New avian protein sequences improve resolution of palaeoproteomic approaches to taxonomic identification & reveal widespread intraspecies variability

Biomolecules offer unparalleled insights into ancient life, from evolutionary pathways of organisms to the health and cultural practices of our ancestors. Biomineral-associated proteins, such as those found in bone, teeth, and eggshell, have become instrumental for studying ancient life, as they often endure post-mortem degradation far longer than DNA. Harnessing advancements in avian genomics, we annotate bone and eggshell protein sequences for 112 Anatidae (ducks, geese and swans) species, a biologically complex group of birds that are also central to many archaeological and ecological questions. While palaeoproteomics (the study of ancient proteins by mass spectrometry) conventionally assumes that protein sequences vary only at the species level or above, our research demonstrates widespread evidence for single amino acid polymorphisms (SAPs) occurring within-species. Furthermore, we construct a phylogenetic tree from 13 proteins that aligns with mtDNA-based phylogenies, while revealing highly variable topologies for individual protein trees, underscoring the need for caution when using fragmented proteins for taxonomic identification and identifying evolutionary relationships. However, with the comprehensive taxonomic coverage of Anatidae proteins, clear taxonomic patterns are evident in the proteins that enable reliable identification of bone and eggshell. We demonstrate this application to archaeological material from Teotihuacan, Mexico, and Shubayqa, Jordan. We highlight that extensive curated protein datasets accompanied by rigorous standards for assessing SAPs as taxonomic biomarkers are fundamental for correct taxonomic identification, setting new benchmarks for palaeoproteomic applications in archaeology, ecology, and evolutionary biology.