Palaeoproteomic evidence identifies archaic hominins associated with the Châtelperronian at the Grotte du Renne

Abstract

In Western Europe, the Middle to Upper Paleolithic transition is associated with the disappearance of Neandertals and the spread of anatomically modern humans (AMHs). Current chronological, behavioral, and biological models of this transitional period hinge on the Châtelperronian technocomplex. At the site of the Grotte du Renne, Arcy-sur-Cure, morphological Neandertal specimens are not directly dated but are contextually associated with the Châtelperronian, which contains bone points and beads. The association between Neandertals and this "transitional" assemblage has been controversial because of the lack either of a direct hominin radiocarbon date or of molecular confirmation of the Neandertal affiliation. Here we provide further evidence for a Neandertal-Châtelperronian association at the Grotte du Renne through biomolecular and chronological analysis. We identified 28 additional hominin specimens through zooarchaeology by mass spectrometry (ZooMS) screening of morphologically uninformative bone specimens from Châtelperronian layers at the Grotte du Renne. Next, we obtain an ancient hominin bone proteome through liquid chromatography-MS/MS analysis and error-tolerant amino acid sequence analysis. Analysis of this palaeoproteome allows us to provide phylogenetic and physiological information on these ancient hominin specimens. We distinguish Late Pleistocene clades within the genus Homo based on ancient protein evidence through the identification of an archaic-derived amino acid sequence for the collagen type X, alpha-1 (COL10α1) protein. We support this by obtaining ancient mtDNA sequences, which indicate a Neandertal ancestry for these specimens. Direct accelerator mass spectometry radiocarbon dating and Bayesian modeling confirm that the hominin specimens date to the Châtelperronian at the Grotte du Renne.

Keywords: Châtelperronian; Neandertal; ZooMS; palaeoproteomics.

Fig. 1.

Identification of damaged ancient proteins. (A) Deamidation values of bone ammonium-bicarbonate extracts for database samples and Arcy bone specimens analyzed using MALDI-TOF-MS. (B) Deamidation frequency based on spectral counts obtained through LC-MS/MS analysis. Cluster analysis provides three clusters (filled triangles, squares, and circles). Open circles represent proteins that have two or fewer spectral matches and were not included in cluster analysis. COL10α1 is indicated in pink; 0% indicates no deamidation and 100% indicates complete deamidation. (Inset) AR30 ppm error distribution by peptide mass for assigned spectra, with spectra matching to COL10α1 in red, open circles and those to COL10α1 128N in red, filled circles.

Fig. 2.

Southeast Asian frequency of the archaic-like allele of rs142463796 in modern human populations. The frequency of the archaic-like allele in modern human populations is displayed in red [Yoruba allele frequency in populations shown in light blue for the 1000 Genomes dataset and dark blue for the Simons Genome Diversity Panel (SGDP) dataset]. The diameter is proportional to the number of individuals in a given population, which ranges between one and 16 individuals in the SGDP and between 66 and 113 individuals in the 1000 Genomes phase 3 data.

Fig. 3.

Bayesian model of radiocarbon dates for the Grotte du Renne. Model constructed including all radiocarbon dates reported in ref. . Archaeological layer boundaries are shown, with the direct dates on the Grotte du Renne (AR-14) and the Saint-Césaire hominins highlighted in red. Posterior/prior outlier probability for AR-14 is shown in square parenthesis (more details in SI Appendix, Radiocarbon Dating and Bayesian Modeling of AR-14).