Proteomic and metagenomic insights into prehistoric Spanish Levantine Rock Art

International audienceThe Iberian Mediterranean Basin is home to one of the largest groups of prehistoric rock art sites in Europe. Despite the cultural relevance of prehistoric Spanish Levantine rock art, pigment composition remains partially unknown, and the nature of the binders used for painting has yet to be disclosed. In this work, we present the first omic analysis applied to one of the flagship Levantine rock art sites: the Valltorta ravine (Castellón, Spain). We used high-throughput sequencing to provide the first description of the bacterial communities colonizing the rock art patina, which proved to be dominated by Firmicutes species and might have a protective effect on the paintings. Proteomic analysis was also performed on rock art microsamples in order to determine the organic binders present in Levantine prehistoric rock art pigments. This information could shed light on the controversial dating of this UNESCO Cultural Heritage, and contribute to defining the chrono-cultural framework of the societies responsible for these paintings. Spanish Levantine rock art is a unique pictorial expression in Prehistoric Europe due to the naturalism of depictions, the narrative component of scenes, and the vast distribution of rock-art shelters in the Iberian Mediterranean basin (Fig. 1a,b). Despite the historical and aesthetic value of Levantine rock art and its unprecedented distribution, its chrono-cultural framework is still unclear, due to the difficulties in radiocarbon dating of pigments 1. Consequently, disparate chronological hypotheses have been proposed and, recently, new arguments have fuelled the debate, resulting in two alternative hypotheses on the chrono-cultural framework for Levantine rock art: one hypothesis proposes a Neolithic origin linked to the Neolithisation process and the spread of the "Neolithic package" in the Iberian Peninsula (from the 6 th to the 3 rd millennium cal. BC) 2,3 while the other argues a Mesolithic affiliation, based mainly on the thematic content of scenes 4,5 , since big game hunting is one of the most frequent activities portrayed in the Levantine graphic tradition. Besides dating, we also lack information on the sequence of technical gestures (chaîne opératoire) of Levantine pigments. Most archaeological research has focused on the characterization of raw materials, rather than the technical processes involved in executing the paintings. This approach is justified by the assumption that Levantine pigments were simple solutions/suspensions rather than complex mixtures, since components such as proteins or lipids, which could act as binders in mixtures, had not been detected at that point. Recently, we developed a new protocol including archaeobotanical microanalysis and experimental archaeology that enabled us to detect binders used to prepare Levantine charcoal pigments; however, we were unsuccessful in identifying them physico-chemically by in situ energy dispersive X-ray spectrometry (EDXRF) and Raman spectroscopy 6. In order to improve the characterization of Levantine pigment composition we also need a better understanding of the microbial communities associated to the rock art patina to assess their putative role in the preservation of the raw materials and binders used. Microbial activity is central to rock-art preservation and degradation 7-9. In particular, microorganisms are known to alter the composition of the mineral patina covering rock art paintings. For instance, the production of oxalic acid and other organic acids as a consequence of microbial metabolism is linked with the reinforcement of the patina, whereas microbial solubilisation of carbonates results in th