Fungal Diversity in Korean Caves and Cave-Inhabiting Bats with Attention to Pseudogymnoascus Species

Abstract

Pseudogymnoascus is a psychrophilic fungus, which is a genus widely distributed in cold regions around the world. Recently, the presence of Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome (WNS) belonging to Pseudogymnoascus, has been reported in neighboring countries of Korea. However, no investigation on Pd has been reported in Korea. In this study, cave-inhabiting bats and their habitats were investigated in terms of the diversity of cave fungi, and we tried to confirm the presence of Pd. Three caves suspected of hosting Pd were selected, and 83 environmental and 53 bat samples were collected. A total of 154 fungal strains belonging to 31 different genera were isolated, and 20 of 154 were confirmed to belong to Pseudogymnoascus. Pd-diagnostic PCR was performed to check whether Pd was present in the isolated Pseudogymnoascus, and seven positives were confirmed. However, phylogenetic analyses revealed that no isolates belonged or were closely related to the clade with Pd. Although samples were collected from limited areas, undescribed Pseudogymnoascus species were isolated, and it was confirmed that Korean isolates were distributed in various clades. In conclusion, it is hypothesized that Korean Pseudogymnoascus presents high diversity.

1. Introduction

Fungi are organotrophic microorganisms and the second-largest eukaryote with an estimated 11.7 million to 13.2 million species [1]. Fungi are most commonly associated with terrestrial ecosystems, but they are present in almost every environment on Earth, from deep-sea deposits to animal skin [2,3,4,5,6]. Among fungal habitats, caves are generally considered an extreme environment for life because of the lack of organic carbon inflow through photosynthesis [7,8]. In these stringent environments, fungal diversity is generally considered to be low. However, several papers underline that caves harbor unexpectedly high diversity [9,10]. Previous studies have suggested that some of the fungal communities in caves were introduced from the outside [11,12,13,14]. They have been reported to be affected by external factors, such as airflow, water movement, and visitors [11,12,15,16]. Among these, cave-inhabiting bats are estimated to be one of the most important factors in the inflow of fungal spores [17,18].

Pseudogymnoascus is one of the fungal genera most closely related to bats [19,20]. In addition, the genus Pseudogymnoascus is a keratolytic and psychrophilic fungus that has a wide geographic distribution in cold regions worldwide [21,22,23]. The genus Pseudogymnoascus was established by Raillo to encompass the species Pseudogymnoascus roseus and Pseudogymnoascus vinaceus; decades later, Samson integrated P. vinaceus into P. roseus [24,25]. Attention to cave fungi has soared since 2009 when the pathogen Geomyces destructans, responsible for the fatal white-nose syndrome (WNS) disease, was identified [19,20,26]. Minnis and Lindner reorganized Geomyces and allied species after a multi-locus analysis. Geomyces destructans has been included in the Pseudogymnoascus genus [26], and many other species have been described in recent years [22,26,27,28].

Traditional classifications in taxonomy and systematics were mainly based on morphological approaches [29]. However, there may be limitations in systematically classifying fungal species because the morphological characteristics are not observed to be as diverse as numerous fungal species [1,30]. To redeem these limitations, molecular methods using a DNA barcode have emerged [31,32,33]. However, each gene marker used has different advantages and limitations [34,35,36,37]. Hence, multi-locus sequence typing, which uses multiple gene markers to redeem for the limitations, is used in fungal phylogeny [38,39].

Research on fungi associated with caves and bats in Korea is insufficient. In addition, the presence of Pd, the causative agent of WNS, has been reported in countries around Korea recently [40,41]. However, no studies on Pseudogymnoascus, including Pd, have been conducted. Therefore, the investigation of fungi present in bats and bat caves in Korea and the diversity of Pseudogymnoascus species is required. In this study, samples collected from bats and bat caves were used to cultivate fungi and to conduct Pd-specific diagnostic PCR and phylogenetic analyses to assess the diversity of Korean Pseudogymnoascus and the presence of Pd.

2. Materials and Methods

2.1. Sample Collection and Laboratory Processing

In 2018, we were provided information about a bat showing WNS-like symptoms in Geum cave by a bat ecologist (Dr. Chung CU, personal communication). Based on this information, we investigated the presence of Pd infection in bat caves and the surrounding environment. Environmental and bat samples were collected from three karst caves (Geum: 36°59′ N, 128°21′ E; Eun: 37°00′ N, 128°21′ E; and Handemy: 36°59′ N, 128°26′ E) in Danyang, Republic of Korea, during September 2019 to April 2020 (Figure 1). The temperature of the caves was 6–15 °C and the humidity was 50–83%. The bat guano and cave wall samples were collected using sterilized cotton swabs. Bats were captured using a bat mist net within the caves and were immediately released after oral and skin swab collection using sterilized cotton swabs. All bats in the caves were examined for the presence of Pd using ultraviolet (UV) light at a wavelength of 365 nm (Analytik Jena, Jena, Germany) [42,43]. All bats showed negative results for the UV light test. Skin swabs were stroked approximately 10 times, focusing on the bat’s wing membrane, ears, nose, and side of the mouth. Furthermore, oral swabs were obtained from the tongue and palate mainly. Bat guano (1 g) was suspended in 10 mL phosphate-buffered saline (PBS), containing 100 μg/mL of gentamycin and chloramphenicol. The swab samples were suspended in 1 mL of the mixture of antibiotics in PBS. A total of 83 environmental samples (Geum: 52; Eun: 14; Handmy: 17) and 53 bat samples (Geum: 15; Eun: 20; Handmy: 18) were collected. All samples were transported to the laboratory in a cooler and stored at 4 °C in the fridge until processed (three days).

2.2. Culture and Isolation of Fungi

To isolate fungi, 83 environmental samples (44 cave wall swabs and 39 bat guano) and 53 bat samples (19 oral swabs and 34 skin swabs) were cultured on Sabouraud dextrose agar (Becton Dickinson Co., Sparks, NV, USA), containing 100 μg/mL gentamycin and chloramphenicol (MBcell, Seoul, Republic of Korea). The supernatants of environmental samples, skin and oral swabs were collected through centrifugation at 3000 rpm for 1 min at 4 °C and then diluted to 1 × 10⁵ fold using PBS. Diluted sample supernatants (100 µL) were inoculated onto the media, and PBS was used as a negative control. The inoculated media were divided into samples at room temperature (20 °C) and at low temperature (6 °C) groups, and dark incubation was performed at room temperature for two weeks and at low temperature for 40 days. Media were checked daily. To obtain fungal cultures, the single-spore isolation method was used [44]. Single fungal cells were washed once with distilled water and harvested in 2 mL of PBS.

2.3. Genomic DNA Extraction, PCR Amplification, and Sequencing

Genomic DNA from pure fungal colonies was extracted using the i-genomic BYF DNA Extraction Mini Kit (iNtron Biotechnology, Seongnam, Republic of Korea) according to the manufacturer’s instructions. The isolates were first identified at genus level by internal transcribed spacer (ITS) sequencing and BLASTn comparison. The strains belonging to the Pseudogymnoascus genus were furtherly processed for the 28S large subunit rRNA gene (LSU), translation elongation factor 1 alpha (TEF1-α), minichromosomal maintenance protein 7 (MCM7), and RNA polymerase II second-largest subunit (RPB2) [22,26,27,28]. In addition, amplification was performed using nu-SSI(1506)-184-5′-Gd and nu5.8S-144-3′-Gd primer pair for Pd diagnosis [45]. The primer sequences used to amplify the markers are listed in Table S2. This amplification was performed using Maxime™ PCR Premix i-StarTaq (iNtron Biotechnology) according to the manufacturer’s instructions. The PCR mixture (20 µL) contained 50 ng of DNA template and 10 pmol of each primer. Sequencing was performed by barcode-tagged sequencing (BTSeqTM; CELEMICS, Seoul, Republic of Korea). All sequences generated in this study were deposited in the GenBank database, and their accession numbers are listed in Table S3.

2.4. Strain Identification and Phylogenetic Analysis

The sequences generated in this study were combined with the ones downloaded from GenBank to generate a sequence dataset (Table S3). Available reference sequences were retrieved from the NBCI database, and fungal sequences were aligned with the reference sequences using MAFFT v7.3113. Both sequence editing and concatenation were performed using MEGA 7.0.26 [46]. Minnis and Lindner observed that LSU and TEF1 introns have limited phylogenetic value because they are present and scattered among unrelated Pseudogymnoascus members [26]. Therefore, homologous gaps corresponding to LSU and TEF1 introns were excluded. In addition, the non-overlapping ends of the sequences in each alignment were trimmed. Phylogenetic analysis was conducted using maximum likelihood (ML) and Bayesian inference (BI) methods. ML analyses were performed using IQ-TREE v 1.6.8 [47]. The best-fit nucleotide substitution model for each locus was estimated using IQ-TREE’s Model Finder function [48] following the Bayesian information criterion (BIC). Bootstrap analyses were performed using ultrafast bootstrap approximation with 1000 replicates [49]. BI analyses were performed using MrBayes v.3.2.6 [50]. The analyses included two independent runs of five million generations with four chains each. The substitution model was set to K2 + I + G, and the first 25% of the samples and trees were discarded as burn-ins. The remaining trees were used to construct a 50% majority rule consensus tree.

3. Results

3.1. Fungus Isolation

Fungal colonies were cultivated from 31 of 83 environmental samples, 17 of 19 oral swab samples, and 13 of 34 skin swab samples. A total of 154 fungi were isolated, including 100 from the environment, 22 from oral swabs, and 32 from skin swabs.

3.2. Identification of Isolated Fungi

In the identification of isolated fungi using the ITS region molecular marker, the occurrence of isolated fungi from environmental samples by family level was as shown in

Table 1. Fungal diversity frequencies by phylum and family level of isolated from cave environmental samples at each cave: (a) Geum; (b) Eun; (c) Handemy. Trichocomaceae was most abundantly isolated.

Phylum	Family	No. Isolates	Isolates %
(a)
Ascomycota	Trichocomaceae	17	27.4
	Pseudeurotiaceae	16	25.8
	Saccharomycetaceae	2	3.2
	Clavicipitaceae	2	3.2
	Hypocreaceae	1	1.6
	Nectriaceae	1	1.6
	Cladosporiaceae	1	1.6
	Myxotrichaceae	1	1.6
	Plectosphaerellaceae	1	1.6
	Sordariomycetes	1	1.6
Basidiomycota	Mrakiaceae	8	12.9
	Trichosporonaceae	7	11.3
	Tremellaceae	1	1.6
Mucoromycota	Mucoraceae	3	4.8
(b)
Basidiomycota	Trichosporonaceae	3	42.9
Ascomycota	Trichocomaceae	2	28.6
	Chaetomiaceae	1	14.3
	Torulaceae	1	14.3
(c)
Ascomycota	Trichocomaceae	10	32.3
	Saccharomycetaceae	9	29.0
	Pseudeurotiaceae	3	9.7
	Cladosporiaceae	2	6.5
	Didymellaceae	1	3.2
	Nectriaceae	1	3.2
	Incertae sedis	1	3.2
	Microascaceae	1	3.2
Basidiomycota	Mrakiaceae	2	6.5
	Trichosporonaceae	1	3.2

. The occurrence of isolated fungi from bat samples by family level was as shown in

Table 2. Fungal diversity frequencies by phylum and family level of isolated from bat samples (oral and skin swab) at each cave: (a) Geum; (b) Eun; (c) Handemy. Saccharomycetaceae was the most frequently isolated.

Phylum	Family	No. Isolates	Isolates %
(a)
Ascomycota	Saccharomycetaceae	14	72.2
	Cladosporiaceae	1	1.9
(b)
Ascomycota	Saccharomycetaceae	4	36.4
	Pseudeurotiaceae	4	36.4
	Trichocomaceae	2	18.2
	Pleosporaceae	1	9.1
(c)
Ascomycota	Saccharomycetaceae	21	75.0
	Dothioraceae	3	10.7
	Trichocomaceae	3	10.7
	Pseudeurotiaceae	1	3.6

. The occurrence frequencies of total isolated fungi by phylum level were as follows: Ascomycota (83.8%); Basidiomycota (14.3%); and Mucoromycota (1.9%). The most abundantly isolated family was Saccharomycetaceae (32.5%), followed by Trichocomaceae (22.1%), Pseudeurotiaceae (15.6%), and Cladosporiaceae (2.6%). The highest-frequency genera were Debaryomyces (40 strains), Penicillium (33 strains), and Pseudogymnoascus (20 strains) (Table S1).

3.3. Pd-Specific Diagnosis

A total of Pseudogymnoascus samples were confirmed from 16 environmental samples, 1 oral sample, and 3 skin samples (Figure 2 and Table S1). Pd-diagnostic PCR results revealed that 7 out of 20 isolates tested positive for a 650 bp size (skin swab: 3 isolates; environment: 4 isolates) (Figure 3).

3.4. Phylogenetic Analysis of Pseudogymnoascus

After excluding non-overlapping ends and LSU and TEF1 introns, the concatenated alignment contained 3199 nucleotides (ITS, 486; LSU, 934; MCM7, 503; RPB2, 526; and TEF1, 750). The BI and ML trees showed identical topologies. Therefore, only the BI tree is shown (Figure 4).

The clades achieved by the BI method were named A to M, as defined by Minis and Lindner [26]. The Pseudogymnoascus isolates fell in clades A (4), B (5), C (1), D (4), H (3), and J (2) (Figure 4). None of the Pseudogymnoascus isolates fell into Pd as clade F.

4. Discussion

A total of 154 fungi were isolated from the bat and habitat environment samples. The most dominant family of isolated fungi from environmental samples was Trichocomaceae, which includes Penicillium and Aspergillus (Table 1 and Table S1). These two genera have been reported as the most common fungi in the cave environment, along with Cladosporidium [51]. The most abundant family of isolated fungi from bat samples was Saccharomycesaceae, which includes Debaryomyces and Candida (Table 2 and Table S1). To begin with, there was a report that Candida and Debaryomyces were identified in bat feces [52,53]. In addition, Debaryomyces is known as a common yeast found in bat skin [54,55]. In conclusion, it is assumed that these fungi continue to circulate in bat-dwelling environments and bats. Except for these two families, the notable one was the family Pseudeurotiaceae (24), which was abundantly isolated in both environmental and bat samples (Table 1 and Table 2). This family contains Pseudogymnoascus (20) and Leuconeurospora (4) (Figure 2 and Table S1). The genus that constituted the most significant proportion of Pseudeurotiaceae was Pseudogymnoascus, which is abundantly present as a keratolytic fungus in cave environments and as a saprophyte in cold soils and tree roots [4,21,23]. In addition, the high abundance of Pseudogymnoascus is assumed to be caused by the fact that the cave environment is suitable for its growth. The optimal temperature for Pseudogymnoascus growth is 15 °C [22], and the average temperature in the cave from which the samples were collected was 11 °C.

A Pd-diagnostic test using Pd-specific primers showed that 7 out of 20 Pseudogymnoascus species were positive. However, the phylogenetic tree revealed they were not located within clade F where Pd belongs. Lorch reported a 100% Pd-specific diagnostic PCR primer specificity [28]. However, in this study, a 35% false positive rate was observed (Figure 3 and Figure 4). The Pd-diagnostic primer described by Lorch contains a portion of the intron of SSU and the ITS1 5.8 s region [28]. SSU is used to analyze high taxonomic levels (family, order, class, and phyla) due to low variation between taxa [56]. In addition, ITS is a region used to analyze low taxonomic levels due to relatively high variations between taxa groups, but it does not work well in some fungal genera, and it was reported that the intergenomic ITS variation does not occurs largely in 3–5% of Ascomycota and Basidiomycota [37,56]. Considering this information, false positive results were presumed to be because Pd and the isolates were of the same genus. Even when isolates were not Pd (Figure 4), because this region is analogous to Pd, it is assumed that the diagnostic PCR result was positive (Figure 3). Further studies, such as improving the false positive rate of diagnostic primers for fungi in the same genus or developing new diagnostic methods using other target regions, are required.

Isolates 19BE01LM3, 20BE01LM1, and 19BE05LM1 fell in clade A. 19BG05LM3, 20BE09LM2, 20BE10LM1, and 20BE20LM3 were located in clade D. However, they were in independent branches of previously known species (Figure 4) [22,26,27,28]. These isolates are new undescribed species, which require future morphological analyses.

Pseudogymnoascus isolates known to date are predominantly from the United States and China, except for some from Antarctica (Table S3). Most studies have been conducted in North America and China, and recent studies in Antarctica have been reported [22,26,27,28]. Pseudogymnoascus species have been isolated several times throughout the United States and constitute an entire clade, most of which have been isolated from bat caves [22,26]. Furthermore, the Pseudogymnoascus species reported from urban soil in various regions of China were located in clades B, E, H, and J [27,28]. In this study, Pseudogymnoascus species were isolated from samples collected from bat caves in limited areas, but isolates were located in various clades (A, B, C, D, H, and J). Some of those isolated are new undescribed species. Samples collected from more regions in the future may contribute more to the study of Pseudogymnoascus.