A library of avian proteins improves palaeoproteomic taxonomic identification and reveals widespread intraspecies variability

Abstract

Biomineral-associated proteins, such as those found in bone, teeth, and egg-shell, have become instrumental for studying ancient life, as they often survive 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 central to many archaeological and ecological questions. While palaeoproteomics conventionally assumes that protein sequences vary only between-species or above, our research demonstrates widespread evidence for single amino acid polymorphisms (SAPs) occurring within-species, particularly within avian eggshell proteins. 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 determining evolutionary relationships. However, with comprehensive taxonomic coverage of Anatidae proteins, clear taxonomic patterns 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 benchmarks for palaeoproteomic applications in archaeology, ecology, and evolutionary biology.