Discovery of novel fungal species and pathogens on bat carcasses in a cave in Yunnan Province, China

ABSTRACT Virulent infectious fungal diseases, in natural and managed landscapes, are increasing. Fungal diseases in humans, animals and plants have caused die-off and extinction events and have become a threat to food security. A caving expedition in Yunnan Province, China, revealed two bat carcasses covered in fungal mycelia. Eleven fungal isolates were obtained from these bat carcasses, and morphological observations and multigene phylogenetic analyses revealed they were Fusarium incarnatum, Mucor hiemalis and Trichoderma harzianum and four new species, Mortierella rhinolophicola, M. multispora, M. yunnanensis and Neocosmospora pallidimors. One of the more alarming findings is that a number of infections related to Neocosmospora, previously associated with human and animal mycotoxicoses, are reported to be increasing, and here we present a new species from this genus, isolated from dead bats. Due to the ecosystem services provided by bats, and the close relationship between bats and humans, future research should focus on the impacts and significance of N. pallidimors to human and animal health, examining its pathogenicity and secondary metabolites. Taxonomic descriptions, color images of the habitat, in situ samples, microstructures and cultures are presented. SEM photographs of microstructures and phylogenetic trees showing the placement of new and known species are also provided.


Introduction
Cave ecosystems are unique windows into microbial and fungal life in the subsurface of the Earth. Living in oligotrophic environments with limited light and nutrients, organisms endemic to cave ecosystems exhibit photoautotrophic and photoperiodic adaptations [1]. Research documented a high level of microbial and bacterial diversity in hypogean systems. Depending on physical, ecological, and environmental characteristics, cave systems host hyper-localized speciation and ecologies [2]. Given our limited knowledge on hypogean biological diversity both at regional and local scales, cave systemsincluding, but not limited to, those winding through limestone karstsare a reservoir of new and undiscovered species.
These caves, and the animals inhabiting them, are also a source of human pathogens and diseases [3]. Severe acute respiratory syndrome coronavirus (SARS-CoV) and the current worldwide coronavirus  epidemic are just two examples that have had far-reaching impacts on society. Although slightly outdated, the review articles by Igreja [4] and Jurado et al. [3] document the different diseases and pathogens found in cave systems. Of subterranean interactions, pathogenic relationships between cave fauna and fungi warrant particular attention. In recent years, fungal and fungal-like diseases have caused die-off and extinctions events of host species at unprecedented scales [5]. For cave fauna, the devastating effect of Pseudogymoascus destructans (known as white-nose disease) is the most notorious example: in 2006, whitenose disease rapidly spread across North America, causing mass mortality and regional population collapse in the little brown myotis (Myotis lucifugus), a hibernacula [6]. As European bats are known to asymptomatically carry P. destructans, it was hypothesized that P. destructans was introduced to susceptible North American bats from the Old World, probably via human transport [7]. Furthermore, Histoplasma capsulatum, a human pathogenic and dimorphic fungus known to cause the disease Histoplasmosis in humans, is found in temperate areas throughout the world [8]. This fungus is found in soil and guano in caves and is carried by bats, although bats are usually asymptomatic. Current epidemics and past research suggest that cave ecosystems and bats pose ideal environments/hosts for the emergence of new pathogens and thus warrant further study.
The logistic difficulty of finding and accessing hypogean habitats has left them relatively undiscovered and unexplored [9]. Fungi are known to fill important ecological niches in cave systems, but there is scant scientific documentation detailing the relationship between the biological role(s) of cave microbes, fungi, flora, and fauna. The earliest cave fungi description was published by Humboldt in 1794 (as described in Dobat) [10]. By 2017, more than 1150 species of fungi, belonging to 550 genera, have been found in mines and caves worldwide [11,12]. The obligate troglobitic taxa, such as Acaulium caviariforme, Aspergillus baeticus, and A. thesauricus have been reported from mines and caves [11]. Interestingly, the majority of the recently discovered fungi from caves have also been discovered from other habitats [11][12][13]. Based on these findings, Zhang et al. [12] mentioned that cave fungi likely originated from non-cave habitats.
Little is known about the ecological origins and roles of hypogean fungi, such as where they originated, how they evolved, or if their evolutionary adaptations are localized. We fill a knowledge gap relating to the study of hypogean fungi with our presentation of new and known fungal species found on bat carcasses in a cave system in Yunnan Province, China. Furthermore, we provide detailed morphological and molecular descriptions of the fungal species isolated from the bat carcasses.

Ethics statement
Bat carcasses were collected in a cave outside of Kunming City, Yunnan Province, China. The collection of material was performed under the following project codes: 41761144055, 41771063 and 2018PC0006, managed through the Kunming Institute of Botany, Chinese Academy of Sciences.

Sample collection, specimen examination and isolation
Two dead bats (Rhinolophus affinis) ( Figure 1) covered with fungi were found on 5th August 2019, in a cave outside of Kunming City, Yunnan Province, China. Temperature, humidity, and carbon dioxide levels were recorded in caverns where the bat carcasses were found using a portable climate station (Xintest, HT-2000). Three climate readings were taken within the cave, and readings were recorded five minutes after the respective data values had stabilised. The fungi growing on the bat carcasses were isolated in Potato Dextrose Agar (PDA) using aseptic techniques. The cultures were placed at 28°C in an incubator. Observation of fungal morphological structures followed Wagner et al. [14], Sandoval-denis et al. [15], and Tibpromma et al. [16]. The pure cultures ( Figure  2

DNA extraction, PCR amplification and DNA sequencing
The mycelia of the cultures grown on PDA at room temperature for 8 weeks were used for DNA extraction. The fungal mycelia were scraped off using a sterile scalpel and transferred to 1.5 ml micro-centrifuge tubes under aseptic conditions and kept at −20°C to avoid contaminations until use. DNA extraction was performed on the fungal cultures using Biospin Fungal Genomic DNA Extraction Kit (BioFlux, P.R. China) following the manufacturer's protocol. To amplify partial gene regions, PCR conditions and primers were set under standard conditions, as shown in Supplementary Table 2. The total volume of PCR mixtures for amplifications was set as described in Tibpromma et al. [17]. Purification and sequencing of PCR products were done by Sangon Biotech Co., Shanghai, China.

Phylogenetic analyses
ITS and TEF1 sequence data produced in this study were used in BLAST searches in the GenBank database (wwwhttp://blast.ncbi.nlm.nih.gov/) to determine their most probable closely related taxa. The sequence data generated in this study were analysed with closely related taxa retrieved from GenBank and recent publications. The single gene sequence dataset was aligned using the MAFFT v. 7.215 website [18]: (http://mafft.cbrc.jp/alignment/server/index.html) and manually edited in BioEdit v. 7.0 when necessary [19]. The single sequence alignment dataset was combined using BioEdit v.7.0 [19]. The alignment of combined datasets in FASTA format was converted to PHYLIP and NEXUS formats by using the ALignment Transformation EnviRonment (ALTER) website (http://sing.ei.uvigo.es/ALTER/). Phylogenetic trees were run in Randomized Accelerated Maximum Likelihood (RAxML) and Bayesian posterior probabilities (BYPP). ML analysis was performed via the CIPRES Science Gateway (http://www.phylo.org/) [20] using RAxML-HPC BlackBox (8.2.4) section [21] with the general time reversible model (GTR) using a discrete gamma distribution as the evolutionary model. To carry out Bayesian analysis, the model of evolution was estimated using MrModeltest 2.2 [22] as nucleotide substitution models selected for combined datasets. Posterior probabilities (PP) [23] were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v 3.0b4 [24]. The parameters were set as six simultaneous Markov chains and ran for 5000000 generations with sample frequency every 100th generations [25]. The first trees representing the burn-in phase of the analyses (20%) were discarded and the remaining (post-burn) trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree (with the critical value for the topological convergence diagnostic values reaching 0.01) [26].
The phylograms were configured in FigureTree v. 1.4 [27] and reorganized using Microsoft Office PowerPoint 2016, Adobe Acrobat XI Pro, and Adobe Photoshop CC 2019 (Adobe Systems Inc., USA). The sequences generated in this study were submitted to MycoBank and are mentioned under the description part.

Pairwise homoplasy index
The pairwise homoplasy index (PHI) test was performed by SplitsTree4 to determine the recombination level within phylogenetically closely related species by using the concatenated dataset of closely related species ( Supplementary Figures 4 and 8) [28]. Pairwise homoplasy index results lower than 0.05 (Φw < 0.05) indicate the presence of significant recombination in the dataset. The relationships between closely related taxa are visualized by constructing splits graphs from concatenated datasets, using the Log-Det transformation and splits decomposition options.

Climatic conditions inside of the cave
The climate inside the cave proved to be highly stable. Climate details of the cavern in which the specimens were collected are provided in Table 1.

Phylogenetic analysis of single and combined sequence data
The single and combined datasets of fungi on bats were analysed using maximum likelihood (ML) and Bayesian analyses (BYPP). Both ML and BYPP trees showed similar results in topology and no significant difference was seen (data not presented).
The phylogram of Fusarium, generated from RAxML analysis and based on the combined dataset of TUB and TEF1 datasets, showed that our strains grouped together with the Fusarium incarnatum clade with relatively high bootstrap support (Supplementary Figure 1).  The Mortierella phylogram, generated from RAxML analysis and based on the combined dataset of 28S and ITS datasets, showed that our five isolates separated into three new species (Mortierella multispora, M. rhinolophicola, and M. yunnanensis), which were well separated from other species in the Mortierella multispora cluster. Our new species clustered with M. pisiformis but were well separated with high bootstrap support (99% ML/ 1 BYPP, Supplementary The Mucor phylogram, generated from RAxML analysis and based on the ITS dataset, showed that our strains grouped together with the Mucor hiemalis clade with relatively high bootstrap support (Supplementary Figure 5).
The Neocosmospora phylogram, generated from RAxML analysis and based on the combined dataset of LSU, ITS, TEF1, and RPB2 datasets, showed that our new species, Neocosmospora pallidimors, was well separated from other Neocosmospora species with relatively high bootstrap supports (100% ML/ 1 BYPP, Supplementary Figure 7).
The Trichoderma phylogram, generated from RAxML analysis and based on the combined dataset of RPB2 and TEF1 datasets, showed that our strains grouped together with the Trichoderma harzianum clade with relatively high bootstrap support (Supplementary Figure 9). Therefore, we introduce Mortierella multispora, M. rhinolophicola, M. yunnanensis, and Neocosmospora pallidimors as new species. In addition, we report 3 known species: Fusarium incarnatum, Mucor hiemalis, and Trichoderma harzianum based on evidence from phylogeny and morphology.
The genus Fusarium (Hypocreales, Nectriaceae), a filamentous fungus, was introduced by Link [29] with distinctive banana-shaped conidia. This fungus is widely distributed in plants (infecting both monocotyledonous and dicotyledonous) soils, and marine environments [30]. Species within Fusarium include the most important plant-pathogenic fungi, causing infections, allergic responses, and acting as pathogens of immunocompromised humans and animals [31]. Fusarium species produce remarkable diversity of secondary metabolites and mycotoxins (most notably trichothecenes and fumonisins) that exhibit toxicity to mammal and human health (especially in immunocompromised individuals) [32,33]. Furthermore, members of Fusarium have previously been reported in cave systems [11,12].
GenBank numbers: TEF1 = MT024982. Notes: Fusarium incarnatum was introduced by Saccardo [34], which belongs to the F. incarnatum-equiseti species complex. This complex is well-known for containing plant, human, and animal diseases [35] (Supplementary Figure 2). In this study, we found Fusarium incarnatum, which was isolated from a bat carcass found in a cave. We assume this fungus can survive in the mild temperature and low oxygen/high carbon dioxide environment of the cave in which they were found. Furthermore, this is the first report of F. incarnatum from bat carcasses as well as caves.
Species of Mortierella (Mortierellales, Mortierellaceae) are frequently isolated from soils, dead or dying plant tissues, freshwater, or animal dung samples [15,36,37]. Many show a strong capacity to decompose plant litter, degrade polyaromatic hydrocarbons, and are also potential producers of c-linolenic acid, polyunsaturated fatty acids and arachidonic acid [38,39]. Several species are known to be indirectly beneficial to humans; for example, they are able to degrade herbicidal residues on crop plants; aid mycorrhizal fungi in phosphorus (P) acquisition; and have the ability to synthesize and secrete oxalic acid [40,41]. There have been reports on Mortierella species being found in cave systems [11,12].
Notes: Based on phylogenetic analyses, our new species is well separated from Mortierella echinosphaera and M. chlamydospore with high statistical support (100% in ML, 0.99 in PP) (Supplementary Figure 3). Our results are consistent with Wagner et al. [14] who mentioned that M. echinosphaera and M. chlamydospora are closely related. Morphological characteristics show M. chlamydospora has globose or elongated chlamydospores, with a varying number of spines that are sometimes smooth when submerged [42,43], and M. echinosphaera has globose to elongated, densely spiny chlamydospores [41,42], while our new species has globose to subglobose, echinulate sporangia. A BLAST search of NCBI's GenBank nucleotide database revealed that the closest hit received for M. rhinolophicola (KUMCC 20-0008), using the ITS sequence, was M. chlamydospora (GenBank KP881466; Identities = 98.64%), and the closest hit received for M. rhinolophicola using the 28S sequence was M. chlamydospora (GenBank KP881466; Identities = 97.15%). Based on a BLAST search of NCBI's Gen-Bank nucleotide database, the closest hit received for M. rhinolophicola (KUMCC 20-0014) using the ITS sequence was M. chlamydospora (GenBank KP881466.1; Identities = 97.15%) and the closest hits received for M. rhinolophicola using the 28S sequence was M. echinosphaera (GenBank KC018382; Identities = 98.57%) and M. chlamydospora (GenBank HQ667430; Identities = 98.17%).
Notes: Based on phylogenetic analysis, this new species is well separated from other closely related species of Mortierella with high statistical support (76% in ML, 0.99 in PP; Supplementary Figure 3). Our new species clustered with M. amoeboidea, but our new species did not sporulate in culture in 5 months (Figure 2). In addition, we compared the cultures of M. amoeboidea and our new species and found that M. amoeboidea colonies are delicate and densely lobed, with some aerial mycelia in the centre, odour faint and sporulation rather poor on all media [44], while our new species shows spreading, with sparse aerial mycelia, velvety and flossy, at first white and later becoming yellow-white. Our morphological data are complemented by the molecular analyses, confirming this as a distinct new species.
GenBank numbers: ITS: MT020421 Notes: Mucor hiemalis, a common soil fungus of phylum zygomycota, produces a diverse array of enzymes and lipids. This species has been reported from organic litter in the Suiyang Cave, Henan, China [12]. Desai et al. [48] reported subcutaneous zygomycosis caused by Mucor hiemalis in an immunocompromised patient.
Neocosmospora E.F. Sm., Bulletin of the U.S. Department of Agriculture 17: 45 (1899) Neocosmospora (Hypocreales, Nectriaceae) was introduced by Smith [49]. This genus was assigned to the Fusarium solani species complex [50]. This genus is widely distributed in soil, plant debris, living plant material, air, and water, in the form of saprobes, plant endophytes, and pathogens, as well as in opportunistic animal pathogens [15]. Neocosmospora species have also been reported to be associated with human and animal mycotoxicoses [51]. Importantly, this genus has never been reported from bats or caves.
Culture characteristics: Colonies on PDA (Figure 2). Colonies on PDA covering 9 cm diam., in 4 weeks at 28°C, spreading, with sparse aerial mycelium with undulate edge, flossy. Mycelium superficial, yellowwhite; reverse yellow, with sporulating on PDA after 4 months.  Notes: Neocosmospora pallidimors is introduced based on evidence from morphology and phylogeny. This is the first report from bats in a cave. Neocosmospora pallidimors is phylogenetically closely related to N. stercicola but is well separated with statistical supports (100% in ML, 1 in PP). The morphological characteristics of N. stercicola include 0-5-septate conidia with colourless drops of liquid, ovoid to ellipsoidal, single or in chains and pale brown chlamydospores [52], while our new species has 0-3-septate conidia with globose to subglobose, septate, often two-celled rough and echinulate chlamydospores that stick together.
Trichoderma Pers., Neues Magazin für die Botanik 1: 92 (1794) Trichoderma (Hypocreales, Hypocreaceae) was introduced by Persoon [53]. Trichoderma forms a large group of microorganisms with global distribution and are known as opportunistic fungi of economic and ecological importance, as they produce toxic secondary metabolites and disease-causing agents of plants, animals, and humans [54]. Several species of Trichoderma have been reported in caves [11,12]. Notes: Trichoderma harzianum is widely recognized as a potential biocontrol agent against several soilborne plant pathogens [55]. This species is reported here for the first time on bats and in caves.

Discussion
In this paper, we present seven species of fungifour of them novelextracted from a pair of bat carcasses in a limestone cave in Yunnan, China. Of these seven species, Fusarium incarnatum is known as a plant pathogen [56], and Neocosmospora pallidimors belongs to a group known to have highly prevalent and aggressive human and animal fungal pathogens [15], while Mucor hiemalis was reported to cause zygomycosis, an opportunistic fungal infection with a high mortality rate infecting across a wide range of substrates, from bread to human skin tissue [48,57]. This density of new species found in an isolated and relatively confined system underscores the exciting potential of subterranea as reservoirs of biological diversity and as a frontier of scientific exploration [9]. This holds particularly true in China, a country thought to contain one of the world's largest cave-containing limestone karsts systems [58]. This point is further emphasized by the work of Deharveng et al. [59], who reported that 90% of species collected during a caving expedition in Mulun, a National Nature Reserve in Guangxi Province, China, were new to science.
Our study answers the call for further research documenting the relationshipsparticularly transmission between fungi and other cave organisms [11]. Discoveries of fungal species on bats are of scientific interest and possible concern, both for humans and for other mammals inhabiting cave systems. The catastrophic effects of Pseudogymnoascus destructans on bat hibernacula (e.g. causing white-nose syndrome) triggered a cascade of research on the societal and environmental importance of bats [6,60]. Difficulties of ecosystem service valuation aside, one well-cited study estimated that a disappearance of North American bats could result in an upward of 3.7 billion dollars in agricultural losses [61]. Prior research on other fungal species within two of the genera we discovered -Mortierella and Neocosmosporasuggest the potential for economic and ecological implications [51,62]. Mortiella wolfi is considered a pathogen solely of animals and is known to induce abortions and cause encephalitis and pneumonia in cattle found in Australia, North America, and Japan [63]. Two reports of human cutaneous infection with M. wolfii have also been reported [63,57]. Neocosmospora have previously been associated with human and animal mycotoxicoses, and infections are thought to be on the rise [51,62]. A decline in Yunnan bat populations could have devastating effects on the loss of ecosystem services and overall ecosystem health. Further research is necessary to understand how bats may act as carriers for these fungi as well as on the ecological and economic implications for such symbiotic relationships.
Our study presents not only new species of pathogens with unknown pathogenicities but also new sources of already-known pathogens with the potential to affect both crops and mammal populations. Further studies investigating more cave systems across a broader region are still required, and this work needs to be coupled with biomedical studies investigating the pathogenicity of these newly discovered species. Y9180822S1), CAS President's International Fellowship Initiative (PIFI) (number 2020PC0009), China Postdoctoral Science Foundation and the Yunnan Department of Human Resources and Social Security for funding her postdoctoral research. Kunming Institute of Botany, Chinese Academy of Science (KIB) is thanked for providing laboratory facilities for morphology and phylogeny work, and Mr Gu Zhi-Jia in KIB is thanked for the assistance in taking the scanning electron microscopy photographs. Stephen Axford and Catherine Marciniak are thanked for their help on field visits, Deping Wei is thanked for her help in depositing fungal cultures and the curator at the Yunnan University fungal herbarium is also thanked. The authors would like to thank Alice C. Hughes for assistance in identifying the bat species. Dr Shaun Pennycook is thanked for help in naming the new fungal species. Austin Smith at World Agroforestry (ICRAF), Kunming Institute of Botany, China, is thanked for English editing.

Disclosure statement
No potential conflict of interest was reported by the authors.