Discovery of a New Lichtheimia (Lichtheimiaceae, Mucorales) from Invertebrate Niche and Its Phylogenetic Status and Physiological Characteristics

Species of Lichtheimia are important opportunistic fungal pathogens in the order Mucorales that are isolated from various sources such as soil, indoor air, food products, feces, and decaying vegetables. In recent years, species of Lichtheimia have become an emerging causative agent of invasive mucormycosis. In Europe and USA, Lichtheimia are the second and third most common causal fungus of mucormycosis, respectively. Thus, the aim of this study was to survey the diversity of species of Lichtheimia hidden in poorly studied hosts, such as invertebrates, in Korea. Eight Lichtheimia strains were isolated from invertebrate samples. Based on morphology, physiology, and phylogenetic analyses of ITS and LSU rDNA sequence data, the strains were identified as L. hyalospora, L. ornata, L. ramosa, and a novel species, L. koreana sp. nov. Lichtheimia koreana is characterized by a variable columellae, sporangiophores arising solitarily or up to three at one place from stolons, and slow growth on MEA and PDA at all temperatures tested. The new species grows best at 30 and 35 °C and has a maximum growth temperature of 40 °C. Detailed descriptions, illustrations, and a phylogenetic tree are provided.

For a long time, Lichtheimia has been treated as a synonym for Absidia based on morphological similarities [11]. Hoffmann et al. [12] revised Absidia based on phylogenetic, physiological, and morphological characteristics and divided its constituent species into three

Phylogenetic Analyses
Sequences of each locus were aligned using MAFFT v. 7 with the L-INS-I algorithm (http://mafft.cbrc.jp/alignment/server, accessed on 2 January 2023) [35], then confirmed manually in MEGA v. 7 [36]. Bayesian inference (BI) and maximum likelihood (ML) analyses were performed for the combined dataset. The most suitable substitution model was determined using jModelTest v. 2.1.10 software [37,38]. ML analyses were conducted using RAxML-HPC2 on XSEDE on the online CIPRES Portal (https://www.phylo.org/ portal2, accessed on 2 January 2023), with a default GTR substitution matrix and 1000 rapid bootstraps. BI analyses were performed using MrBayes v. 3.2.6 [39]. Four Markov chain Monte Carlo (MCMC) chains were run from a random starting tree for 5 million generations, and trees were sampled every 100th generation. The first 25% of the trees were removed as burn-in, and the remaining trees were used to calculate posterior probabilities. A PP value ≥ 0.95 was considered significant. Fennellomyces linderi CBS 158.54 was chosen as the outgroup. The newly obtained sequences were deposited in the GenBank database (http://www.ncbi.nlm.nih.gov, accessed on 5 February 2023) under the accession numbers provided in Table 1.
The multigene analysis contained 60 taxa, including Fennellomyces linderi CBS 158.54 as the outgroup taxon. The concatenated alignment consisted of 1589 characters (including alignment gaps), with 939 and 650 characters used in the ITS and LSU, respectively. The isolates CNUFC ISS71, CNUFC S724, and CNUFC CY2204 formed an independent branch that was well-supported (97% MLBS, 0.99 PP) and clearly distinct from the other Lichtheimia species. CNUFC CY2219 clustered with strains of L. ramosa, while CNUFC S871 and CNUFC CY2232 clustered with strains of L. ornata, and CNUFC CY2246 and CNUFC CY2248 clustered with strains of L. hyalospora (Figure 1).

Mating Experiments
Zygospores were not produced under any conditions between any of the mating pairs.

Growth Experiments
The growth experiments using plates with PDA, MEA, and SMA showed that the choice of media affected the growth of the studied isolates ( Figure 4). All isolates grew at temperatures between 20 to 40 • C. Maximum growth was recorded for different species at temperatures ranging from 40 to 47 • C ( Table 2). The highest growth rates at all temperatures tested were recorded for Lichtheimia ramosa (CNUFC CY2219) and L. ornata (CNUFC CY2232 and CNUFC S817), respectively. The most favourable growth media for all species was SMA. Lichtheimia koreana grew slower on SMA, PDA and MEA than L. hyalospora, L. ornata and L. ramosa. Lichtheimia hyalospora (CNUFC CY2246 and CNUFC CY2248) were able to grow at 45 • C, while none of the tested L. koreana grew at this temperature. Maximum growth temperature for L. koreana is 40 • C. Lichtheimia ramosa (CNUFC CY2219) and L. ornata (CNUFC CY2232 and CNUFC S817) grew well at 45 • C. However, L. ornata (CNUFC CY2232 and CNUFC S817) could be distinguished from Lichtheimia ramosa (CNUFC CY2219) by its ability to grow at 47 • C, since the maximal growth temperature for Lichtheimia ramosa (CNUFC CY2219) was at 46 • C.

Discussion
The genus Lichtheimia contains six accepted species. In this study, Lichtheimia isolates obtained from invertebrates in Korea were studied. A new species is described based on evidence from a polyphasic approach.
The data from the combined sequence analysis of two loci (ITS and LSU rDNA) showed that L. koreana formed well-supported clades (MLBS: 97%, PP: 0.99) (Figure 1).

Discussion
The genus Lichtheimia contains six accepted species. In this study, Lichtheimia isolates obtained from invertebrates in Korea were studied. A new species is described based on evidence from a polyphasic approach.
The data from the combined sequence analysis of two loci (ITS and LSU rDNA) showed that L. koreana formed well-supported clades (MLBS: 97%, PP: 0.99) (Figure 1). Lichtheimia koreana was embedded among the clade of L. brasiliensis and clade containing L. sphaerocystis and L. hyalospora. Lichtheimia koreana shares several similarities with L. brasiliensis, including optimal growth at 30 to 35 • C, restricted growth at 40 • C, and rhizoid production [8]. However, this species differs from L. brasiliensis in forming columellae with projections, sporangiophores arising solitarily or up to three at a single place from stolons, and smaller sporangia, while L. brasiliensis forms columellae with no projections, sporangiophores arising solitary or in pairs from stolon, and sporangia up to 55 µm in diameter [8]. Lichtheimia sphaerocystis produces giant cells, whereas this structure is not observed in L. koreana. Lichtheimia hyalospora differs from L. koreana in its larger sporangia (20-56 µm) and sporangiospores [5.5-9 (-13) µm diameter]) [40]. Lichtheimia koreana can also be distinguished from L. corymbifera, L. ornata, and L. ramosa by its maximum growth temperature. The maximum growth temperature for L. corymbifera, L. ornata, and L. ramosa as determined by Alastruey-Izquierdo et al. [7] is 49 • C, 46 • C and 49 • C, respectively, while in our study, L. koreana exhibited a maximum growth temperature of 40 • C.
The temperature factor for maximum growth is useful to distinguish between species of Lichtheimia [7]. For example, at 43 • C, L. ramosa has higher growth rate than L. corymbifera and L. ornata, while L. hyalospora and L. sphaerocystis did not grow at this temperature [7]. However, two strains of L. hyalospora (CNUFC CY2246 and CNUFC CY2248) in this study were able to grow at 43 • C and have a maximum growth temperature of 45 • C. These discrepancies could be attributed to different hosts, seasons of sample collection, and geographical regions. Interestingly, both species, L. ramosa (CNUFC CY2219) and L. ornata (CNUFC S871 and CNUFC CY2232) did not grow above 46 and 47 • C, respectively.
All species of Lichtheimia grow well at 37 • C, but only three species, namely L. corymbifera, L. ornata, and L. ramosa, have been reported to cause human infections [7,15]. Lichtheimia koreana is embedded among clade of L. brasiliensis, L. sphaerocystis and L. hyalospora, which are not human pathogens [15]. Thus, the pathogenic potential of this new species is probably limited.
Lichtheimia corymbifera and L. ramosa, which represent the most important pathogenic species of Lichtheimia, are also isolated from Asian food productions such as meju (soybean based fermented products) and nuruk (a traditional starter culture for brewing alcoholic beverages in Korea) [42,54]. In this study, we isolated L. ramosa and L. corymbifera from invertebrates, suggesting that we need to consider the natural environments of these species alongside their ability to infect humans.
Members of Lichtheimia are thermotolerant and can grow at a wide range of temperatures from 24 to 50 • C [7]. The ability to grow at high temperatures makes these species valuable in industrial processes. Thus, the potential biological activities of species of Lichtheimia obtained from this study should be further examined. It is also necessary to better understand the distribution of these species and their relevance in human and animal diseases.

Conclusions
A new species, L. koreana, and three new host records, L. hyalospora, L. ornata, L. ramosa, isolated from invertebrates, were classified based on polyphasic approaches including molecular, morphological, and physiological works. Our findings may contribute to the current knowledge of the species diversity of Lichtheimia in Korea. Using poorly studied substrates or hosts for isolation of the fungal species will increase our knowledge of their biodiversity and lead to a better understanding of their specific habitats or niches.  Data Availability Statement: All sequences generated in this study were submitted to GenBank.