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Département Systématique & Evolution, Unité Taxonomie-Collections, Muséum national d’histoire naturelle, CP 39, 57 rue Cuvier, 75231 Paris Cedex 05, France
Faisl Bousta
Geneviève Orial
Laboratoire de recherche des monuments historiques, Pôle microbiologie, 29 rue de Paris, 77420 Champs sur Marne, France
Corinne Cruaud
Arnaud Couloux
Genoscope. Centre National de Sequençage. 2, rue Gaston Crémieux, CP5706, 91057 Evry Cedex, France2
Marie-France Roquebert
Département Systématique & Evolution, Unité Taxonomie-Collections, Muséum national d’histoire naturelle, CP 39, 57 rue Cuvier, 75231 Paris Cedex 05, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A major fungal invasion was discovered in the prehistoric painted cave of Lascaux in France in Sep 2001. At least three species of the Fusarium solani complex were isolated and identified with a portion of the translation elongation factor 1
gene (EF-1), a portion of the nuclear large subunit rDNA (LSU) and nuclear ribosomal intergenic spacer region (ITS). This study represents the first time that Fusarium species have been reported from a cave containing prehistoric paintings. Significant interspecific molecular variability was observed, suggesting that there might have been repeated introduction of the species, possibly carried by water from soils above the cave.
Key words: Fusarium, identification, mural paintings, prehistoric cave art
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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It was discovered in 1940 and shortly after World War II construction improved access to the cave. The entrance was enlarged considerably and floors lowered to aid visitor access. In 1955 the first indication of deterioration of the paintings was observed. Research has shown that carbon dioxide generated by the visitors (sometimes more than 1000/d) acidified the increased water vapor that when condensed corroded the calcareous rock face resulting in the loss of pigments (Vouvé et al 1983
). Artificial ventilation was installed in 1958 to reduce carbon dioxide. The temperature was fixed at 14 C, 1.5 C above the mean, and electric lighting was installed for use during visiting hours. Within a few years green patches developed on the wall, which signaled the presence of an alga, Bracteacoccus minor (Lefevre 1974). Repetitive treatments with formaldehyde have been necessary to stop its development. The cave was closed in 1963 and a replica was constructed nearby to accommodate visitors and to maintain the cave and its art.
In 1966 new installations were made for climatic regulation of the cave and the temperature was lowered to its natural temperature, 12.5 C. Vouvé et al (1983) noted that the cave’s climatic environment was stable in 1979.
Early in 2000 the air conditioning and percolating water recovery systems were replaced. In addition to changes associated with opening the cave for construction, intensive rain fell during this period resulting in the accumulation of water and mud at the entrance of the Great Hall of the Bulls. Significant thermic and hygrometric disturbances followed.
In Mar 2000 bacterial mats and fungal colonies developed in the machinery room. In summer 2001 a white fungal growth was discernible on the floor and the banks of the Great Hall of the Bulls. Fusarium appeared as the main contaminant, but there also were colonies of Chrysosporium, Gliocladium, Gliomastix, Paecilomyces, Trichoderma and Verticillium. The natural ecosystem of the cave is poorly known because it was opened to the public soon after its discovery. Only partial records of the original microbial flora exist. For example Pseudomonas fluorescens was replaced by Bacillus brevis after an antibiotic treatment in 1964 (Billy and Chalvignac 1976).
Although an exhaustive microbiological investigation of the "green disease" of Lascaux was not conducted, Lefevre (1974) mentioned an abundance of fungal spores and bacteria, principally Actinomycetes and cyanobacteria, numerous green algae, diatoms, amoebae, ciliates, rotifers and nematodes. Other studies provide more complete data, but these represent caves without paintings (Dickson and Kirk 1976, Seigle-Murandi et al 1977, Northup et al 1994, Rutherford and Huang 1994, Cunningham et al 1995).
There are few reports on microbial colonization of mural paintings from prehistoric caves, and most focus on bacteria. Cultured-independent methods based on 16S rDNA analysis from Spanish paleolithic caves have revealed complex bacterial communities with biocorrosive potential (Gurtner et al 2000, Schabereiter-Gurtner et al 2002a, b, 2003, 2004, Zimmermann et al 2005) and demonstrate a significantly more diverse group of microbes (including undescribed species) compared to previous culture-based reports (Groth and Saiz-Jimenez 1999, Groth et al 2001, Laiz et al 1999). However comparison of bacterial communities from caves extensively visited in the past (Tito Bustillo, Spain) with those from caves never opened to the public (La Garma, Spain) led to the conclusion that bacteria are mainly intrinsic inhabitants of the caves that are affected little, or not at all, by anthropogenic activities (Schabereiter-Gurtner et al 2004).
This study is the first to investigate a sudden fungal colonization within a prehistoric painted cave with the main objective of analyzing the phylogenetic diversity of the main colonizers, Fusarium, to address the question of the origin of the contamination.
Fusarium isolates were identified by BLAST comparison of partial translation elongation factor 1 gene (EF-1), partial nuclear large subunit rDNA (LSU) and nuclear ribosomal intergenic spacer region (ITS) sequences to the FUSARIUM-ID database (Geiser 2004). Their relationships within the Nectria haematococca-Fusarium solani complex were assessed with reference to the phylogenies proposed by O’Donnell (2000), Aoki et al (2003, 2005) and Zhang et al (2006).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) in 2000 (Hall of the Bulls only), 2004 (Mar, Sep) and 2005 ( Jan).
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) Petri dishes, which incubated at 24 C for 5 d. Fusarium colonies were picked under the stereomicroscope, transferred to MA (2% malt-extract agar) augmented with 0.01% chloramphenicol to eliminate bacteria. Morphological identification were based on SNA cultures (synthetischer nährstoffarmer agar, Nirenberg 1976) incubated at 24 C for 7 d. The strains (TABLE I) are maintained at the LCP collection (Laboratoire Cryptogamie, Muséum national d’histoire naturelle, Paris).
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PCR was performed in 50 µL reactions, with 25 µL (50–100 ng) of template DNA, 1.25 units of Taq DNA polymerase (Q-BIOgene, Illkirch, France), 5 µL of 10x Taq DNA Polymerase buffer, 5 µL of 50% glycerol, 2 µL of 5 mM dNTPs (Eurogentec, Seraing, Belgium) and 2 µL of each 10 µM primer. The oligonucleotide primer set EF-1H and EF-2T from O’Donnell (1998) was used to amplify a ~700 bp portion of the EF-1 gene. ITS 4/ITS 5 (White et al 1990) and LROR/LR6 (Vilgalys, Duke University) were used to amplify portions of the rDNA. Amplifications were performed on a Perkin Elmer Cetus thermal cycler model 2400 with these parameters: a 4 min step at 94 C, followed by 30 cycles of 30 s at 94 C, 30 s at 55 C (TEF, ITS), 50 C (LSU) and 40 s at 72 C and then a final 10 min extension step at 72 C.
Ava II and Rsa I restriction enzymes were used for a preliminary evaluation of the genomic variability of the Fusarium strains. Aliquots of 10 µL of the amplified DNAs were digested with 2 units of Ava II (Qbiogène, Illkirch, France) or Rsa I (Fermentas/Euromedex, Mundolsheim, France) following the manufacturers’ directions. After digestion of the EF-1 amplicons in the PCR buffer, restriction fragments and a molecular size marker, the "100 bp Ladder" (Eurogentec), were separated on 2% "small fragments" agarose gels (Eurogentec), stained with ethidium bromide (10 µg/µL) and photographed under UV light.
DNA sequencing was performed on both strands with the ABI PRISM TM Dye terminator cycle sequencing ready reaction kit (Applied Biosystems) with the amplification primer set. Sequencing assays were analysed on an automated ABI PRISM 377 DNA sequencer (GenoScreen, Lille-France and Genoscope-Centre National de Sequençage, Evry-France). The nucleotide sequences were aligned with the BioEdit program.
Restriction profiles analysis.— –
Restriction patterns obtained experimentally were compared to the theoretical patterns of representative strains, deduced from the sequences with the BioEdit program. Results for all the isolates examined are reported (TABLE I
).
Phylogenetic analysis.— –
Twenty-one sequences (EF-1, LSU, ITS) were newly obtained and deposited at GenBank (accession Nos. DQ792839
[GenBank]
–DQ792845
[GenBank]
, EF579651
[GenBank]
–EF579657
[GenBank]
, EF583844
[GenBank]
–EF583850
[GenBank]
). They were aligned with sequences obtained from K. O’Donnell (2000) and from D. Geiser (pers comm) with Fusarium illudens and Nectria plagianthi as outgroup. Preliminary alignments were generated with GeneDoc (Nicholas et al 1997) and optimized manually.
Maximum parsimony trees were calculated with a heuristic search in PAUP version 4.0b10. Clade stability was evaluated with 10 000 bootstrap replications with random taxon addition sequence and TBR branch swapping (FIG. 2).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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amplicon suggested the existence of three groups (TABLE I).
Representatives of each of the three PCR-RFLP groups were sequenced (Sp1, isolates 2-1, 2-4, 2-8, 2-10, 3-1, 5-1, 5-5, 6-1, and 9-2; Sp2, isolates 7-4 and 8-1; Sp3, isolates 7-5, 8-3, 9-1, 10-3 and 04.4553) within the three regions, EF-1, LSU and ITS. LSU and ITS sequencing distributed the isolates in three groups identical to those obtained by PCR-RFLP (with an exception in Sp3, where ITS sequences diverged at one position, separating 7.5 and 10.3 isolates from the others). More variability was observed within EF-1 sequences, showing seven haplotypes. Three haplotypes were found within Sp1, and were labeled Sp11 (2-1, 2-4, 2-8, 5-1 and 6-1*), Sp12 (3-1* = 2-10) and Sp13 (5-5 = 9-2*) (*deposited in GenBank). The last two differed respectively from Sp11 at one and five nucleotide positions. Two haplotypes were found in each of the two other taxa, Sp2 and Sp3. Sp21 (7-4*) and Sp22 (8-1*) differed in four positions while Sp31 (9-1 = 04.4553*) and Sp32 (10-3 = 8-3 = 7-5*) differed in three positions. These sequences were used as a query to BLAST the "FUSARIUM-ID v. 1.0" database (Geiser 2004) accessed at http://fusarium.cbio.psu.edu. All matched members of the Fusarium solani complex (FSSC). The sequences from Lascaux and those reported as the closest from the BLAST option were used for their phylogenetic assessment (FIG. 2).
Phylogenetic analysis of the combined dataset, representing 1563 bp (483 for ITS, 604 for EF-1 and 476 for LSU) yielded 66 most parsimonious trees (MPT) of 532 steps. One of the MPT is shown (FIG. 2). All Lascaux isolates nested with 99% bootstrap support within clade 3, as defined in O’Donnell (2000) and Aoki et al (2003). Sp1, Sp2 and Sp3 isolates segregated in three distinct clades strongly supported (more than 97%) by bootstrap analysis. Sp1 isolates clustered within the f. sp. pisi/f. sp. mori/f. sp. robiniae/f. sp. cucurbitae and homo-thallic NNRL 22389 monophyletic clade with 100% bootstrap support. Internal nodes are not strongly supported, as shown in O’Donnell (2000). Sp2 isolates shared sequences with environmental (soil: s373, s231, soil debris: s572, s231, plants: s1578, s1408, s1383, NRRL25083, s1710) and clinical isolates (NRRL32810, NRRL32488, NRRL32763, NRRL 32842) and the two haplotypes are grouped in a clade with a high bootstrap support of 97%. Sp3 is related to environmental isolates of an other clade (among them s1233, recovered from subterranean termites, NRRL22823 from Eustoma russelliana, s338 from air and s616 from soil) with a high bootstrap support of 99%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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gene, a portion of the LSU and the ITS rDNA to identify the Fusarium strains isolated from the Lascaux cave 2002–2005. All strains were identified as members of the Fusarium solani species complex, actually comprising more than 45 phylogenetic and/or biological species (Zhang et al 2006). Curiously no other Fusarium species was recovered in the cave. This is the first time that members of the FSSC have been reported as responsible for a major colonization of a Paleolithic painted cave. FSSC are mainly plant pathogens, causing root and stem rots (Domsch et al 1983). They survive in soils after harvest or after the decay of plants that they have parasitized. Members of the FSSC are also human and animal pathogens, increasingly implicated as the causative agents of human mycoses (Zhang et al 2006) and mostly causing keratitis (de Hoog et al 2000). Isolates that cause infections in both humans and plants are common in the environment (Zhang et al 2006). FSSC also are reported from food and indoor environments and often found in wounds (Pitt and Hocking 1999, Samson et al 2002). Thus in Lascaux FSSC might have entered by two possible ways, either percolating from the upper soils by water or brought from outside soils by people, small animals or running water, or by both routes.
With reference to the phylogenies of the Fusarium solani complex published by O’Donnell (2000) and Zhang et al (2006) we identified at least three distinct phylogenetic species in the cave, which were resolved in seven haplotypes with the EF-1 sequences, all nesting in clade 3. We have retained the informal names Sp1, 2 and 3 rather than formally describing these species pending further taxonomic studies of this group. Members of clade 3 frequently are isolated from soil and as saprotrophs in other environments and include many human pathogens sharing multi-locus haplotypes with environmental isolates, indicating widespread ability to cause infection in this diverse species complex (Zhang et al 2006). Whereas members of clades 1 and 2 are known exclusively from diseased or dead plants (Zhang et al 2006). Sp1 was related to plant isolates of the formae speciales pisi, mori, robiniae and cucurbitae (of the mating population V), cucurbitae isolates being members of group 1 of clinical isolates identified by Zhang et al (2006). Sp2 and Sp3 were related to unnamed taxa from clinical and/or environmental isolates.
Distribution of the three groups in the cave was not uniform (FIG. 1). Sp1 and Sp3 were found in the Great Hall of the Bulls and in the Chamber of Engravings, whereas Sp2 and Sp3 were found in the Lateral Passage. The galleries farthest from the entrance each harbor only one species: Sp1 in the Main Gallery and Sp3 in the Moonmilk Gallery.
The same EF-1 haplotypes were found in different places in the cave over several years (FIG. 1, TABLE I). Sp11, found in 2004 in the Great Hall of the Bulls (2-1, 2-4 and 2-8) and in the Main Gallery (5-1) was recovered at the same spot in the Main Gallery 4 mo later (6-1), Sp13 was found in the Main Gallery in Sep 2004 (5-5) and in the Chamber of Engravings in Jan 2005 (9-2). Sp31 was isolated in 2002 from the Great Hall of the Bulls (04.4553) and recovered 3 y later in the Chamber of Engravings (9-1). Sp32 was isolated in the Lateral Passage (7-5) and in the Moonmilk Gallery (10-3) in Mar 2004 and recovered in the Lateral Passage in Jan 2005 (8-3). The most probable cause of this distribution could be the transport of spores (by humans or small animals) from the entrance of the cave, possibly washed down from outside by the extensive rain in Mar 2000. Thus the highest diversity would be expected in the Great Hall of the Bulls near the entrance. Admittedly the results are not completely convincing in this respect because Sp13, Sp32 and Sp2 were not observed at this location. However the topology of the site is irregular with many microhabitats, which made thorough sampling particularly difficult.
We found representatives of six genera in the cave: Chrysosporium, Gliocladium, Gliomastix, Paecilomyces, Trichoderma and Verticillium. Comparison of our findings with those of others is difficult because no exhaustive microbiological inventory has been made of Lascaux. Moreover research on the microbial colonization of art cave is scant and mainly focused on bacteria (Gurtner et al 2000, Schabereiter-Gurtner et al 2002a, b, 2003, 2004, Zimmermann et al 2005). However fungi have colonized unpainted caves (Dickson and Kirk 1976, Seigle-Murandi et al 1977, Northup et al 1994, Rutherford and Huang 1994, Cunningham et al 1995). Fusarium species were mentioned in all these papers. Fusarium sp. was isolated from condensation-corrosion deposits on the wall of Lechuguilla Cave, along with Aspergillus, Cylindrocladium, Epicoccum, Mucor, Paecilomyces, Penicillium, Rhizopus and sterile isolates (Cunningham et al 1995). The fungal diversity and density was much higher when mud and pools were considered (including representatives of additional genera: Bipolaris, Cladosporium, Cylindrocladium, Gliocladium, Monilia, Sepedonium, Ulocladium [Northup et al 1994]). All fungi identified were common telluric, epigeous species, mostly associated with decaying matter in soils.
Fungi cause weathering and biotransformation of rocks and minerals in subaerial or subsoil environments through biomechanical and biochemical processes (Budford et al 2003a, b). Most fungi mentioned by these authors are ubiquitous, rapidly growing saprophytes, including Alternaria, Aspergillus, Cladosporium, Paecilomyces, Penicillium, Trichoderma, Ulocladium and Verticillium. In addition to these common fungi, microcolonial rock fungi have been discovered in extreme environmental conditions on desert rocks (Staley et al 1982). Their role in the blackening and deterioration of stone monuments and natural rock surfaces, even in temperate regions, is particularly significant (Taylor-George et al 1983, Palmer et al 1987, Wollenzien et al 1995, Gorbushina and Krumbein 2000). However to our knowledge microcolonial fungi, which are adapted to drastic changes in temperature and hydration have not been reported from caves with constant temperature and humidity. Lithobiotic communities often colonize mineral surfaces where they form biofilms (Decho 2002). In the Lechuguilla Caves, fungi may have been observed in the network covering calcite concretions but they seem to be dependent on the chemolithoautotrophic bacteria (Cunningham et al 1995). Biofilms are under study in Lascaux (Laboratoire de recherche des monuments historiques).
Once introduced into the cave, spores of the FSSC might have found climatic conditions and nutrients adequate for germination and for subsequent mycelial development observed in fall 2001. Water, which is essential for germination, is abundant in the cave and, the temperature is 12.5 C year-round. A marl bed above the cave stops rainwater, which accumulates in a thin layer. Water percolates through microfissures in the marl and drips from the roof of the cave, especially in the Hall of the Bulls, resulting in a high and constant relative humidity. Although the cave temperature is low for the growth of FSSC (optimum range 27–31 C according to Domsch et al 1993) germination has been observed at temperatures as low as 12 C (Chi and Hanson 1964), and it is likely that the cave temperature rose when it was open early in 2000.
The nutrition of Fusarium in caves is not well understood. Caves are thought to be nutrient-poor biotopes with the absence of light preventing the growth of plants and phototrophic microorganisms. The food chain in caves is thought to be based mainly on detritus (dead organic material, bat guano), although weak concentration of organic compounds may be introduced from groundwater infiltrations. In Virginia Caves the distribution of filamentous fungi was shown to be highly dependant on nonuniform deposits of organic matter, whereas the distribution of bacteria was more uniform (Dickson and Kirk 1976). In the Lechuguilla Caves an interdependency was observed between bacteria and fungi with chemolithoautotrophic bacteria supporting populations of chemoheterotrophic bacteria and many varieties of fungi (Cunningham et al 1995).
In conclusion this study points to human activity as the probable cause in the alteration of the cave environment and to the introduction of fungi. These two actions likely led the contamination observed in Lascaux. We also have demonstrated that the major contaminant, members of the FSSC, still is present in the cave 5 y after the alterations. The persistence of these fungi raises questions as to how the microflora changes over time. The uncomfortable conclusion is that caves are complex, poorly known ecosystems that may be fragile and that restoring a cave to its initial condition, before human activity, may prove extremely difficult.
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ACKNOWLEDGMENTS |
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and J. Taylor for kindly reviewing the manuscript before submission. |
FOOTNOTES |
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1 Corresponding author. E-mail: jdupont{at}mnhn.fr
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