Palaeoproteomic insights into the deep roots of the cave bear lineage in Europe

Palaeoproteomics has emerged as a powerful tool for reconstructing the evolutionary history of extinct species, particularly when ancient DNA is poorly preserved or beyond recovery. Here, we present the first large-scale enamel proteomic study focused on the cave bear, spanning specimens from the Early to Late Pleistocene. A primary objective was to determine whether the ursid population from level TD4 of Gran Dolina (Sierra de Atapuerca, Spain) belongs to the cave or the brown bear lineage, a long-standing taxonomic debate. We analyzed specimens from the Atapuerca sites, alongside comparative material from other southwestern European localities. Using LC-MS/MS and an acid demineralization protocol without enzymatic digestion, we successfully recovered enamel proteomes from all fossil samples, including the oldest specimens. Protein profiles were obtained for each extinct ursid, enabling the identification of taxonomically informative peptides across multiple individuals per taxon. Notably, two novel single amino acid polymorphisms (SAPs), found in ameloblastin (AMBN) and alpha-1 antitrypsin (SERPINA1), were restricted to Middle and Late Pleistocene cave bears and may represent new phylogenetic markers for this clade. This study provides the first molecular phylogenetic placement of Ursus dolinensis, supporting its basal position within the speloid lineage, consistent with its proposed ancestral status. Our results highlight the strong phylogenetic signal preserved in dental enamel and the exceptional biomolecular preservation at Atapuerca, providing a robust framework for reconstructing the evolutionary history of Ursidae. Moreover, the consistent recovery of systemic proteins, such as serpins, underscores the potential of enamel proteomes to capture not only evolutionary relationships but also physiological signals relevant to extinct ursids. SignificanceThe phylogenetic position of Ursus dolinensis within the Ursidae family has been a subject of long-standing debate. While some authors place it within the speloid lineage, others suggest affinities with arctoid bears. Here, we analyze the bear population from level TD4 of Gran Dolina (Sierra de Atapuerca, Spain) using palaeoproteomic methods for the first time. Molecular evidence supports the inclusion of U. dolinensis within the speloid lineage, in a basal position, supporting its potential ancestral position. These findings extend the evolutionary depth of the speloid clade in Europe and demonstrate the power of enamel palaeoproteomics to resolve deep-time relationships beyond the limits of ancient DNA, revealing the deep evolutionary roots of the lineage.