Palaeoproteomic insights into the evolutionary history of cave bears from southwestern Europe.

Dental enamel proteomics provides access to molecular information from time periods far beyond the reach of ancient DNA, offering a powerful means to investigate evolutionary dynamics in deep time. Here we present the first large-scale enamel proteomic dataset for 55 cave bears to date, analysing fossils spanning the Early to Late Pleistocene across southwestern Europe, including key specimens from Atapuerca sites. Using a non-enzymatic demineralization workflow and LC-MS/MS analysis we consistently recovered rich enamel proteomes and identified taxonomically informative peptides alongside two previously unknown amino acid variations in AMBN-249 and SERPINA1-341 positions. Phylogenetic analyses are consistent with a basal position of Ursus dolinensis within the speleoid clade, providing new molecular evidence relevant to its debated taxonomy. Together, these findings show that enamel proteomes preserve phylogenetic structure and lineage-specific molecular signals, highlighting the potential of palaeoproteomics to inform evolutionary relationships within the speleoid clade across the Pleistocene.