Expanding the Zooarchaeology by Mass Spectrometry reference library for North American mammals: new collagen fingerprints from extinct and extant taxa

Abstract

Robust zooarchaeological analysis of Pleistocene-Holocene faunal assemblages is fundamental for reconstructing past environments, species distributions, human subsistence and faunal turnovers in North America. However, faunal remains from archaeological and palaeontological sites are commonly highly fragmented or modified, often precluding morphological identification and limiting the information they can provide about faunal communities and their relationships with human populations and changing climates. Zooarchaeology by Mass Spectrometry (ZooMS) provides an effective biomolecular means of overcoming this limitation, enabling the identification of unidentifiable bone fragments, but its resolution depends on the availability of empirically validated reference datasets, which remain incomplete for North American fauna. Here, we expand the North American ZooMS reference library by characterizing and validating collagen peptide markers for 22 extant and extinct mammal taxa across four mammalian orders: Artiodactyla, Perissodactyla, Carnivora and Rodentia, integrating Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry fingerprinting with Liquid Chromatography-Tandem Mass Spectrometry peptide sequencing to provide sequence-level validation of diagnostic peptide markers. The resulting markers enable robust family-level discrimination across a range of taxa with genus- or species-level identification possible for several groups. This expanded reference framework enhances the taxonomic resolution and applicability of ZooMS across North American faunal assemblages, supporting its use for advancing archaeological, palaeontological and conservation-related research on the continent.