Stochastic and deterministic assembly processes of microbial communities in relation to natural attenuation of black stains in Lascaux Cave.

Community assembly processes are complex and understanding them represents a challenge in microbial ecology. Here, we used Lascaux Cave as a stable, confined environment to quantify the importance of stochastic vs deterministic processes during microbial community dynamics across the three domains of life in relation to an anthropogenic disturbance that had resulted in the side-by-side occurrence of a resistant community (unstained limestone), an impacted community (present in black stains), and a resilient community (attenuated stains). Metabarcoding data showed that the microbial communities of attenuated stains, black stains, and unstained surfaces differed, with attenuated stains being in an intermediate position. We found four scenarios to explain community response to disturbance in stable conditions for the three domains of life. Specifically, we proposed the existence of a fourth, not-documented yet scenario that concerns the always-rare microbial taxa, where stochastic processes predominate even after disturbance but are replaced by deterministic processes during post-disturbance recovery. This suggests a major role of always-rare taxa in resilience, perhaps because they might provide key functions required for ecosystem recovery.IMPORTANCEThe importance of stochastic vs deterministic processes in cave microbial ecology has been a neglected topic so far, and this work provided an opportunity to do so in a context related to the dynamics of black-stain alterations in Lascaux, a UNESCO Paleolithic cave. Of particular significance was the discovery of a novel scenario for always-rare microbial taxa in relation to disturbance, in which stochastic processes are replaced later by deterministic processes during post-disturbance recovery, i.e., during attenuation of black stains.

Dark-zone alterations expand throughout Paleolithic Lascaux Cave despite spatial heterogeneity of the cave microbiome.

BACKGROUND: Cave anthropization related to rock art tourism can lead to cave microbiota imbalance and microbial alterations threatening Paleolithic artwork, but the underpinning microbial changes are poorly understood. Caves can be microbiologically heterogeneous and certain rock wall alterations may develop in different rooms despite probable spatial heterogeneity of the cave microbiome, suggesting that a same surface alteration might involve a subset of cosmopolitan taxa widespread in each cave room. We tested this hypothesis in Lascaux, by comparing recent alterations (dark zones) and nearby unmarked surfaces in nine locations within the cave. RESULTS: Illumina MiSeq metabarcoding of unmarked surfaces confirmed microbiome heterogeneity of the cave. Against this background, the microbial communities of unmarked and altered surfaces differed at each location. The use of a decision matrix showed that microbiota changes in relation to dark zone formation could differ according to location, but dark zones from different locations displayed microbial similarities. Thus, dark zones harbor bacterial and fungal taxa that are cosmopolitan at the scale of Lascaux, as well as dark zone-specific taxa present (i) at all locations in the cave (i.e. the six bacterial genera Microbacterium, Actinophytocola, Lactobacillus, Bosea, Neochlamydia and Tsukamurella) or (ii) only at particular locations within Lascaux. Scanning electron microscopy observations and most qPCR data evidenced microbial proliferation in dark zones. CONCLUSION: Findings point to the proliferation of different types of taxa in dark zones, i.e. Lascaux-cosmopolitan bacteria and fungi, dark zone-specific bacteria present at all locations, and dark zone-specific bacteria and fungi present at certain locations only. This probably explains why dark zones could form in various areas of the cave and suggests that the spread of these alterations might continue according to the area of distribution of key widespread taxa.

Metabarcoding of the Three Domains of Life in Aquatic Saline Ecosystems.

High-throughput amplicon sequencing, known as metabarcoding, is a powerful technique to decipher exhaustive microbial diversity considering specific gene markers. While most of the studies investigating ecosystem functioning through microbial diversity targeted only one domain of life, either bacteria, or archaea or microeukaryotes, the remaining challenge in microbial ecology is to uncover the integrated view of microbial diversity occurring in ecosystems. Indeed, interactions occurring between the different microbial counterparts are now recognized having a great impact on stability and resilience of ecosystems. Here, we summarize protocols describing sampling, molecular, and simultaneous metabarcoding of bacteria, archaea, and microeukaryotes, as well as a bioinformatic pipeline allowing the study of exhaustive microbial diversity in natural aquatic saline samples.

Microscale dynamics of dark zone alterations in anthropized karstic cave shows abrupt microbial community switch.

Strong anthropization of karstic caves may result in formation of various wall alterations including dark zones, whose microbial community differs from that of non-altered surfaces nearby. Dark zones grow quickly and without gradual visual changes, leading to the hypothesis of a simple process rather than complex microbial successions, but this is counter-intuitive as underground microbial changes are typically slow and dark zones are microbiologically very distinct from unmarked surfaces. We tested this hypothesis in Paleolithic Lascaux Cave, across two years of microscale sampling. Indeed, Illumina MiSeq metabarcoding evidenced only three community stages for bacteria, fungi and all microeukaryotes together (i.e. unmarked surfaces, newly-formed dark zones and intermediate/old dark zones) and just two stages for archaea (unmarked surfaces vs dark zones), indicating abrupt community changes. The onset of dark zone formation coincided with the development of Ochroconis fungi, Bacteroidota and the bacterial genera Labrys, Nonomuraea and Sphingomonas, in parallel to Pseudomonas counter-selection. Modeling of community assembly processes highlighted that the dynamics of rare taxa in unmarked surfaces adjacent to dark zones and in newly-formed dark zones were governed in part by deterministic processes. This suggests that cooperative relationships between these taxa might be important to promote dark zone formation. Taken together, these findings indicate an abrupt community switch as these new alterations form on Lascaux cave walls.

Microbial ecology of tourist Paleolithic caves.

Microorganisms colonize caves extensively, and in caves open for tourism they may cause alterations on wall surfaces. This is a major concern in caves displaying Paleolithic art, which is usually fragile and may be irremediably damaged by microbial alterations. Therefore, many caves were closed for preservation purposes, e.g. Lascaux (France), Altamira (Spain), while others were never opened to the public to avoid microbial contamination, e.g. Chauvet Cave (France), etc. The recent development of high-throughput sequencing technologies allowed several descriptions of cave microbial diversity and prompted the writing of this review, which focuses on the cave microbiome for the three domains of life (Bacteria, Archaea, microeukaryotes), the impact of tourism-related anthropization on microorganisms in Paleolithic caves, and the development of microbial alterations on the walls of these caves. This review shows that the microbial phyla prevalent in pristine caves are similar to those evidenced in water, soil, plant and metazoan microbiomes, but specificities at lower taxonomic levels remain to be clarified. Most of the data relates to Bacteria and Fungi, while other microeukaryotes and Archaea are poorly documented. Tourism may cause shifts in the microbiota of Paleolithic caves, but larger-scale investigation are required as these shifts may differ from one cave to the next. Finally, different types of alterations can occur in caves, especially in Paleolithic caves. Many microorganisms potentially involved have been identified, but diversity analyses of these alterations have not always included a comparison with neighboring unaltered zones as controls, making such associations uncertain. It is expected that omics technologies will also allow a better understanding of the functional diversities of the cave microbiome. This will be needed to decipher microbiome dynamics in response to touristic frequentation, to guide cave management, and to identify the most appropriate reclamation approaches to mitigate microbial alterations in tourist Paleolithic caves.