Characterization of Two Undescribed Mucoralean 2 Species with Specific Habitats in Korea 3

The order Mucorales, the largest in number of species within the Mucoromycotina, 10 comprises typically fast-growing saprotrophic fungi. During a study of the fungal diversity of 11 undiscovered taxa in Korea, two mucoralean strains, CNUFC-GWD3-9 and CNUFC-EGF1-4, were 12 isolated from specific habitats including freshwater and fecal samples, respectively, in Korea. The 13 strains were analyzed both for morphology and phylogeny based on the internal transcribed 14 spacer (ITS) and large subunit (LSU) of 28S ribosomal DNA regions. On the basis of their 15 morphological characteristics and sequence analyses, isolates CNUFC-GWD3-9 and CNUFC16 EGF1-4 were confirmed to be Gilbertella persicaria and Pilobolus crystallinus, respectively.To the 17 best of our knowledge, there are no published literature records of these two genera in Korea. 18


Introduction
Previously, taxa of the former phylum Zygomycota were distributed among the phylum Glomeromycota and four subphyla incertae sedis, including Mucoromycotina, Kickxellomycotina, Zoopagomycotina, and Entomophthoromycotina [1].Recently, Spatafora et al. [2] proposed two new phyla, Mucoromycota and Zoopagomycota, on the basis of phylogenetic analyses of a genome-scale data set for 46 taxa, including 25 zygomycetes and 192 proteins.According to these results, Mucoromycota and Zoopagomycota were newly formalized phyla of fungi and comprised six subphyla.The phylum Mucoromycota comprises the subphyla Mucoromycotina, Mortierellomycotina, and Glomeromycotina, whereas the phylum Zoopagomycota comprises the subphyla Entomophthoromycotina, Zoopagomycotina, and Kickxellomycotina.
Mucorales is the largest order within the Mucoromycotina and comprises 15 families, 57 genera, and approximately 334 species [3].Most mucoralean species are saprotrophic and grow on different organic substrates, such as fruits, soil, dung, and plants [4,5].Several species are parasites or pathogens of animals, plants, and fungi [4,5].Among these, a few species cause human and animal diseases called mucormycosis, as well as allergic reactions [6].The traditional classification of Mucorales has been determined on the basis of morphological characteristics, such as the size and shape of the sporangium, sporangiophore, sporangiospore (asexual reproduction), and zygospore (sexual reproduction) [4,5].Recently, several molecular studies evaluating mucoralean species had indicated that some of the genera may be polyphyletic [4,5].
The genus Gilbertella belongs to the subphylum Mucoromycotina, order Mucorales, family Choanephoraceae.It was named Choanephora persicaria by E.D. Eddy in 1925 [7] and then renamed as the genus Gilbertella by C.W. Hesseltine in 1960 [8].Species of this genus are characterized as having sporangia with a persistent wall dehiscing via a longitudinal suture; sporangiospores with apical, hyaline appendages; and Mucor-type zygospores [9].Previously, the genus Gilbertella was assigned within the Choanephoraceae because it had not been seen since its original description [7].Hesseltine placed the genus within the Mucoraceae because the zygospores are of Mucor-type [8].Later, Gilbertella was confirmed through studies of DNA sequence data as in fact belonging to the family Choanephoraceae [10].
Genus Pilobolus Tode (Pilobolaceae, Mucorales) is characterized by positive phototropism and its method of spore dispersal; that is, through the ballistic discharge caused by the elevated pressure generated by subsporangial swelling of the sporangiophore [16,17].Pilobolus species are attached to the substrate by an absorptive structure, the swollen trophocyst, which is semi-immersed in the substrate [17].The trophocysts are generally ovoid to globose, whereas the rhizoidal extension is long and cylindrical [17].The sporangiophores are straight, unbranched, and positively phototropic, with two rings of orange pigment at the base and near the subsporangial vesicle [17].The sporangia are hemispherical and contain the spores, which are globose or ellipsoidal depending on the species [17].Zygospores are formed in the substrate and have apposed suspensors [18].
Until now, 8 new species from Korea have been registered in Index Fungorum and dozens of unreported species have been discovered in Korea, but information about the species diversity of mucoralean fungi is still lacking.In Korea, within the Choanephoraceae, only 3 species have been described, whereas species belonging to the Piloboloceae have not yet been described.
The aim of the present study was to perform molecular and morphological analyses to characterize two unrecorded mucoralean species from specific habitats such as freshwater and animal feces in Korea: Gilbertella persicaria and Pilobolus crystallinus.

Morphological Characterization
Taxonomic descriptions of the morphological structures for the two species (G.persicaria CNUFC-GWD3-9 and P. crystallinus CNUFC-EGF1-4) are shown in details below.

CNUFC-GWD3-9 Gilbertella persicaria
Colonies grew rapidly at 25°C on SMA, filling the Petri dish after 2 days of incubation.The colony color was initially white and later grayish yellow.The colony reverse side was white and later pale yellow.Sporangiophores were 10.5-50.0μm wide, variable in length, hyaline, light brown to grayish, sometimes branched, and uncommonly had a septum under the sporangia.The sporangia separated longitudinally into two halves, were globose to subglobose, many-spored, initially whiteyellowish and then turning brown or black at maturity, and measured 36.5-250.5 × 37.2-253.5 μm.Columellae were variable in shape, ovoid to pyriform, subglobose, and measured 20.5-110.7 × 25.2-139.0μm.Sporangiospores were irregular in shape, mainly ellipsoidal, and measured 5.9-15.5 × 4.5-8.9μm.Chlamydospore formations were well defined on the medium.Zygospores were not observed.Subsidiarily, colonies grew slowly on SMA, PDA, and MEA at 5°C.Among these, the best mycelial growth and sporulation were on PDA at 5°C.

Distinguishing Characters
The CNUFC-GWD3-9 isolate was similar to the description of G. persicaria as detailed by Hesseltine [8], in terms of the shape, size of the sporangiospores (5.9-15.5 × 4.5-8.9μm).However, some morphological features differed.The size of columellae described by Hesseltine [8] was larger (40-119 × 20-170 μm) than that (20.5-110.7 × 25.2-139.0μm) observed in our isolate.Our G. persicaria isolate presented sporangiophores that were sometimes branched, which was not described by Hesseltine [8].Moreover, our G. persicaria isolate had a septum under the sporangia.In conclusion, comparing the morphology of the isolate with previous descriptions [8], our present isolate was similar to G. persicaria, with some exceptions (Table 1).a From the description by Hesseltine [8].

Distinguishing Characters
The sporangiospores of isolate CNUFC-EGF1-4 were morphologically similar to the description for those of P. crystallinus by Boedijn [32], some differences in other morphological characteristics were found (Table 2).

Zygospores Not observed Unknown
a From the description by Boedijn [32].

Discussion
The finding of such species belonging to Piloboloceae has not yet been described in Korea.Thus, it is of great scientific significance not only in fungal diversity belonging to undiscovered taxa but also in ecology.
Order Mucorales has been distinguished on the basis of morphological characteristics [4,5].Similarly, Gilbertella and Pilobolus have been identified on the basis of morphology in the past.However, many of the morphological features used to classify species of Pilobolus are problematic [28,29].
According to Foos et al. [28], although the size and shape of the sporangiospores have been detected to be stable within species [30], species descriptions typically give a large range of sporangiospore sizes.Moreover, the sizes of many structures used for species identification vary greatly depending on changes in the environmental conditions [28,31].In 2011, Foos et al. [28] conducted sequence analysis of the ITS region of rRNA, small subunit of 18S rRNA, and LSU (23S) of mitochondrial rRNA, and showed that the genus Pilobolus is polyphyletic.The results revealed that molecular phylogenetic identification of Pilobolus species based on sequence analysis of pure culture isolates was more reliable than the traditional method of identification [28,29].
In the phylogenetic trees for species within the Choanephoraceae based on ITS and LSU sequences, our strains CNUFC-GWD3-9 and CNUFC-GWD3-10 were clustered with other G. persicaria species in a well-supported clade with high bootstrap values (100% and 99%).In the trees for species within the Piloboloceae, the two investigated strains CNUFC-EGF1-4 and CNUFC-EGF1-5 were clustered with other P. crystallinus species in a well-supported clade.
Despite the wide intraspecific variation found among some taxa, the ITS and D1/D2 regions have been used as appropriate barcode markers for identifying mucoralean fungi at the species level [4,5].Currently, the traditional method of fungal identification is still mainly in use, as further studies are required to reconcile the molecular and morphological conceptions of families and genera.In the present study, we also used the molecular strategy for fungal identification at the level of species, specifically utilizing of ITS rDNA gene sequence and phylogenetic analysis.

Sampling and Isolation of Fungal Strain
Water deer dung samples were collected on Eulsukdo Island (35°6'17.92"N, 128°56'24.52"E; located in Busan, Korea) in June 2017.The samples were transferred to sterile 50-mL conical tubes (SPL Life Sciences Co., Pocheon, Korea), and stored at 4°C until examination.The fecal samples were placed onto sterile moist Whatman's filter paper in a Petri dish using sterile forceps, and incubated in a moist chamber at 25°C for 6-9 days.
Freshwater samples were collected from the Geum River (36°27'47.32"N, 127°6'3.24"E; located in Gongju, Korea) in August 2017.These samples were transported in sterile 50-mL conical tubes, and stored at 4°C until examination.Fungi were isolated by the direct plating method.In brief, plant debris in the freshwater samples was placed onto synthetic mucor agar (SMA; 40 g of dextrose, 2 g of asparagine, 0.5 g of KH2PO4, 0.25 g of MgSO4•7H2O, 0.5 g of thiamine chloride, and 15 g of agar in 1 L of deionized water) using sterile forceps and incubated at 25°C for 1-3 days.To isolate pure cultures, individual colonies of varied morphologies were picked up, transferred to potato dextrose agar (39 g of PDA in 1 L of deionized water; Becton, Dickinson and Co., Sparks, MD, USA) plates, and subcultured until pure mycelia were obtained.All pure isolates, including those of G. persicaria and P. crystallinus, were stored in 20% glycerol at −80°C at the Environmental Microbiology Laboratory Fungarium (Chonnam National University, Gwangju, Korea), as CNUFC-GWD3-9 and CNUFC-EGF1-4, respectively.Strain CNUFC-EGF1-4 was also deposited at the Culture Collection of the National Institute of Biological Resources (NIBR, Incheon, Korea), whereas strain CNUFC-GWD3-9 was also deposited at the Culture Collection of the Nakdonggang National Institute of Biological Resources (NNIBR, Sangju, Korea).

Morphological Studies
For detailed morphological studies, strain CNUFC-GWD3-9 was cultured on SMA, PDA, and malt extract agar (33.6 g of MEA in 1 L of deionized water; Becton, Dickinson and Co.).The plates were incubated at 5°C, 15°C, 25°C, 35°C, and 40°C in the dark for 2-3 days.Samples were mounted in distilled water and observed using an Olympus BX51 microscope with differential interference contrast (DIC) optics (Olympus, Tokyo, Japan).CNUFC-EGF1-4 strain was cultured on dung agar medium (2 g of water deer dung and 2 g of agar in 100 mL of deionized water) and the plates were incubated at 20°C, 25°C, and 35°C in the dark for 7-14 days.Additionally, fungal spores of strain CNUFC-EGF1-4 were inoculated on surface-sterilized pieces of water deer dung by touching with a sterile needle, and the plates were then incubated at 25°C in the dark for 7-14 days.Samples were observed under an Olympus BX51 microscope with DIC optics.

Conclusions
In present work, by using morphological and phylogenetic analyses (ITS and LSU rDNA), the two mucoralean strains CNUFC-GWD3-9 and CNUFC-EGF1-4 were identified as G. persicaria and P. crystallinus, respectively.There is no species of G. persicaria and P. crystallinus described in Korea, therefore, they can be considered as new record.
Our findings contribute to the current knowledge of diversity of the order Mucorales in Korea.However, data regarding the diversity of the order Mucorales in Korea are still lacking, further studies on the classification of different orders and families within the Mucoromycotina are required to expand our knowledge of undiscovered taxa with specific habitats in Korea.

Figure 1 .
Figure 1.Phylogenetic tree based on neighbor-joining analysis of internal transcribed spacer rDNA sequences for Gilbertella persicaria CNUFC-GWD3-9 and G. persicaria CNUFC-GWD3-10.Hyphomucor assamensis was used as an outgroup.Bootstrap support values of ≥50% are indicated at the nodes.The bar indicates the number of substitutions per position.

Figure 2 .
Figure 2. Phylogenetic tree based on neighbor-joining analysis of internal transcribed spacer rDNA sequences for Pilobolus crystallinus CNUFC-EGF1-4 and P. crystallinus CNUFC-EGF1-5.Umbelopsis isabellina was used as an outgroup.Bootstrap support values of ≥50% are indicated at the nodes.The bar indicates the number of substitutions per position.

Table 3 .
Primers used in this study, with sequences and sources.

Table 4 .
Taxa, collection numbers, sequences, and GenBank accession numbers used in this study.Chonnam National University, Gwangju, South Korea); NRRL (Agricultural Research Service Culture Collection, Peoria, IL, USA); T, ex-type strain.