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Characterization of the archaeal and fungal diversity associated with gypsum efflorescences on the walls of the decorated Sorcerer’s prehistoric cave

Characterization of the archaeal and fungal diversity associated with gypsum efflorescences on... Purpose This study focuses on analysis of the archaeal and fungal diversity associated with gypsum efflorescences damaging the walls of the Sorcerer’s prehistoric cave registered as a world cultural heritage site. Method Archaeal 16S rDNA and fungal internal transcribed spacer (ITS) clone libraries were constructed and analysed. Results Two thaumarchaeotal OTUs belonging to the Nitrososphaeraceae family dominated the archaeal community (100% of clones). Nitrososphaeraceae are obligate aerobic, chemolithoautotrophic organisms that derive their energy from the oxidation of ammonia and may contribute to primary productivity in the cave. Seven fungal OTUs belonging to Ascomycota and one belonging to Basidiomycota were present. The Cordycipitaceae family, mainly represented by entomophilous fungi, dominated the analysis (66.7% of clones). Conclusion We show that archaeal and fungal OTUs are associated with gypsum efflorescences damaging the walls of the Sorcerer’s cave. The role of these microorganisms in the deterioration of the walls of the cave remains to be determined. . . . . . Keywords Cave Gypsum Biodeterioration Archaea Fungi Diversity Introduction environmental factors that can alter the rock substrate accord- ing to their mineralogical composition and structure (Chamley Characterization of the microorganisms associated with bio- 2003;Saiz-Jimenez 2015). deterioration processes encountered in rock art caves and shel- Decorated caves and shelters are natural cavities subjected ters is needed (Saiz-Jimenez et al. 2011, 2012; Lepinay et al. to natural deterioration processes. Natural cavities are 2017, 2018), as also in mural painting from catacombs and nutrient-poor environments containing an abundant and di- tombs (Sanchez-Moral et al. 2005;Laizetal. 2009; verse sessile biomass (Barton and Jurado 2007;Lavoieet al. Vasanthakumara et al. 2013; Krakova et al. 2015). These 2010). Representatives of the 3 domains of life are recovered works of art are constantly threatened by decay resulting from in these subterranean environments. As in most environments, the interaction of biological and physicochemical the focus in the caves was on the diversity of bacteria. Among them, the most abundant and frequently retrieved phyla are as follows: Acidobacteria, Actinobacteria, Bacteroidetes, * Agnès Mihajlovski Cyanobacteria, Firmicutes, Nitrospirae and Proteobacteria agnes.mihajlovski@u-cergy.fr (Saiz-Jimenez 2015). Previous work in the Sorcerer’s cave showed that Actinobacteria, Alphaproteobacteria, Université de Cergy-Pontoise – ERRMECe-EA1391 – Site de Gammaproteobacteria, Bacteroidetes and Planctomycetes Neuville-sur-Oise, 1 rue Descartes, 95000 Neuville-sur-Oise were the main bacterial inhabitants of areas of salt efflores- Cedex, France cences damaging the walls (Lepinay et al. 2018). Laboratoire de Recherche des Monuments Historiques, Ministère de Actinobacteria was the most prevalent phylum, mainly repre- la Culture et de la Communication, Centre de Recherche sur la sented by members of the Pseudonocardiaceae family, data Conservation (CRC-USR 3224), Muséum national d’Histoire consistent with other findings (Barton and Jurado 2007; naturelle, CNRS, Sorbonne Universités, 29 Rue de Paris, 77420 Champs-sur-Marne, France Porca et al. 2012; Riquelme et al. 2015; Wu et al. 2015). 1072 Ann Microbiol (2019) 69:1071–1078 Conversely, a limited number of investigations were carried The gypsum deposits are located on the outer layer of the out on Archaea, particularly for sites of cultural heritage pres- stone and make it a kind of extremely fragile “molasses” ervation. However, many Archaea are adapted to nutrient- (Pigeaud et al. 2012). The same samples as those collected poor conditions and may contribute to nutrient cycling in ol- by Lepinay et al. (2018) in April 2015 (SCApr15) have been igotrophic environments through nitrogen fixation, analysed. Sterile scalpels were used to collect 3 efflorescence methanogenesis, sulphur oxidation, nitrification and ammonia areas (each ~ 2 cm ) and the underlying rock from a depth of oxidation (Ettenauer et al. 2010; Jarrell et al. 2011;Meng etal. ~ 2 mm, (Fig. 1b, c). Samples were mixed together and ho- 2016, 2017). To date, the most frequently recovered archaeal mogenized in sterile mortars. Samples were stored at − 20 °C phylum is Thaumarchaeota (Northup et al. 2003; Chelius and awaiting molecular analysis. Scanning electron microscopy Moore 2004;Legatzkiet al. 2011;Miller etal. 2012;Ortiz observations of the samples and elemental and mineralogical et al. 2013). Members of the Crenarchaeota have also been analyses of the corresponding saline efflorescences have been retrieved from steam vents and caves from volcanic national previously published (Lepinay et al. 2018). This indicated that parks in the USA (Benson et al. 2011) and Euryarchaeota in the formation of gypsum on the walls of the cave was mainly caves (Macalady et al. 2006; Reitschuler et al. 2014, 2016). In related to geochemical phenomena, efflorescences being addition to Bacteria and Archaea, fungi are very well repre- mainly composed of calcium, sulphur and oxygen, with addi- sented in cave environments due to their high rate of spore tional small amounts of nitrogen and carbon. production and air dispersion. They are important in the feed- ing strategies of cave fauna because they are important de- composers providing food for many organisms (Nováková DNA extraction and purification 2009). As reviewed by Vanderwolf et al. (2013) and recently completed by Zhang et al. (2017), > 1150 fungal species in DNA extraction was done as previously described (Zhou et al. 550 genera have been discovered in caves and mines world- 1996; Lepinay et al. 2018). The protocol combines the use of wide by 2017. The most frequently encountered genera (e.g. enzymatic (proteinase K) and chemical (CTAB and SDS) Aspergillus, Penicillium, Mucor, Fusarium, Trichoderma, components to ensure efficient cell lysis. Extracted, nucleic Cladosporium, Alternaria, Paecilomyces, Acremonium, acids were purified with a Power Biofilm™ DNA Isolation Engyodontium) are air-borne and soil-borne fungi that come Kit (MoBio) and were stored at − 20 °C until use. from outside the cave cavities due to air circulation (Saiz- Jimenez 2015). Several efforts have been made to characterize the diversity and distribution of fungi in caves, but there is PCR amplification of fungal ITS and archaeal 16S little information on taxa associated with specific niches, such rDNA genes as oligotrophic conditions and mineral formations (Saiz- Jimenez 2015). Fungal ITSs were amplified using primers ITS1-F and ITS4. We have focused on the French Sorcerer’s prehistoric cave A25-μl reaction comprising 20 ng of DNA, 100 μMof (“La grotte du Sorcier”). The cave undergoes alternations of each dNTP, 0.4 μM of each primer, 1× PCR buffer and humidification and desiccation phases which induce cycles of 1.5 U of Accusure DNA Polymerase (Bioline, France) was dissolution and recrystallization of the salts leading to efflo- performed. Cycling conditions were as follows: 5 min at rescence development on the walls that threaten the engrav- 95 °C, followed by 30 cycles of 20s at 95 °C, 15 s at ings (Pigeaud et al. 2012). This work completes the previously 54 °C and 1 min and 30 s at 68 °C, and a final extension published data corresponding to bacterial diversity following at 68 °C for 7 min. Amplicon was extracted from an agarose analysis of the archaeal and fungal diversity associated with gel after electrophoresis using the Nucleospin Gel and PCR saline efflorescences (Lepinay et al. 2018). clean up kit (Macherey Nagel, France). Archaeal 16S rRNA gene sequences were amplified using a semi-nested PCR approach. Archaeal universal primers 340F-pSTC (5′- Materials and methods CCTTCgCCgACTgACCCTAY-GGGGYGCASCAG-3′) and 1000R (5′-GGCCATGCACYWCYTCTC-3′) (Gantner Site description and sampling et al. 2011) were used as outer primers. Then, a semi- nested PCR was carried out using 340F-pSTC and 915R The Sorcerer’s cave, the Saint-Cirq cave, dug in a Turonian (5′-GTGCTCCCCCGCCAATTCCT-3′) (Stahl and Amann siliceous limestone, harbours several prehistoric engravings 1991). The PCR reaction was carried out as previously de- already described by different archaeologists (Fig. 1)(Delluc scribed, using an annealing temperature of 57 °C (Lepinay et al. 1987;Karnayet al. 1999; Pigeaud et al. 2012). et al. 2017). One microlitre of the first PCR was used in the Formation of gypsum efflorescences are present absolutely nested PCR, and an amplicon of ~ 580 bp was purified as everywhere in the cave, from the entrance to the bottom. described above. Ann Microbiol (2019) 69:1071–1078 1073 Fig. 1 The Sorcerer’s cave (Saint-Cirq-du-Bugue, Dordogne, France): a entrance (white arrow); b sampling areas 1, 2 and 3; c enlarged view of the sampling area no. 2 showing gypsum efflorescences. Credit photo: LRMH Clone libraries Good’s C index of coverage were calculated as described by Kemp and Aller (2004). Purified PCR products were inserted into the pSTC1.3 vector of the StabyCloning kit (Eurogentec, Belgium), and recombi- Nucleotide sequence accession numbers nant plasmids were electrotransformed in E. coli. Transformed cellswereplatedontoLBagarcontaining50 μg/ml ampicillin The nucleotide sequence data reported herein have been de- and incubated overnight at 37 °C. PCR amplifications of in- posited in the NCBI nucleotide sequence database under ac- serts using primers targeting the pSTC1.3 vectors: pSTC-F cession numbers MK212376 to MK212382, MK590246 to (5′-AATGCAGCGCGTTAGAA-3′) and pSTC-R (5′-CGCC MK590262 for fungi, and MK226534 to MK226535 and CGGTTTATTGAAA-3′), followed by agarose electrophore- MK607017 to MK607025 for Archaea. sis in 1.5% agarose gel, allowed to select clones presenting inserts at the expected sizes for archaeal 16S rDNA and ITS libraries, respectively. Restriction fragment length polymor- Results phism (RFLP) was used to screen these clones. Each PCR product was digested using the restriction enzymes, AluI and OTU richness and its reliability RsaI (Fermentas, France) for 16S rDNA amplicons or with the restriction enzyme BshfI (Fermentas, France) for ITS Analysis of RFLP patterns allowed grouping 178 archaeal amplicons. At least one clone per RFLP profile was partially 16S rDNA clones into 4 restriction patterns and 91 fungal sequenced (Eurofins Genomics, Germany). ITS clones into 10 restriction patterns. When the number of clone inside a RFLP pattern was higher than 2, several ran- Sequence analysis domly chosen clones were partially sequenced. After elimina- tion of chimeric and low quality sequences, 172 archaeal 16S Sequences were first checked using the software FinchTV rDNA clones and 81 fungal ITS clones remained in the banks. v1.4 (Geospiza, Inc.). Chimeric sequences identified with Pairwise alignments of randomly chosen clone sequences the Decipher program (Wright et al. 2012) were excluded. using 98% similarity as the cut-off allowed discrimination of ITS and 16S rDNA sequences were clustered into OTUs (op- 2 and 8 OTU in the archaeal 16S rDNA and the fungal ITS erational taxonomic unit) at an overlap percentage identity libraries, respectively (Tables 1 and 2). Estimated OTU richness (S-Chao1 estimator) was com- cut-off of 98% using ClustalW (Thompson et al. 1994). BLASTn was then used to compare DNA sequences with pared to the values of cloned libraries Kemp and Aller (2004). Predicted S-Chao1 values were of 2 and 8 phylotypes those in the GenBank database (Basic Local Alignment Search Tool, http://blast.ncbi.nih.gov/Blast.cgi). Sequences for the archaeal 16S rDNA and the fungal ITS libraries, re- with no significant similarities were excluded from the spectively. Calculation of Good’s C indices showed that most analysis. The affiliation of each cloned sequence to a genus of the sample diversity was represented in the clone libraries. or species was based on a similarity ≥ 95 or ≥ 98%, Indeed, coverage was 0.99 for both libraries, which indicates respectively, with the closest identified phylogenetic that the analysis of an increasing number of clones would have sequence in GenBank (Yarza et al. 2008). Archaeal sequences shown only a little further richness. were also classified using the Naïve Bayesian Classifier from the RDP project with a confidence threshold of 80% (https:// Archaeal diversity rdp.cme.msu.edu/classifier/classifier.jsp). We assessed whether DNA clone libraries were large enough to be Only the Thaumarchaeota phylum was recovered in the ar- representative of OTU richness from samples. S-Chao1 and chaeal 16S rDNA clone library (Table 1); this phylum was 1074 Ann Microbiol (2019) 69:1071–1078 Table 1 Assignment of archaeal 16S rDNA sequences to their closest cultured representative OTU Representative clones* % of RDP family*** Closest cultured Closest Candidatus clones** representative organism (percentage (percentage similarity) similarity) Thaumarchaeota OTU-ARC-01 SC-Arc-09 (MK607017); 99.4% Nitrososphaeraceae Nitrososphaera Candidatus SC-Arc-30 (MK226534); (171/172) (100%) viennensis Nitrosocosmicus SC-Arc-47 (MK607019); strain EN76 oleophilus strain MY3 SC-Arc-64 (MK607018); (95.8 to 96.3%) (99.4 to 100%) SC-Arc-69 (MK607020); SC-Arc-95 (MK607021); SC-Arc-B2-12 (MK607022); SC-Arc-B2-35 (MK607023); SC-Arc-B2-56 (MK607024); SC-Arc-B2-74 (MK607025) OTU-ARC-02 SC-Arc-54 (MK226535) 0.6% Nitrososphaeraceae Nitrosopumilus - (1/172) (100%) maritimus SCM1 strain SCM1 (96.2%) *Number in brackets indicates the GenBank accession number **In parentheses: ratio between the number of clones of the corresponding OTU and the total number of clones ***Bacterial family determined using the Naïve Bayesian Classifier from the RDP project website. The confidence threshold is 80% represented by 2 phylotypes belonging to the archaeal family, family closely related to Trametes hirsuta isolate BHI-F579a Nitrososphaeraceae. One of them dominated the library and (1.2% of clones, 99.8% SS). presented as closest cultured representative: Nitrososphaera viennensis strain EN76 (99.4% of clones, 95.8 to 96.3% se- quence similarity, SS); and as closest Candidatus organism: Discussion Candidatus Nitrosocosmicus oleophilus strain MY3 (99.4 to 100% SS). The second one presented as closest cultured rep- This study aims to improve the understanding of biological resentative: Nitrosopumilus maritimus SCM1 strain SCM1 phenomena associated with the formation of gypsum efflores- (0.6% of clones, 96.2% SS). cences on the walls of the French Sorcerer’s prehistoric cave. We have previously shown that these areas of gypsum efflo- Fungal diversity rescences harbour a variety of bacterial communities and a dense subjacent biofilm (Lepinay et al. 2018). We continued ITS sequences mainly belonged to the phylum Ascomycota the investigations of the microbial communities living in these (Table 2), with most represented family being the zones of efflorescence by analysing Archaea and fungi. Cordycipitaceae (66.7% of clones). Cordycipitaceae were rep- Concerning Archaea, all the clones belonged to the phylum resented by 2 phylotypes, one being affiliated with Isaria Thaumarchaeota, a phylum, previously referred to fumosorosea strain BCMU PF01 (51.9% of clones, 99.8 to “mesophilic Crenarchaeota”, being proposed by Brochier- 100% SS), and the other related to Engyodontium album iso- Armanet et al. (2008). Thaumarchaeota have been detected late MC_A31 (14.8% of clones, 95.5 to 95.6% SS). The sec- by molecular studies in many subterranean habitats (Takei ond most abundant OTU belonged to the Mycosphaerellaceae et al. 2001; Northup et al. 2003;CheliusandMoore 2004; family, being distantly related to Pseudocercosporella sp. Legatzki et al. 2011; Miller et al. 2012; Ortiz et al. 2013; 09CT02 (18.5% of clones and 92.9 to 93.1% SS). The fourth Barton et al. 2014;Anda et al. 2017) and in many different most abundant OTU belonged to the Cladosporiaceae family environments, such as seawater (e.g. Church et al. 2003), and corresponded to Cladosporium pulvericola CPC: 22403 freshwater sediments (e.g. Schleper et al. 1997) and soils (6.2% of clones, 100% SS). Three other ascomycotal families (e.g. Ochsenreiter et al. 2003). All known ammonia- were recovered in the library, each representing 2.5% of clones oxidizing archaea (AOA) belong to Thaumarchaeota and that had sequence identities between 92 and 95% with their Thaumarchaeota are receiving much attention regarding their closest relative in Genbank, namely the Ophiocordycipitaceae, ability to proliferate in environments with low nutrient avail- Diatrypaceae and Teratosphaeriaceae. Finally, Basidiomycota ability (Martens-Habbena et al. 2009; Pester et al. 2011;Ortiz were represented by one clone belonging to the Polyporaceae et al. 2014). This phylum can represent the main source of Ann Microbiol (2019) 69:1071–1078 1075 Table 2 Assignment of fungal ITS sequences to their closest cultured representatives and closest sequence matches OTU Representative clones* % of Fungal family Closest cultured Closest clones** representative sequence match (percentage (percentage similarity) similarity)*** Ascomycota OTU-FUNG-01 SC-ITS-04 (MK590248); SC-ITS-14 51. 9% Cordycipitaceae Isaria fumosorosea - (MK590250); SC-ITS-28 (MK590251); (42/81) strain BCMU SC-ITS-38 (MK212376); SC-ITS-42 PF01 (99.8 to (MK590252); SC-ITS-48 (MK590253); 100%) SC-ITS-49 (MK590254); SC-ITS-58 (MK590258); SC-ITS-74 (MK590260) OTU-FUNG-02 SC-ITS-02 (MK590246); SC-ITS-50 18.5% Mycosphaerellaceae Acrodontium Pseudocercosporella (MK212378); SC-ITS-76 (MK590261) (15/81) crateriforme sp. 09CT02 strain CPC 11509 (92.9 to 93.1%) (91.3to91.5%) OTU-FUNG-03 SC-ITS-39 (MK212377); SC-ITS-57 14.8% Cordycipitaceae Engyodontium Uncultured fungus (MK590257); SC-ITS-79 (MK590262) (12/81) album isolate clone MC_A31 RS-Apr-ITS37 (95.5to95.6%) (99.9 to 100%) OTU-FUNG-04 SC-ITS-13 (MK590249); SC-ITS-55 6.2% Cladosporiaceae Cladosporium (MK590256) (5/81) pulvericola CPC: 22403 (100%) OTU-FUNG-05 SC-ITS-30 (MK212379) 2.5% Ophiocordycipitaceae Thyronectria - (2/81) asturiensis strain MA3 (92.5%) OTU-FUNG-06 SC-ITS-44 (MK212380) 2.5% Diatrypaceae Eutypa consobrina - (2/81) culture-collection CBS: 122678 (95.1%) OTU-FUNG-07 SC-ITS-33 (MK212381) 2.5% Teratosphaeriaceae Teratosphaeria Uncultured fungus (2/81) knoxdaviesii strain clone CBS 122898 RS-Apr-ITS18 (94.7%) (99.3%)- Basidiomycota OTU-FUNG-08 SC-ITS-52 (MK590255) 1.2% Polyporaceae Trametes hirsuta Uncultured fungus (1/81) isolate BHI-F579a clone S98 (100%) (99.8%) *Number in parenthesis indicates the GenBank accession number **In parentheses: ratio between the number of clones of the corresponding OTU and the total number of clones ***The “-” indicates that the closest sequence corresponds to the one of the closest cultured representatives nutrient in closed or semi-closed environments, e.g. caves Nitrosocosmicus oleophilus strain MY3, a Candidatus organ- (Hathaway et al. 2014). ism retrieved from contaminated soils. No archaea with met- The library we reached was dominated by Thaumarchaeota abolic functions associated with sulphur cycling were detect- of the Nitrososphaeraceae family and was closely related to ed, showing that Thaumarchaeota were not directly involved genera Nitrososphaera and Nitrosopumilus. Members of the in the formation of gypsum efflorescences. Meng et al. (2017) Nitrososphaeraceae family are AOA that derive energy from showed a high occurrence of AOA closely related to genera the oxidation of ammonia to nitrite that can be a source of Nitrososphaera, Nitrosopumilus and Nitrosotalea on Angkor nitrogen for other organisms in close and semi-close environ- monuments. AOA may play an important role in the biodete- ments, such as the Sorcerer’s cave. Unlike rosy saline efflo- rioration of Angkor monuments by nitrogen cycling and nitric rescences found in many subterranean environments acid production. (reviewed by Piñar et al. 2014), the archaeal diversity associ- The Cordycipitaceae family was the most represented ated with gypsum efflorescences did not correspond to the fungal family in the cave. Fungi from this family are partic- halophilic archaeal genera Halococcus, Halobacterium and ularly known to have a significant negative impact on global Halalkalicoccus. Our most abundant Thaumarchaeota se- human and animal health (Menzies and Turkington 2015). quence showed similar percentage > 99% with Candidatus The most abundant OTU recovered in the library was related 1076 Ann Microbiol (2019) 69:1071–1078 to the entomophilous fungi, I. fumosorosea, a pathogen of 7 substrates in caves, such as plant debris and animal excre- insect orders (Humber and Hansen 2005). Entomophilous ments (Vanderwolf et al. 2013; Zhang et al. 2017). fungi are the most abundant fungal phylotypes in the Lascaux cave, where they may have an important ecological role (Bastian et al. 2009; Martin-Sanchez et al. 2015). Isaria Conclusion fumosorosea was identified as a facultative oligotrophic spe- cies able to adapt to particular conditions of caves by Jiang This study highlighted that gypsum efflorescences damaging et al. (2017). Martin-Sanchez et al. (2015) described the the walls and the engravings of the French Sorcerer’scave presence of entomophilous fungal phylotypes in caves, harbour several archaeal and fungal phylotypes. Analyses in- which may be due to cave-dwelling arthropods feeding on dicated the dominance of ammonia-oxidizing archaea belong- fungal mycelia and disseminating their spores in their excre- ing to the Nitrososphaeraceae archaeal family and the ento- ment during their movements, and by their bodies acting as mophilous member of the Cordycipitaceae fungal family. a support for the attachment of spores. Moreover, as Equivalent results were obtained from other cave environ- I. fumosorosea is used as a microbial insecticide to control ments not associated with gypsum efflorescences (Northup several pests around the world (Zimmermann 2007a, b, et al. 2003; Chelius and Moore 2004; Bastian et al. 2009; 2008; Gurulingappa et al. 2011), its abundance in the cave Legatzki et al. 2011; Miller et al. 2012; Ortiz et al. 2013; could also be the consequence of the air circulation into the Martin-Sanchez et al. 2015). With the exception of the 2 most cave. The second most abundant Cordycipitaceae phylotype abundant Cordycipitaceae phylotypes, most of the other ar- is distantly related to Engyodontium album a widespread chaeal and fungal phylotypes had similarity percentages be- species that can also be harmful to the health of humans low 97% with their closest cultured representatives. This and mammals (Siegel and Shadduck 1990; Goettel et al. showed that they correspond to yet uncultured microorgan- 2001; Nucle Tucker et al. 2004; Balasingham et al. 2011). isms. Effort to characterize the physiology of these microor- Interestingly, this phylotype has already been retrieved from ganisms is needed to gain more insight into their roles and areas of gypsum crystallization on the walls of a decorated behaviour in this particular ecosystem. shelter located only some kilometres from the Sorcerer’s cave (Lepinay et al. 2017). Acknowledgements We thank Jean-Max Touron (owner of the The second most represented OTU corresponded to an un- Sorcerer’s Cave) and Jean-Christophe Portais, who allowed us access cultivated Pseudocercosporella sp. from the and sample the cave. We thank Alexandre François and Mareva Sandou for the technical assistance. The final manuscript has been improved by Mycosphaerellaceae family. Mycosphaerellaceae include thou- BioMedES UK (www.biomedes.biz). sands of species of phytopathogenic fungi (Aguilera-Cogley et al. 2017). An OTU related to Pseudocercosporella sp. pre- Funding This study was funded, in part, by a grant from the Labex viously identified in the air of the carbonate cave located in Patrima and by a financial support of the “Ministere de la Culture et de China’s Shuanghe National Geographic Park is well adapted to la Communication”. This work was supported by the French minister of culture and communication and by the foundation PATRIMA. these oligotrophic conditions (Jiang et al. 2017). The next most represented OTU was the cultivated Compliance with ethical standards Cladosporium pulvericola species, already recognized as in- habitants of caves. Indeed, Pusz et al. (2015) showed that Conflict of interest The authors declare that they have no conflict of Cladosporium spp. were the dominant fungi of the internal interest. atmosphere of the Jarkowicka cave in Poland. All other ascomycotal OTUs had similar percentages < Research involving human participants and/or animals N/A 96% with any other existing Genbank sequence and thus Informed consent N/A corresponded to fungal species never previously recovered in any other environment. 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Characterization of the archaeal and fungal diversity associated with gypsum efflorescences on the walls of the decorated Sorcerer’s prehistoric cave

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Publisher
Springer Journals
Copyright
Copyright © 2019 by Università degli studi di Milano
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-019-01506-2
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See Article on Publisher Site

Abstract

Purpose This study focuses on analysis of the archaeal and fungal diversity associated with gypsum efflorescences damaging the walls of the Sorcerer’s prehistoric cave registered as a world cultural heritage site. Method Archaeal 16S rDNA and fungal internal transcribed spacer (ITS) clone libraries were constructed and analysed. Results Two thaumarchaeotal OTUs belonging to the Nitrososphaeraceae family dominated the archaeal community (100% of clones). Nitrososphaeraceae are obligate aerobic, chemolithoautotrophic organisms that derive their energy from the oxidation of ammonia and may contribute to primary productivity in the cave. Seven fungal OTUs belonging to Ascomycota and one belonging to Basidiomycota were present. The Cordycipitaceae family, mainly represented by entomophilous fungi, dominated the analysis (66.7% of clones). Conclusion We show that archaeal and fungal OTUs are associated with gypsum efflorescences damaging the walls of the Sorcerer’s cave. The role of these microorganisms in the deterioration of the walls of the cave remains to be determined. . . . . . Keywords Cave Gypsum Biodeterioration Archaea Fungi Diversity Introduction environmental factors that can alter the rock substrate accord- ing to their mineralogical composition and structure (Chamley Characterization of the microorganisms associated with bio- 2003;Saiz-Jimenez 2015). deterioration processes encountered in rock art caves and shel- Decorated caves and shelters are natural cavities subjected ters is needed (Saiz-Jimenez et al. 2011, 2012; Lepinay et al. to natural deterioration processes. Natural cavities are 2017, 2018), as also in mural painting from catacombs and nutrient-poor environments containing an abundant and di- tombs (Sanchez-Moral et al. 2005;Laizetal. 2009; verse sessile biomass (Barton and Jurado 2007;Lavoieet al. Vasanthakumara et al. 2013; Krakova et al. 2015). These 2010). Representatives of the 3 domains of life are recovered works of art are constantly threatened by decay resulting from in these subterranean environments. As in most environments, the interaction of biological and physicochemical the focus in the caves was on the diversity of bacteria. Among them, the most abundant and frequently retrieved phyla are as follows: Acidobacteria, Actinobacteria, Bacteroidetes, * Agnès Mihajlovski Cyanobacteria, Firmicutes, Nitrospirae and Proteobacteria agnes.mihajlovski@u-cergy.fr (Saiz-Jimenez 2015). Previous work in the Sorcerer’s cave showed that Actinobacteria, Alphaproteobacteria, Université de Cergy-Pontoise – ERRMECe-EA1391 – Site de Gammaproteobacteria, Bacteroidetes and Planctomycetes Neuville-sur-Oise, 1 rue Descartes, 95000 Neuville-sur-Oise were the main bacterial inhabitants of areas of salt efflores- Cedex, France cences damaging the walls (Lepinay et al. 2018). Laboratoire de Recherche des Monuments Historiques, Ministère de Actinobacteria was the most prevalent phylum, mainly repre- la Culture et de la Communication, Centre de Recherche sur la sented by members of the Pseudonocardiaceae family, data Conservation (CRC-USR 3224), Muséum national d’Histoire consistent with other findings (Barton and Jurado 2007; naturelle, CNRS, Sorbonne Universités, 29 Rue de Paris, 77420 Champs-sur-Marne, France Porca et al. 2012; Riquelme et al. 2015; Wu et al. 2015). 1072 Ann Microbiol (2019) 69:1071–1078 Conversely, a limited number of investigations were carried The gypsum deposits are located on the outer layer of the out on Archaea, particularly for sites of cultural heritage pres- stone and make it a kind of extremely fragile “molasses” ervation. However, many Archaea are adapted to nutrient- (Pigeaud et al. 2012). The same samples as those collected poor conditions and may contribute to nutrient cycling in ol- by Lepinay et al. (2018) in April 2015 (SCApr15) have been igotrophic environments through nitrogen fixation, analysed. Sterile scalpels were used to collect 3 efflorescence methanogenesis, sulphur oxidation, nitrification and ammonia areas (each ~ 2 cm ) and the underlying rock from a depth of oxidation (Ettenauer et al. 2010; Jarrell et al. 2011;Meng etal. ~ 2 mm, (Fig. 1b, c). Samples were mixed together and ho- 2016, 2017). To date, the most frequently recovered archaeal mogenized in sterile mortars. Samples were stored at − 20 °C phylum is Thaumarchaeota (Northup et al. 2003; Chelius and awaiting molecular analysis. Scanning electron microscopy Moore 2004;Legatzkiet al. 2011;Miller etal. 2012;Ortiz observations of the samples and elemental and mineralogical et al. 2013). Members of the Crenarchaeota have also been analyses of the corresponding saline efflorescences have been retrieved from steam vents and caves from volcanic national previously published (Lepinay et al. 2018). This indicated that parks in the USA (Benson et al. 2011) and Euryarchaeota in the formation of gypsum on the walls of the cave was mainly caves (Macalady et al. 2006; Reitschuler et al. 2014, 2016). In related to geochemical phenomena, efflorescences being addition to Bacteria and Archaea, fungi are very well repre- mainly composed of calcium, sulphur and oxygen, with addi- sented in cave environments due to their high rate of spore tional small amounts of nitrogen and carbon. production and air dispersion. They are important in the feed- ing strategies of cave fauna because they are important de- composers providing food for many organisms (Nováková DNA extraction and purification 2009). As reviewed by Vanderwolf et al. (2013) and recently completed by Zhang et al. (2017), > 1150 fungal species in DNA extraction was done as previously described (Zhou et al. 550 genera have been discovered in caves and mines world- 1996; Lepinay et al. 2018). The protocol combines the use of wide by 2017. The most frequently encountered genera (e.g. enzymatic (proteinase K) and chemical (CTAB and SDS) Aspergillus, Penicillium, Mucor, Fusarium, Trichoderma, components to ensure efficient cell lysis. Extracted, nucleic Cladosporium, Alternaria, Paecilomyces, Acremonium, acids were purified with a Power Biofilm™ DNA Isolation Engyodontium) are air-borne and soil-borne fungi that come Kit (MoBio) and were stored at − 20 °C until use. from outside the cave cavities due to air circulation (Saiz- Jimenez 2015). Several efforts have been made to characterize the diversity and distribution of fungi in caves, but there is PCR amplification of fungal ITS and archaeal 16S little information on taxa associated with specific niches, such rDNA genes as oligotrophic conditions and mineral formations (Saiz- Jimenez 2015). Fungal ITSs were amplified using primers ITS1-F and ITS4. We have focused on the French Sorcerer’s prehistoric cave A25-μl reaction comprising 20 ng of DNA, 100 μMof (“La grotte du Sorcier”). The cave undergoes alternations of each dNTP, 0.4 μM of each primer, 1× PCR buffer and humidification and desiccation phases which induce cycles of 1.5 U of Accusure DNA Polymerase (Bioline, France) was dissolution and recrystallization of the salts leading to efflo- performed. Cycling conditions were as follows: 5 min at rescence development on the walls that threaten the engrav- 95 °C, followed by 30 cycles of 20s at 95 °C, 15 s at ings (Pigeaud et al. 2012). This work completes the previously 54 °C and 1 min and 30 s at 68 °C, and a final extension published data corresponding to bacterial diversity following at 68 °C for 7 min. Amplicon was extracted from an agarose analysis of the archaeal and fungal diversity associated with gel after electrophoresis using the Nucleospin Gel and PCR saline efflorescences (Lepinay et al. 2018). clean up kit (Macherey Nagel, France). Archaeal 16S rRNA gene sequences were amplified using a semi-nested PCR approach. Archaeal universal primers 340F-pSTC (5′- Materials and methods CCTTCgCCgACTgACCCTAY-GGGGYGCASCAG-3′) and 1000R (5′-GGCCATGCACYWCYTCTC-3′) (Gantner Site description and sampling et al. 2011) were used as outer primers. Then, a semi- nested PCR was carried out using 340F-pSTC and 915R The Sorcerer’s cave, the Saint-Cirq cave, dug in a Turonian (5′-GTGCTCCCCCGCCAATTCCT-3′) (Stahl and Amann siliceous limestone, harbours several prehistoric engravings 1991). The PCR reaction was carried out as previously de- already described by different archaeologists (Fig. 1)(Delluc scribed, using an annealing temperature of 57 °C (Lepinay et al. 1987;Karnayet al. 1999; Pigeaud et al. 2012). et al. 2017). One microlitre of the first PCR was used in the Formation of gypsum efflorescences are present absolutely nested PCR, and an amplicon of ~ 580 bp was purified as everywhere in the cave, from the entrance to the bottom. described above. Ann Microbiol (2019) 69:1071–1078 1073 Fig. 1 The Sorcerer’s cave (Saint-Cirq-du-Bugue, Dordogne, France): a entrance (white arrow); b sampling areas 1, 2 and 3; c enlarged view of the sampling area no. 2 showing gypsum efflorescences. Credit photo: LRMH Clone libraries Good’s C index of coverage were calculated as described by Kemp and Aller (2004). Purified PCR products were inserted into the pSTC1.3 vector of the StabyCloning kit (Eurogentec, Belgium), and recombi- Nucleotide sequence accession numbers nant plasmids were electrotransformed in E. coli. Transformed cellswereplatedontoLBagarcontaining50 μg/ml ampicillin The nucleotide sequence data reported herein have been de- and incubated overnight at 37 °C. PCR amplifications of in- posited in the NCBI nucleotide sequence database under ac- serts using primers targeting the pSTC1.3 vectors: pSTC-F cession numbers MK212376 to MK212382, MK590246 to (5′-AATGCAGCGCGTTAGAA-3′) and pSTC-R (5′-CGCC MK590262 for fungi, and MK226534 to MK226535 and CGGTTTATTGAAA-3′), followed by agarose electrophore- MK607017 to MK607025 for Archaea. sis in 1.5% agarose gel, allowed to select clones presenting inserts at the expected sizes for archaeal 16S rDNA and ITS libraries, respectively. Restriction fragment length polymor- Results phism (RFLP) was used to screen these clones. Each PCR product was digested using the restriction enzymes, AluI and OTU richness and its reliability RsaI (Fermentas, France) for 16S rDNA amplicons or with the restriction enzyme BshfI (Fermentas, France) for ITS Analysis of RFLP patterns allowed grouping 178 archaeal amplicons. At least one clone per RFLP profile was partially 16S rDNA clones into 4 restriction patterns and 91 fungal sequenced (Eurofins Genomics, Germany). ITS clones into 10 restriction patterns. When the number of clone inside a RFLP pattern was higher than 2, several ran- Sequence analysis domly chosen clones were partially sequenced. After elimina- tion of chimeric and low quality sequences, 172 archaeal 16S Sequences were first checked using the software FinchTV rDNA clones and 81 fungal ITS clones remained in the banks. v1.4 (Geospiza, Inc.). Chimeric sequences identified with Pairwise alignments of randomly chosen clone sequences the Decipher program (Wright et al. 2012) were excluded. using 98% similarity as the cut-off allowed discrimination of ITS and 16S rDNA sequences were clustered into OTUs (op- 2 and 8 OTU in the archaeal 16S rDNA and the fungal ITS erational taxonomic unit) at an overlap percentage identity libraries, respectively (Tables 1 and 2). Estimated OTU richness (S-Chao1 estimator) was com- cut-off of 98% using ClustalW (Thompson et al. 1994). BLASTn was then used to compare DNA sequences with pared to the values of cloned libraries Kemp and Aller (2004). Predicted S-Chao1 values were of 2 and 8 phylotypes those in the GenBank database (Basic Local Alignment Search Tool, http://blast.ncbi.nih.gov/Blast.cgi). Sequences for the archaeal 16S rDNA and the fungal ITS libraries, re- with no significant similarities were excluded from the spectively. Calculation of Good’s C indices showed that most analysis. The affiliation of each cloned sequence to a genus of the sample diversity was represented in the clone libraries. or species was based on a similarity ≥ 95 or ≥ 98%, Indeed, coverage was 0.99 for both libraries, which indicates respectively, with the closest identified phylogenetic that the analysis of an increasing number of clones would have sequence in GenBank (Yarza et al. 2008). Archaeal sequences shown only a little further richness. were also classified using the Naïve Bayesian Classifier from the RDP project with a confidence threshold of 80% (https:// Archaeal diversity rdp.cme.msu.edu/classifier/classifier.jsp). We assessed whether DNA clone libraries were large enough to be Only the Thaumarchaeota phylum was recovered in the ar- representative of OTU richness from samples. S-Chao1 and chaeal 16S rDNA clone library (Table 1); this phylum was 1074 Ann Microbiol (2019) 69:1071–1078 Table 1 Assignment of archaeal 16S rDNA sequences to their closest cultured representative OTU Representative clones* % of RDP family*** Closest cultured Closest Candidatus clones** representative organism (percentage (percentage similarity) similarity) Thaumarchaeota OTU-ARC-01 SC-Arc-09 (MK607017); 99.4% Nitrososphaeraceae Nitrososphaera Candidatus SC-Arc-30 (MK226534); (171/172) (100%) viennensis Nitrosocosmicus SC-Arc-47 (MK607019); strain EN76 oleophilus strain MY3 SC-Arc-64 (MK607018); (95.8 to 96.3%) (99.4 to 100%) SC-Arc-69 (MK607020); SC-Arc-95 (MK607021); SC-Arc-B2-12 (MK607022); SC-Arc-B2-35 (MK607023); SC-Arc-B2-56 (MK607024); SC-Arc-B2-74 (MK607025) OTU-ARC-02 SC-Arc-54 (MK226535) 0.6% Nitrososphaeraceae Nitrosopumilus - (1/172) (100%) maritimus SCM1 strain SCM1 (96.2%) *Number in brackets indicates the GenBank accession number **In parentheses: ratio between the number of clones of the corresponding OTU and the total number of clones ***Bacterial family determined using the Naïve Bayesian Classifier from the RDP project website. The confidence threshold is 80% represented by 2 phylotypes belonging to the archaeal family, family closely related to Trametes hirsuta isolate BHI-F579a Nitrososphaeraceae. One of them dominated the library and (1.2% of clones, 99.8% SS). presented as closest cultured representative: Nitrososphaera viennensis strain EN76 (99.4% of clones, 95.8 to 96.3% se- quence similarity, SS); and as closest Candidatus organism: Discussion Candidatus Nitrosocosmicus oleophilus strain MY3 (99.4 to 100% SS). The second one presented as closest cultured rep- This study aims to improve the understanding of biological resentative: Nitrosopumilus maritimus SCM1 strain SCM1 phenomena associated with the formation of gypsum efflores- (0.6% of clones, 96.2% SS). cences on the walls of the French Sorcerer’s prehistoric cave. We have previously shown that these areas of gypsum efflo- Fungal diversity rescences harbour a variety of bacterial communities and a dense subjacent biofilm (Lepinay et al. 2018). We continued ITS sequences mainly belonged to the phylum Ascomycota the investigations of the microbial communities living in these (Table 2), with most represented family being the zones of efflorescence by analysing Archaea and fungi. Cordycipitaceae (66.7% of clones). Cordycipitaceae were rep- Concerning Archaea, all the clones belonged to the phylum resented by 2 phylotypes, one being affiliated with Isaria Thaumarchaeota, a phylum, previously referred to fumosorosea strain BCMU PF01 (51.9% of clones, 99.8 to “mesophilic Crenarchaeota”, being proposed by Brochier- 100% SS), and the other related to Engyodontium album iso- Armanet et al. (2008). Thaumarchaeota have been detected late MC_A31 (14.8% of clones, 95.5 to 95.6% SS). The sec- by molecular studies in many subterranean habitats (Takei ond most abundant OTU belonged to the Mycosphaerellaceae et al. 2001; Northup et al. 2003;CheliusandMoore 2004; family, being distantly related to Pseudocercosporella sp. Legatzki et al. 2011; Miller et al. 2012; Ortiz et al. 2013; 09CT02 (18.5% of clones and 92.9 to 93.1% SS). The fourth Barton et al. 2014;Anda et al. 2017) and in many different most abundant OTU belonged to the Cladosporiaceae family environments, such as seawater (e.g. Church et al. 2003), and corresponded to Cladosporium pulvericola CPC: 22403 freshwater sediments (e.g. Schleper et al. 1997) and soils (6.2% of clones, 100% SS). Three other ascomycotal families (e.g. Ochsenreiter et al. 2003). All known ammonia- were recovered in the library, each representing 2.5% of clones oxidizing archaea (AOA) belong to Thaumarchaeota and that had sequence identities between 92 and 95% with their Thaumarchaeota are receiving much attention regarding their closest relative in Genbank, namely the Ophiocordycipitaceae, ability to proliferate in environments with low nutrient avail- Diatrypaceae and Teratosphaeriaceae. Finally, Basidiomycota ability (Martens-Habbena et al. 2009; Pester et al. 2011;Ortiz were represented by one clone belonging to the Polyporaceae et al. 2014). This phylum can represent the main source of Ann Microbiol (2019) 69:1071–1078 1075 Table 2 Assignment of fungal ITS sequences to their closest cultured representatives and closest sequence matches OTU Representative clones* % of Fungal family Closest cultured Closest clones** representative sequence match (percentage (percentage similarity) similarity)*** Ascomycota OTU-FUNG-01 SC-ITS-04 (MK590248); SC-ITS-14 51. 9% Cordycipitaceae Isaria fumosorosea - (MK590250); SC-ITS-28 (MK590251); (42/81) strain BCMU SC-ITS-38 (MK212376); SC-ITS-42 PF01 (99.8 to (MK590252); SC-ITS-48 (MK590253); 100%) SC-ITS-49 (MK590254); SC-ITS-58 (MK590258); SC-ITS-74 (MK590260) OTU-FUNG-02 SC-ITS-02 (MK590246); SC-ITS-50 18.5% Mycosphaerellaceae Acrodontium Pseudocercosporella (MK212378); SC-ITS-76 (MK590261) (15/81) crateriforme sp. 09CT02 strain CPC 11509 (92.9 to 93.1%) (91.3to91.5%) OTU-FUNG-03 SC-ITS-39 (MK212377); SC-ITS-57 14.8% Cordycipitaceae Engyodontium Uncultured fungus (MK590257); SC-ITS-79 (MK590262) (12/81) album isolate clone MC_A31 RS-Apr-ITS37 (95.5to95.6%) (99.9 to 100%) OTU-FUNG-04 SC-ITS-13 (MK590249); SC-ITS-55 6.2% Cladosporiaceae Cladosporium (MK590256) (5/81) pulvericola CPC: 22403 (100%) OTU-FUNG-05 SC-ITS-30 (MK212379) 2.5% Ophiocordycipitaceae Thyronectria - (2/81) asturiensis strain MA3 (92.5%) OTU-FUNG-06 SC-ITS-44 (MK212380) 2.5% Diatrypaceae Eutypa consobrina - (2/81) culture-collection CBS: 122678 (95.1%) OTU-FUNG-07 SC-ITS-33 (MK212381) 2.5% Teratosphaeriaceae Teratosphaeria Uncultured fungus (2/81) knoxdaviesii strain clone CBS 122898 RS-Apr-ITS18 (94.7%) (99.3%)- Basidiomycota OTU-FUNG-08 SC-ITS-52 (MK590255) 1.2% Polyporaceae Trametes hirsuta Uncultured fungus (1/81) isolate BHI-F579a clone S98 (100%) (99.8%) *Number in parenthesis indicates the GenBank accession number **In parentheses: ratio between the number of clones of the corresponding OTU and the total number of clones ***The “-” indicates that the closest sequence corresponds to the one of the closest cultured representatives nutrient in closed or semi-closed environments, e.g. caves Nitrosocosmicus oleophilus strain MY3, a Candidatus organ- (Hathaway et al. 2014). ism retrieved from contaminated soils. No archaea with met- The library we reached was dominated by Thaumarchaeota abolic functions associated with sulphur cycling were detect- of the Nitrososphaeraceae family and was closely related to ed, showing that Thaumarchaeota were not directly involved genera Nitrososphaera and Nitrosopumilus. Members of the in the formation of gypsum efflorescences. Meng et al. (2017) Nitrososphaeraceae family are AOA that derive energy from showed a high occurrence of AOA closely related to genera the oxidation of ammonia to nitrite that can be a source of Nitrososphaera, Nitrosopumilus and Nitrosotalea on Angkor nitrogen for other organisms in close and semi-close environ- monuments. AOA may play an important role in the biodete- ments, such as the Sorcerer’s cave. Unlike rosy saline efflo- rioration of Angkor monuments by nitrogen cycling and nitric rescences found in many subterranean environments acid production. (reviewed by Piñar et al. 2014), the archaeal diversity associ- The Cordycipitaceae family was the most represented ated with gypsum efflorescences did not correspond to the fungal family in the cave. Fungi from this family are partic- halophilic archaeal genera Halococcus, Halobacterium and ularly known to have a significant negative impact on global Halalkalicoccus. Our most abundant Thaumarchaeota se- human and animal health (Menzies and Turkington 2015). quence showed similar percentage > 99% with Candidatus The most abundant OTU recovered in the library was related 1076 Ann Microbiol (2019) 69:1071–1078 to the entomophilous fungi, I. fumosorosea, a pathogen of 7 substrates in caves, such as plant debris and animal excre- insect orders (Humber and Hansen 2005). Entomophilous ments (Vanderwolf et al. 2013; Zhang et al. 2017). fungi are the most abundant fungal phylotypes in the Lascaux cave, where they may have an important ecological role (Bastian et al. 2009; Martin-Sanchez et al. 2015). Isaria Conclusion fumosorosea was identified as a facultative oligotrophic spe- cies able to adapt to particular conditions of caves by Jiang This study highlighted that gypsum efflorescences damaging et al. (2017). Martin-Sanchez et al. (2015) described the the walls and the engravings of the French Sorcerer’scave presence of entomophilous fungal phylotypes in caves, harbour several archaeal and fungal phylotypes. Analyses in- which may be due to cave-dwelling arthropods feeding on dicated the dominance of ammonia-oxidizing archaea belong- fungal mycelia and disseminating their spores in their excre- ing to the Nitrososphaeraceae archaeal family and the ento- ment during their movements, and by their bodies acting as mophilous member of the Cordycipitaceae fungal family. a support for the attachment of spores. Moreover, as Equivalent results were obtained from other cave environ- I. fumosorosea is used as a microbial insecticide to control ments not associated with gypsum efflorescences (Northup several pests around the world (Zimmermann 2007a, b, et al. 2003; Chelius and Moore 2004; Bastian et al. 2009; 2008; Gurulingappa et al. 2011), its abundance in the cave Legatzki et al. 2011; Miller et al. 2012; Ortiz et al. 2013; could also be the consequence of the air circulation into the Martin-Sanchez et al. 2015). With the exception of the 2 most cave. The second most abundant Cordycipitaceae phylotype abundant Cordycipitaceae phylotypes, most of the other ar- is distantly related to Engyodontium album a widespread chaeal and fungal phylotypes had similarity percentages be- species that can also be harmful to the health of humans low 97% with their closest cultured representatives. This and mammals (Siegel and Shadduck 1990; Goettel et al. showed that they correspond to yet uncultured microorgan- 2001; Nucle Tucker et al. 2004; Balasingham et al. 2011). isms. Effort to characterize the physiology of these microor- Interestingly, this phylotype has already been retrieved from ganisms is needed to gain more insight into their roles and areas of gypsum crystallization on the walls of a decorated behaviour in this particular ecosystem. shelter located only some kilometres from the Sorcerer’s cave (Lepinay et al. 2017). Acknowledgements We thank Jean-Max Touron (owner of the The second most represented OTU corresponded to an un- Sorcerer’s Cave) and Jean-Christophe Portais, who allowed us access cultivated Pseudocercosporella sp. from the and sample the cave. We thank Alexandre François and Mareva Sandou for the technical assistance. The final manuscript has been improved by Mycosphaerellaceae family. Mycosphaerellaceae include thou- BioMedES UK (www.biomedes.biz). sands of species of phytopathogenic fungi (Aguilera-Cogley et al. 2017). An OTU related to Pseudocercosporella sp. pre- Funding This study was funded, in part, by a grant from the Labex viously identified in the air of the carbonate cave located in Patrima and by a financial support of the “Ministere de la Culture et de China’s Shuanghe National Geographic Park is well adapted to la Communication”. This work was supported by the French minister of culture and communication and by the foundation PATRIMA. these oligotrophic conditions (Jiang et al. 2017). The next most represented OTU was the cultivated Compliance with ethical standards Cladosporium pulvericola species, already recognized as in- habitants of caves. Indeed, Pusz et al. (2015) showed that Conflict of interest The authors declare that they have no conflict of Cladosporium spp. were the dominant fungi of the internal interest. atmosphere of the Jarkowicka cave in Poland. All other ascomycotal OTUs had similar percentages < Research involving human participants and/or animals N/A 96% with any other existing Genbank sequence and thus Informed consent N/A corresponded to fungal species never previously recovered in any other environment. 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