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Use of Biocides for the Control of Fungal Outbreaks in Subterranean Environments: The Case of the Lascaux Cave in France Pedro M. Martin-Sanchez,† Alena Nováková,‡ Fabiola Bastian,§ Claude Alabouvette,§ and Cesareo Saiz-Jimenez†,* †

Instituto de Recursos Naturales y Agrobiologia de Sevilla, IRNAS-CSIC, Apartado 1052, 41080 Sevilla, Spain Institute of Soil Biology, Biology Centre AS CR, v.v.i., Na Sádkách 7, CZ-370 05 Č eské Budějovice, Czech Republic § UMR INRA-Université de Bourgogne, Microbiologie du Sol et de l’Environment, BP 86510, 21065 Dijon Cedex, France ‡

S Supporting Information *

ABSTRACT: The Lascaux Cave in France suffered an outbreak of the fungus Fusarium solani in 2001. Biocides were applied for three years to control this outbreak. Four months after the initial biocide application, a new outbreak appeared in the form of black stains that progressively invaded the cave. The black stains on the ceiling and passage banks were so evident by 2007 that they became one of the cave’s major problems. Therefore, biocides were used again in 2008. The present study investigated the fungal communities associated with the black stains and the effectiveness of the biocides applied, by using cloning, denaturing gradient gel electrophoresis, and culture-dependent methods. A novel species, Ochroconis lascauxensis, was the most abundant fungus in samples collected between 2007 and 2008, and the biocides applied were not effective in eliminating this fungus; on the contrary, they appeared to increase the fungal diversity. The fungal communities represented in the samples collected in 2010 were quite different from those collected in 2008 and 2009: the major OTUs corresponded to black yeasts belonging to the Herpotrichiellaceae family. The origin and evolution of these microorganisms are probably linked to the intensive biocide treatments and to the anthropogenic changes introduced by cave management.

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INTRODUCTION In the last few decades, many caves depicting rock art have been discovered.1,2 Some of them were protected from visitors, but many others were adapted for tourism. European show caves are suffering the impact of massive tourism, with some caves receiving up to 500 000 visitors/year or more. This interrupted the delicate cave microclimate. The visits also represent a considerable input of organic matter and detritus, changing the cave ecosystem. Today, many caves are examples of ecologically disturbed sites3−5 and the curators are attempting to mitigate the deleterious effects of past policies of inappropriate control. Of all cave microorganisms, fungi are by far the most worrying, due to their high rate of spore dissemination and colonization ability when organic carbon is available. These abilities have led to serious outbreaks in some show caves, but little data are available on the origin and evolution of most fungal colonizations. Recently, a number of papers have reported the presence of fungi in caves of France, Spain and the U.S.5−7 Lascaux Cave, France, suffered a high level of anthropogenic impacts from its discovery in 1940 until 1963, when the cave was closed to the public after the invasion of a green alga that flourished on the walls due to the continuous lighting system. During that time, the cave underwent several adaptations and structural work, as well as the installation of a climate control © 2012 American Chemical Society

system to avoid water condensation and facilitate as many visitors as possible.8 In July 2001, the first evidence of a fungal outbreak of Fusarium solani appeared in the Lascaux Cave. Dupont et al.9 studied the colonization of the cave by a F. solani species complex. F. solani covered the ground and walls with dense white mycelial overgrowth. Fungal biomass, water percolation from the forest topsoil and the application of biocides, such as benzalkonium chloride (BC), between 2001 and 2004, increased the level of organic carbon content in the cave. Years of BC treatments in the Lascaux Cave have selected for a mixed population of Ralstonia and Pseudomonas, as well as freeliving amoebae, both highly resistant to BC.10,11 After the outbreak of F. solani, sparse, patchily distributed black stains appeared in the cave in December 2001, four months after the treatment with BC. In 2007, the black stains on the ceiling, walls, and passage banks were so obvious that they became, and remain one of the cave’s major problems.8 Two new species of the genus Ochroconis, O. lascauxensis, and O. anomala were isolated from black stains and described by Received: Revised: Accepted: Published: 3762

November 14, 2011 February 9, 2012 March 1, 2012 March 1, 2012 dx.doi.org/10.1021/es2040625 | Environ. Sci. Technol. 2012, 46, 3762−3770

Environmental Science & Technology

Article

Table 1. Samples Used in This Study analysis performed samples A08

features

sampling date

extraction amplification

DGGE profiles

passageway, left side

26/08/2008

DNA

X

location

clone libraries

no. of clonesa

isolation of melanized fungi b

passageway, left side

26/08/2008

DNA

X

X

81

A09 C09 M1c

black stain before cleaning and biocide treatments black stain before cleaning and biocide treatments A08 after treatments C08 after treatments black stain

passageway, left side passageway, left side passageway, right side

DNA DNA DNA/RNA

X X X

X X

104 104/156

M3c

pink-violet stain

passageway, right side

DNA/RNA

X

M6c

black stain forming circular ring black stain in area with constant humidity

Upper Apse, left side

17/02/2009 17/02/2009 21/09/2010; 01/02/2011 21/09/2010; 01/02/2011 21/09/2010; 01/02/2011 21/09/2010; 01/02/2011

DNA/RNA

X

X

104/104

X

DNA/RNA

X

X

104/104

X

C08

M8c

Great Hall of Bulls, entrance to Painted Gallery, right side

b

b

X X

a Total number of clones sequenced, but fewer sequences were used in the analysis due to our stringent quality checks. bSome Ochroconis lascauxensis strains were isolated from these samples in a previous study (Martin-Sanchez et al.12). cTwo samples were collected from the same stains in different dates: September 2010 for molecular analysis and February 2011 for fungal isolations.

Martin-Sanchez et al.12 The descriptions are based on the morphology and the phylogenetic relationships of two of its gene regions (ITS and RPB2). While O. lascauxensis is one of the major contributors to the black stain formation and is widely distributed all over the cave, the distribution of O. anomala currently seems restricted. Here we describe the microbiology of the black stains that threaten the rock art paintings in the Lascaux Cave. The aims of this study were to identify the fungal communities associated with the black stains from the Lascaux Cave (especially the metabolically active communities), to characterize the evolution of these communities in recent years, and to determine the effect of biocide treatments on the origin and development of the fungal communities. For this purpose, we decided to use both culture-independent methods (DNA and RNA cloning libraries and DGGE profiling studies) and culture-dependent methods to isolate the most representative melanized fungal species.

An additional four stain samples were taken in September 2010, three of them from three different-looking black stains in the Passageway, the Apse, and the Great Hall of Bulls (M1, M6, and M8, respectively), and one from a pink-violet stain from the Passageway (M3), which was included in this sampling for comparison. These samples were subjected to a DNA-RNA comparative analysis in order to identify the metabolically active fungi. Five samples collected from the black stains were selected to further study the fungal communities by construction of clone libraries (Table 1; Figure S1 of the Supporting Information, SI). The fungal communities of all samples were characterized by denaturing gradient gel electrophoresis (DGGE). In February 2011, a new sampling from stains M1, M3, M6, and M8, was performed for the isolation of melanized fungi. Further details on material and methods are included in the SI. The ITS nucleotide sequences from clone libraries and isolated strains were submitted to GenBank under accession nos. HE605212-HE605286 and HE575202.

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MATERIALS AND METHODS To study the fungi associated with the black stains on the walls and ceiling of the Lascaux Cave, twelve samples of stains or sediments were collected between 2008 and 2011. Their features and the different analyses performed are detailed in Table 1. From each sampling point, several Eppendorf tubes with 100−200 mg of sample were collected using a sterile scalpel. All tubes were kept on ice during transport to the laboratory. The samples for isolation of fungi were processed immediately, and those for molecular characterization were stored at −80 °C for later processing. To assess the effectiveness of the antifungal treatments, we sampled two black stains from the Passageway on 26 August 2008 before any treatment (A08 and C08). The area was then subjected to cleaning and treatments with the biocide Devor Mousse (5% concentration), which contained a mixture of quaternary ammonium (10% to 25% benzalkonium chloride, 10% to 25% miristalkonium chloride) and 2.5% of the fungicide 2-octyl-2H-isothiazol-3-one, and additional applications of 3% Parmetol DF12 (solution of isothiazoline derivatives). On 17 February 2009, we collected samples from the two treated areas (A09 and C09). Strains of O. lascauxensis had been isolated from these two sampling points in a previous study.12

RESULTS Clone Libraries. To study the fungal communities from the black stains, we constructed eight DNA and RNA clone libraries (Table 2). From 861 sequenced clones, a total of 664 high-quality ITS sequences were used for the final analysis. The DNA and RNA libraries were independently analyzed for each sample. The sequences were clustered in Operational Taxonomic Units (OTUs) by DOTUR at evolutionary distances of 5% because at smaller distances the number of OTUs increased considerably without providing any useful taxonomic information. The additional OTUs were singletons with the same BLAST identification as that of existing OTUs, which may be due to sequencing errors or other potential artifacts. In Table 2, only the OTUs with clone abundance higher than 5% were included. Rarefaction curves are shown in Figure S2 of the SI. Only the rarefaction curve of sample C08, collected before treatments in 2008, reached a clear plateau, confirming community composition coverage. The rarefaction curves from the rest of the samples did not reach a plateau. This shows that the fungal communities from the black stains of the Lascaux Cave are quite diverse (see especially the RNA analysis of sample M8).

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dx.doi.org/10.1021/es2040625 | Environ. Sci. Technol. 2012, 46, 3762−3770


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Table 2. Summary of the ITS Clones Representative of Major OTUs identified in Lascaux Cave stains DNAb clones

GenBank accession

closest relative sequence from GenBank (Similarity %)

no. clones

%

no. clones

%

Ochroconis lascauxensis (100)

65

97

Ochroconis lascauxensis (100) Aspergillus versicolor (100) Cladosporium cladosporioides (100) Trichoderma sp. (99)

46 12 10 8

52.3 13.6 11.4 9.1

Capronia coronata (= Exophiala sp.) (90) Acremonium nepalense (96)

37 17

48.7 22.4

13 22

14.1 23.9

Exophiala moniliae (96)

7

9.2

27

29.3

Chrysosporium pseudomerdarium (97)

5

6.6

9

9.8

Thysanorea papuana (91)

5

6.6

9

9.8

Ochroconis lascauxensis (100) Capronia coronata (= Exophiala sp.) (90) Acremonium nepalense (96)

32 31 9

35.2 34.1 9.9

20 2

24.7 2.5

Exophiala moniliae (96)

4

4.4

20

24.7

Thysanorea papuana (91) Physarum melleum (85); low coverage 35%c

3 3

3.3 3.3

16 11

19.7 13.6

Capronia kleinmondensis (82) Uncultured eukaryote (99) Prunus avium (100); low coverage 13%d Apedinella radians (90); low coverage 29%c Capronia coronata (= Exophiala sp.) (99)

34 16 9 9 7

41.5 19.5 11 11 8.5

15

17.2

37 5 2

42.5 5.7 2.3

taxonomic identificationa

C08DNA analysis (67 clones) C08−1 HE605223 Ascomycota (Mitosporic); Ochroconis C09DNA analysis (88 clones) C09−2 HE605225 Ascomycota (Mitosporic); Ochroconis C09−63 HE605226 Ascomycota; Trichocomaceae; Aspergillus C09−64 HE605227 Ascomycota; Davidiellaceae; Cladosporium C09−9 HE605228 Ascomycota; Hypocreaceae; Trichoderma M1DNA/RNA analysis (76/92 clones) M1D-1 HE605235 Ascomycota; Herpotrichiellaceae M1D-13 HE605236 Ascomycota; Plectosphaerellaceae; Acremonium M1D-53 HE605237 Ascomycota; Herpotrichiellaceae; Exophiala M1D-17 HE605238 Ascomycota; Eurotiomycetidae; Chrysosporium M1D-64 HE605239 Ascomycota; Herpotrichiellaceae M6DNA/RNA analysis (91/81 clones) M6D-7 HE605251 Ascomycota (Mitosporic); Ochroconis M6D-44 HE605252 Ascomycota; Herpotrichiellaceae M6D-32 HE605253 Ascomycota; Plectosphaerellaceae; Acremonium M6D-66 HE605254 Ascomycota; Herpotrichiellaceae; Exophiala M6D-39 HE605255 Ascomycota; Herpotrichiellaceae M6D-6 HE605256 Eukaryota; Amoebozoa M8DNA/RNA analysis (82/87 clones) M8D-2 HE605268 Ascomycota; Herpotrichiellaceae M8D-1 HE605269 Eukaryota M8D-7 HE605271 Eukaryota M8D-28 HE605272 Eukaryota M8D-12 HE605273 Ascomycota; Herpotrichiellaceae; Exophiala

RNAb

a

When possible, sequences were assigned to genus and species. However, in some cases only was possible an assignation to higher taxonomic levels. Only were included the OTUs with percentages of clones higher than 5% cCoverage located into 5.8S gen. dCoverage located in the beginning of 28S gen. b

Black Stains in 2010. The Lascaux Cave is a dynamic ecosystem with rapidly changing fungal communities. The fungal communities identified from the stain samples collected in 2010 were indeed quite different from those observed in 2008 and 2009, as shown by their DGGE profiles (Figure 1b) and clone libraries (Table 2). In this approach, we investigated the fungi present in the samples (DNA), as well as those that were metabolically active (RNA). DNA analysis provides information from currently active fungi, as well as fungi that are no longer active, which complicates the accurate identification of the current fungal spectrum. However, RNA analysis provides a picture of the fungal activity in the cave at the time of sampling. Some DGGE bands that were common to different samples were sequenced and compared with those from isolated strains (Table 3) and representative clones of OTUs. All studied black stains showed, in both DNA and RNA analyses, a band with a similar migration point (band 1; Figure 1b). One of these bands, from M8-RNA sample, was identified as a species of the family Herpotrichiellaceae (100% identity with clone M8D-2; Table 2). The black stains M1 and M6 and the pink-violet stain (M3 sample) showed bands with the same migration point (band 2). Two of them were excised from M3RNA and M6-RNA samples, respectively, and were identified as

However, enough clones were studied to reliably characterize the majority of the fungal communities. Antifungal Treatments. To halt the dissemination of the black stains, biocide treatments had been intensively applied in the cave since 2001, with little success, as described by Bastian et al.3,10,11 To confirm the negative effect of biocide treatments, we performed DGGE analysis. The profiles of the fungal communities from stains collected before treatments (A08 and C08) coincide with the O. lascauxensis profile, showing that this fungus is dominant in these stains (Figure 1a). This fact was confirmed by the results of the clone library from sample C08 (Table 2), in which the major OTU corresponded to O. lascauxensis (97% of clones). After cleaning and biocide treatments (A09 and C09), the number of clones of O. lascauxensis was reduced but still represented the main OTU (52.3%) in the clone library from sample C09. Interestingly, after biocide treatments, the fungal communities were more diverse, as shown by the DGGE profiles. In C09, 10 OTUs were obtained (percentage of clones: 96.6% for Ascomycota, 3.4% for Basidiomycota) which corresponded to the frequent airborne genera Aspergillus, Cladosporium, Trichoderma, and Alternaria, also found in other caves.13,14 DGGE patterns from representative clones of OTUs belonging to these fungal genera also occurred in the profiles from samples A09 and C09 (Figure 1a). 3764



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Figure 1. ITS DGGE profiles of environmental samples, representative clones of the major OTUs and reference isolates from the Lascaux Cave. (a) Analysis of samples taken in 2008−09, before and after treatments. Reference isolates are Ochroconis lascauxensis (Ochr. 1 and Ochr. 2). Representative clones from C09: O. lascauxensis (C09−2), Aspergillus sp. (C09−63), Trichoderma sp. (C09−9), Cladosporium sp. (C09−64), Alternaria sp. (C09−1), Rhodotorula sp. (C09−14), and Gymnascella sp. (C09−48). (b) Analysis of stain samples taken in 2010. Numbers indicate bands that were identified by sequencing: Herpotrichiellaceae sp. (1); Exophiala moniliae (2); and Acremonium nepalense (3). (c) Analysis of the M1 black stain. Representative clones: Herpotrichiellaceae sp. (M1D-1), Acremonium nepalense (M1D-13), Exophiala moniliae (M1D-53), Chrysosporium sp. (M1D-17), and Herpotrichiellaceae spp. (M1D-64 and M1R-10). *Asterisk indicates samples analyzed by cloning.

Exophiala moniliae (100% identity with strain LX M1−4 and clone M6D-66; Tables 2 and 3). Finally, band 3, from black stain samples (M1 and M6) in both DNA and RNA analyses, appeared at the bottom of the gel. Band 3 was identified as Acremonium nepalense (100% identity with all strains and clones belonging to this species). DGGE analysis thus illustrates changes in the composition of the fungal communities of the Lascaux Cave, which suggest the need to focus on metabolically active black yeasts belonging to the Herpotrichiellaceae family and on the fungus A. nepalense, both of which have been active in the last year. The clone libraries from the black stain M1, located in the Passageway, yielded nine OTUs for DNA analysis (percentage of clones: 96.1% for Ascomycota, 2.6% for Basidiomycota, 1.3% for Zygomycota) and 12 OTUs for RNA analysis (percentage of clones: 94.6% for Ascomycota, 1.1% for Basidiomycota, 1.1% for Zygomycota, 3.2% for other eukaryotes). The majority of the clones corresponded to the family Herpotrichiellaceae (64.5% of DNA clones and 56.5% of RNA clones), comprising five metabolically active OTUs; the three most abundant are detailed in Table 2. Moreover, two Herpotrichiellaceae spp.

with only 1−2 clones, for which the representative clones were M1R-10 and M1R-3, were detected among the omitted OTUs. The fungus A. nepalense was also detected as being metabolically active in this sample, representing 22.4% of DNA clones and 23.9% of RNA clones. However, no O. lascauxensis clones were detected. Also significant was the detection of a Chrysosporium sp. (6.6% of DNA clones and 9.8% of RNA clones), whose closest relative sequence belongs to C. pseudomerdarium (97% similarity). The DGGE patterns from representative clones of these major OTUs are coincident with the profiles from the DNA-RNA analysis of M1, allowing the identification of each band (Figure 1c). The clone libraries from the black stain M6 located in the Apse yielded 11 OTUs for DNA analysis, consisting of Ascomycota (90%), Basidiomycota (2.2%), and other eukaryotes (7.8%), and 13 OTUs for RNA analysis, consisting of Ascomycota (76.6%), Basidiomycota (3.7%), and other eukaryotes (19.7%) (Table 2). Most of the metabolically active fungi corresponded again to the family Herpotrichiellaceae (41.8% of DNA clones and 71.6% of RNA clones), forming five OTUs. 3765



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Table 3. Isolation of Melanized Fungi from Stain Samples Collected in February 2011 Ochroconis spp.a

black yeastsa

Acremonium spp.a

+++

+

+++

M3 (pink-violet)

+

+++

+

M6 (black)

+

++

+++

M8 (black)

+

++

+

stain samples M1 (black)

strains−molecular identificationb LX LX LX LX LX LX LX LX LX LX LX LX

M1−1 Acremonium nepalense 96% M1−3 Ochroconis anomala 100% M1−4 Exophiala moniliae 96% M3−2 Ochroconis lascauxensis 100% M3−3 Exophiala castellanii 94% M6−3 Ochroconis lascauxensis 100% M6−4 Exophiala castellanii 94% M6−10 Acremonium nepalense 96% M6−11 Capronia coronata 90% M8−1 Acremonium nepalense 96%, M8−5 Ochroconis lascauxensis 100% M8−6 Capronia coronata 99%

Genbank accessionc HE605212 HE575202 HE605213 HE605214 HE605215 HE605216 HE605217 HE605218 HE605219 HE605220 HE605221 HE605222

a

Detection based on colony morphology. The abundance was estimated as absent (−), weak presence (+), significant presence (++) or abundant presence (+++). bIdentification based on comparison of ITS sequences with GenBank by BLAST algorithm (NCBI). The closest relative sequences from GenBank and their similarity percentage are detailed. cGenBank accession number corresponding to ITS sequences of strains isolated from Lascaux Cave.

and several species of black yeasts. The relative abundance of each group and the strains that were identified according to their morphological and molecular characteristics are shown in Table 3. The isolates belonging to the genus Acremonium were identified as A. nepalense, with 96% similarity, based on the ITS region sequence, and their morphological features were consistent with those described for this species.15 These colonies, while initially hyaline, became melanized within a few days of incubation in ECA medium (Figure S3a of the SI). However, the melanization of the colonies grown on MEA and PDA was weak or negligible (Figure S3b,c of the SI). Among the black yeasts isolated, we found four species belonging to the Herpotrichiellaceae family, as follows: Exophiala castellanii (96% similarity), with morphology consistent with that described for this species.16 Abundant colonies of this species were isolated from a black stain (M6) and from a pink-violet stain (M3) (Figure S3d−f of the SI). Exophiala sp. showed high similarity (99%) with C. coronata (anamorph: Exophiala sp.) and Exophiala angulospora. The morphology did not match that of E. angulospora17 but showed some similarities with that of E. jeanselmei, although the data were not conclusive. This species was abundant in the black stain M8 (Figure S3g−i of the SI). Exophiala moniliae (96%) showed morphology consistent with that described for this species.16 Only one strain of this species was isolated from the black stain M1 (Figure S3j,k of the SI). Herpotrichiellaceae sp. (90% similarity with C. coronata). A morphological study was not conclusive at the genus level. Only one strain of this species was isolated from the black stain M6 (Figure S3l,m of the SI). Phylogenetic Relationships of Black Yeasts. The phylogenetic relationship between isolated strains and representative OTUs belonging to the Herpotrichiellaceae family showed eight different species associated with the black stains from the Lascaux Cave (Figure 2). All of them were metabolically active, as indicated by data from the clone libraries (Table 2). To elucidate their identity, ITS sequences from GenBank for the closest related species were included in this analysis, selecting type strains whenever possible. The black stains M1 and M6 showed a similar fungal composition. Three species in particular were widely distributed in both samples: Herpotrichiellaceae sp. 1, whose OTUs exhibited

The three most abundant OTUs were the same species that were identified in M1. Two additional species were detected, each represented by one clone in the RNA analysis: Exophiala sp. (clone M6R-57), whose closest relative sequence belongs to E. castellanii, and Herpotrichiellaceae sp. (clone M6R-96). The major OTU identified by M6-DNA analysis (35.2% of clones) was O. lascauxensis, with 99−100% similarity with the isolated strains.12 However, this OTU was not metabolically active because there were no clones found in the RNA analysis, which likely indicates the presence of conidia of this fungus, quite common in the cave in past years. A. nepalense also appeared in M6 sample, although in significantly lower numbers than in M1 (9.9% of DNA clones and 2.5% of RNA clones). Interestingly, nonfungal eukaryotes appeared in this sample: one metabolically active OTU was identified as Amoebozoa (3.3% of DNA clones and 13.6% of RNA clones). The presence of Amoebozoa was previously reported by Bastian et al.11 Clone libraries from the black stain M8 located in the Great Hall of Bulls yielded 10 OTUs (percentage of clones: 53.6% Ascomycota, 1.2% unidentified fungi, and 45.2% other eukaryotes) for DNA analysis and 15 OTUs (percentage of clones: 22.9% Ascomycota, 1.2% Basidiomycota, 10.4% unidentified fungi, and 65.5% other eukaryotes) for RNA analysis (Table 3). In this case, the majority of the clones corresponded to six OTUs assigned to unknown eukaryotes, which were present in DNA and/or RNA analyses. The major metabolically active OTU was an unknown eukaryote, which represented 11% of DNA clones and 42.5% of RNA clones. However, 41.5% of DNA clones and 17.2% of RNA clones belonged to an OTU identified as Herpotrichiellaceae, identical to clone M1R-3, whose closest related sequence in GenBank is from Capronia kleinmondensis (82%). Another OTU representing 8.5% of DNA clones and 2.3% of RNA clones corresponded to the isolated member of the genus Exophiala whose closest related sequence is from Capronia coronata (99%). No O. lascauxensis or A. nepalense clones were detected in this sample. Black Stains in 2011. Owing to the prevalence of clones belonging to the family Herpotrichiellaceae, in February 2011 we sampled the same spots previously sampled in September 2010 for molecular studies, this time for the purpose of isolating strains. On the basis of colony morphology, the melanized fungi isolated from the four stained samples could be classified into three main groups: Ochroconis spp., Acremonium spp., 3766



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Figure 2. Phylogenetic tree derived from the ITS1−5.8S−ITS2 regions of rRNA gene sequences showing the relationships between isolated strains and OTU representative clones from the black stains in the Lascaux Cave. Some reference strains from GenBank are also included. For each OTU, the percentage of clones identified by DNA-RNA analyses is shown in parentheses. The tree was constructed using the Neighbor-Joining method applying the Kimura 2 parameter model. All positions containing gaps and missing data were eliminated. There were a total of 460 positions in the final data set. The tree was rooted with Phialophora sessilis CBS 243.85 T as outgroup. The tree was bootstrapped 1000 times and values above 65% are indicated at nodes. These same nodes were also recovered using Maximum-Likelihood and Maximum-Parsimony treeing algorithms. Bar, 0.02 substitutions per nucleotide position.

clone M6R-57 was identified as E. castellanii, as it was identical to the strains isolated from M6 and M3 samples (LX M3−3 and LX M6−4). E. castellanii forms a homogeneous clade with the type strain CBS 158.58, confirming their identification (Figure 2). Exophiala sp. 2 was detected in the M1-RNA clone library (only two clones, of which M1R-10 is the representative clone); its sequence showed a 99% similarity with Exophiala sp. strain JCM 16194. Both form a homogeneous clade with the type strains E. salmonis and E. pisciphila. Finally, Exophiala sp. 3 was detected in M6-RNA sample (only one clone, M6R-96), and their closest related sequence was the strain CPC 12173 of Exophiala sp., showing 84% similarity. Metabolism of Selected Strains. To compare the data reported in the literature18−25 with the behavior of the fungi isolated, several strains (O. lascauxensis, O. anomala, A. nepalense, E. moniliae, E. castellanii, Herpotrichiellaceae sp., and Exophiala sp.) were grown on different carbon sources (vanillic acid, sodium p-toluene sulfonate, and toluene). Vanillic acid was selected as lignin phenol, sodium p-toluene sulfonate as detergent product and

99−100% similarity with the isolated strain LX M6−11; E. moniliae, whose OTUs exhibited 98−100% similarity with the strain LX M1−4 and which form a homogeneous clade with the type strain CBS 520.76, confirming the identity of the species; and Herpotrichiellaceae sp. 2, composed of two identical OTUs, most closely related to Thysanorea papuana (91%). Despite the abundance of Herpotrichiellaceae sp. 2 in both samples, it could not be isolated. The sample M8 had two main species: Herpotrichiellaceae sp. 3, which was also found in M1-RNA (only the clone M1R-3) and formed an independent clade clearly separated from the closest species, C. kleinmondensis (82%) and Capronia leucadendri (81%); and Exophiala sp. 1, whose OTU was identical to strain LX M8−6 and was very closely related to C. coronata and E. angulospora (99% in both cases). Despite the morphological similarity of Exophiala sp. 1 with E. jeanselmei, this species could not be identified as such, as shown in the phylogenetic tree. Three other Herpotrichiellaceae species were found at lower abundance, based on the clone library data. Representative 3767



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ease; numerous colonies were isolated, although their percentages in clone libraries were low. However, for E. moniliae and Herpotrichiellaceae sp. the opposite occurs, despite having the highest percentages in the libraries, only one colony of each species could be isolated. The other four species were not cultivable, so it was impossible to elucidate their identification, two of them with significant representation in clone libraries. The other key fungal species in the black stains was identified as A. nepalense. It was isolated from the four 2010 samples, being particularly abundant in the black stains M1 and M6. The abundance of this fungus is consistent with the DGGE profiles and clone libraries (Figure 1b; Table 2). In previous samplings, this fungus has been repeatedly isolated from black stains, but we have not paid much attention to this because it always developed white colonies on the media used (MEA, PDA, and DRBC). However, when we used the ECA medium for isolation, this fungus developed melanized colonies within a few days taking a characteristic gray-green color (Figure S3a-c of the SI). Kiyuna et al.26 have described different species of the genus Acremonium associated to black stains on Japanese murals paintings. The main OTUs detected in the sample M8 corresponded to unknown species of eukaryotes (41.5% and 59.6% for DNA and RNA). Although the group of black yeasts was still abundant (50% for DNA and 19.5% for RNA), the majority of species were different from those present in M1 and M6 samples. These remarkable differences in the composition of communities may be due to the characteristic constant humidity of this area. Impact of Biocides on the Cave. BC was used for controlling the F. solani outbreak. BC has no lasting effect, was not tested previously for such a wide spectrum of fungal species present in the cave, and its efficacy is questionable in a cave system (biodegradation, presence of dense biofilms, adsorption of BC to rocks, sediments and clays, etc.). The extensive biocide treatments between 2001 and 2004 resulted in a decrease of F. solani and other fungi, because this fungus represented only 1% of the clones in 11 samples investigated all over the cave, which were collected between April 2006 and January 2007.6 However, the scarce presence of F. solani and the high number of other phylotypes encountered in the cave demonstrates that controlling fungal dispersion in the cave was not a matter of only one fungal species (F. solani), and the consequence was an increasing in biodiversity. This biodiversity is further supported by the appearance in the past few years of Ochroconis spp., A. nepalense, and the members of the Herpotrichiellaceae family. A few genera of black yeasts, mainly belonging to the Herpotrichiellaceae family,27,28 have been reported to cause black stains on monuments. Additionally, more often than any other fungal group, these organisms, particularly the species of Exophiala, have been found in environments rich in aromatic compounds, in which they assimilate alkylbenzenes as the sole source of carbon.18−22 These reports described the Herpotrichiellaceae family as the most important alkylbenzenedegrading fungal group worldwide, based on the number of isolates and the diversity of habitats. In addition, some Exophiala strains assimilated 54 of 63 aromatic compounds tested, including a high number of lignin phenols.23 Black yeasts were also abundant inside washing machines and bathrooms.24,25 Exophiala and Scolecobasidium (=Ochroconis) are thought to utilize detergents as nutrients, and the addition of sodium dodecylbenzenesulfonate promotes their growth on

toluene as alkylbenzene. The data (Figure S4 of the SI) showed that vanillic acid was an excellent carbon source providing better growth than the positive control with sucrose in all cases; growth in a medium without a carbon source was similar to the medium with sodium p-toluene sulfonate in all cases. Regarding the growth in an atmosphere of toluene (Figure S5 of the SI), O. lascauxensis, A. nepalense, E. moniliae, and Herpotrichiellaceae sp. showed similar growth in a medium with sucrose or toluene but O. anomala and Exophiala sp. exhibited a better growth on sucrose than on toluene.

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DISCUSSION Composition of Black Stains. Clone libraries constructed to characterize the fungal communities of the untreated and biocide treated samples from the black stains showed a different clone composition for each type of sample (Table 2). The untreated sample contained a very large proportion of O. lascauxensis (97% of clones) and a comparatively low proportion of Pochonia sp. (3%). These data indicated that O. lascauxensis was actively participating in the colonization of the cave and in the formation of the black stains during the years 2007 and 2008, as observed by Martin-Sanchez et al.12 The clone library constructed from the treated black stain exhibited remarkable changes with respect to the initial library. In fact, a much more diverse library was obtained. The proportion of O. lascauxensis was reduced but still represented half of the clones, which puts into question the effectiveness of the applied treatments, as suggested by Bastian et al.10 In areas treated with biocides, when O. lascauxensis is partially removed and other bacteria and fungi killed, there is a rapid succession in the ecological niche, which is occupied by airborne fungal spores. The rest of the library is composed by species of the genera Aspergillus, Trichoderma, Cladosporium, Alternaria, etc., frequently found in cave air,4,13,14 so they could colonize quickly the treated areas as saprophytic fungi specialized in the decomposition of organic matter. Dead microbial biomass and degradation products from biocides likely trigged this secondary colonization and conducted to an increasing in microbial diversity. The DGGE profiles obtained from the four samples included in this test (Figure 1a) allowed us to extrapolate this clone library data from samples C08 and C09 to other samples collected under the same conditions from a nearby area (A08 and A09). According to the molecular characterization of black stains collected in September 2010, O. lascauxensis was only detected in the M6 library. A 35.2% of clones from DNA analysis were identified as belonging to this species, but was not metabolically active because no clone was found in the RNA analysis. However, Ochroconis spp. were recovered from all stain samples by culture methods, which means that O. lascauxensis is still present in the stains and air from Lascaux14 (as conidia) and it is easily isolated using the specific media and conditions. However, this does not necessarily indicate that in the last year this fungus was the most active species in black stains formation, but their conidia were distributed in the stains all over the cave. The most abundant fungi corresponded to black yeasts species affiliated to the Herpotrichiellaceae family, with a percentage of clones for DNA analysis of 64.5% for M1, 41.8% for M6, and 50% for M8 samples. For RNA analysis, the percentages were of 56.5% for M1, 71.6% for M6, and 19.5% for M8 (Table 2). Four of the eight species identified at the molecular level could be isolated in culture media. E. castellanii and Exophiala sp. 1 was recovered in culture media with relative 3768



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culture media.24 Alkylbenzenesulphonates and alkylbenzenes are ubiquitous components of commercial laundry detergents and surfactants.29 The quaternary ammonium biocides used in the Lascaux Cave are based on an alkylbenzene structure. In addition, alkylbenzenes are quite common in soils and sediments, and aliphatic and aromatic compounds were found in the molecular structure of humic substances, mainly composed of humic and fulvic acids.30,31 Dissolved organic carbon and fulvic acids from soils also contain alkylbenzenes, and it has been reported that aquatic and soil humic substances contain lignin phenols and alkylbenzenes of up to 22 carbon atoms.32,33 The main sources of lignin phenols and alkylbenzenes in soils and humic substances were discussed and summarized by Saiz-Jimenez.29,32,34 Percolating water and dissolved organic carbon that may enter the cave from the forest topsoil are presumably also rich in these compounds, as reported earlier for a similar cave.35 Therefore, Ochroconis spp., A. nepalense and the members of the Herpotrichiellaceae family found in the cave can metabolize a variety of organic carbon nutrients containing lignin phenols and alkylbenzenes, and their specialization in the selective degradation of these structures19,23 could explain their abundance. Considering the features of the Herpotrichiellaceae family discussed here, the origin and dominance of black yeasts, Ochroconis spp., and A. nepalense in the Lascaux Cave is likely related both to the previous intensive biocide treatments and to the introduction of dissolved organic carbon (e.g., lignin phenols) by percolating water, particularly during periods of heavy rain.

A solution to the black stain problem in Lascaux Cave is not easy, nor is it easy to ask for remedial strategies when the cave was extremely disturbed by continuous years of treatments with aggressive biocides, chemicals, and occasional construction/ repairing works. Although this work provides essential information regarding the composition and evolution of an irreversibly damaged ecosystem, further research targeting the newly detected species of black yeasts and A. nepalense seems necessary for investigating the factors triggering the fungal outbreaks. This knowledge is important for discerning the behavior of cave microorganisms and discovering whether preventive and sustainable control of the continuous blackening of the cave walls is possible.

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ASSOCIATED CONTENT

S Supporting Information *

This section presents all supporting figures and tables referenced in the text as well as detailed description of additional methods. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +34 954624909; fax: +34 954624002; e-mail: saiz@ irnase.csic.es. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Culture and Communication, France, project “Ecologie microbienne de la grotte de Lascaux” and the Spanish Ministry of Science and ́ para la Innovation, “Programa de investigación en tecnologias valoración y conservación del patrimonio cultural”, TCP CSD2007-00058. The collaboration and facilities of Lascaux staff, restoration team and DRAC Aquitaine are acknowledged.

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CONCLUSIONS This work shows that a complete description of the fungal biodiversity of the black stains from the Lascaux Cave required the combination of molecular techniques and culture-dependent methods. In this study, the most practical approach to establish the composition of the fungal communities was to study DNA-RNA clone libraries. However, using this approach, the groups of fungi that were not abundant may not have been detected, as happened with O. lascauxensis and A. nepalense in some samples; however, these strains could be detected by culture methods. In contrast, the predominant fungal species, as identified by molecular methods, may have been difficult to detect by culturing only, as observed for the black yeast species. Significant research has been conducted on the effects of BC on prokaryotes,36 but very little has been done for eukaryotes.37 The sole use of BC during three years in a subterranean environment was a great error that marked the fate of the microbial communities in Lascaux Cave. To avoid microbial resistance, a second biocide or a rotation of different biocides should have been included in the treatment protocol,38−41 which was not the case. In general, a careful preliminary study on the possible advantages and disadvantages of applying biocides in subterranean environments is required. Furthermore, any treatment should be planned after testing new molecules in laboratory as well as under field conditions in order to find an effective biocide treatment, although this type of treatment is not recommended for caves with rock art paintings, because both the presence of complex biofilms, inaccessible to biocides and the fast inactivation of the biocides due to biotic10,36,39 and abiotic42 factors are expected, and the treatment, if not ineffective, can cause further and irreversible damage to the ecosystem.

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