and is known as the "Alps of Chungnam" and a place that needs to be preserved. In 2019, we found a new species of Mucor, M. cheongyangensis from the surface of Lycorma delicatula in this region [25]. Here, three new species to genus Backusella from the Cheongyang area are described and are compared with those of their most closely related species.
Most species of Backusella are saprobic and commonly isolated from soil and leaf litter [4,9]. Some species have been found in excrement of humans, herbivores, or insects [4,8,13]. However, the genus Backusella has not been involved in human infections [28]. Interestingly, the new species (B. chlamydospora, B. koreana, and B. thermophila), and two newly recorded species (B. oblongielliptica and B. oblongispora) of Backusella were first discovered from invertebrate and toad samples. The presence of Backsuella species in different substrates reflects their ecological importance in ecosystems. Further studies on the fungal diversity of the niches are needed to understand the relationships between these organisms in ecosystems.