Ancient biomolecule analyses are proving increasingly useful in the study of evolutionary patterns, including extinct organisms. Proteomic sequencing techniques complement genomic approaches, having the potential to examine lineages further back in time than achievable using ancient DNA, given the less stringent preservation requirements. In this study, we demonstrate the ability to use collagen sequence analyses via proteomics to provide species delimitation as a foundation for informing evolutionary patterns. We uncover biogeographic information of an enigmatic and recently extinct lineage of Nesophontes across their range on the Caribbean islands. First, evolutionary relationships reconstructed from collagen sequences reaffirm the affinity of Nesophontes and Solenodon as sister taxa within Solenodonota. This relationship helps lay the foundation for testing geographical isolation hypotheses across islands within the Greater Antilles, including movement from Cuba towards Hispaniola. Second, our results are consistent with Cuba having just two species of Nesophontes (N. micrus and N. major) that exhibit intrapopulation morphological variation. Finally, analysis of the recently described species from the Cayman Islands (N. hemicingulus) indicates that it is a closer relative to the Cuban species, N. major rather than N. micrus as previously speculated. Our proteomic sequencing improves our understanding of the origin, evolution, and distribution of this extinct mammal lineage, particularly with respect to approximate timing of speciation. Such knowledge is vital for this biodiversity hotspot, where the magnitude of recent extinctions may obscure true estimates of species richness in the past.

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Sloths were among the most diverse groups of land vertebrates that inhabited the Greater Antilles until their extinction in the middle-late Holocene following the arrival of humans to the islands. Although the fossil record of the group is well known from Quaternary deposits in Cuba, Hispaniola, and Puerto Rico, remains from older units are scarce, limiting our understanding of their evolution and biogeographic history. Here we report the oldest known fossil ground sloth from Hispaniola, represented by an unassociated partial tibia and scapula that are recognized as a single taxon from the late Miocene-early Pliocene of the Dominican Republic. The combination of characters observed on the tibia suggests a close relationship with Megalocnus, otherwise only known from the Pleistocene–Holocene of Cuba. These fossils fill a temporal gap between those previously known from the early Miocene of Cuba and those from Pleistocene–Holocene deposits in the region and provide additional support for a continuous presence of the group in the Greater Antilles since the Oligocene.

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