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.