A paleoproteomic approach to environmental reconstruction through identification of waterfowl eggshells

In the Near East, the Pleistocene-Holocene transition coincides with the Epipaleolithic, an archaeological period marked by significant cultural and climatic changes. This thesis attempts to characterize Epipaleolithic environments at the archaeological site of Shubayqa (Northeast Jordan), focussing on information inferred from avian remains, since many birds have narrow and specific ecological requirements. More specifically, the work was conducted by applying paleoproteomic techniques to identify eggshells, as eggs represent direct evidence of the environmental characteristics necessary for the reproductive phase of birds, e.g. water depth, vegetation types and distribution. Eggshells recovered from archaeological sites are poorly preserved, therefore their identification is challenging. However, molecular methods based on Peptide Mass Fingerprinting (PMF), i.e. the analysis of ancient protein by mass spectrometry, may achieve family or sub-family identification. Here we attempted to improve this approach by focusing on the Anatidae group (ducks, geese and swans family). By obtaining PMFs of 33 Anatidae reference eggshell samples, we characterised 30 marker peptide sequences that are taxon specific, some to the genus level, and we applied these to identify 54 archaeological eggshell samples from Shubayqa 1 and 6 from the Early Natufian (14,400–13,100 cal BP), Late-Final Natufian (13,100–11,600 cal BP) and Pre Pottery Neolithic A (PPNA, 11,600–10,600 cal BP) periods. The analyses showed stable presence of breeding populations of Anatidae at Shubayqa throughout the studied periods. In particular, a preponderance of the genus Tadorna (shelduck) was observed, along with Anser (geese), Cygnus (swans), Oxyura (ruddy ducks) and Grus (cranes). This suggests that diverse types of water bodies with abundant fringe vegetation were present during the Early Natufian. In the Late-Final Natufian, a decrease in Anatidae populations, especially Tadorna, was observed, indicating potential changes in the environment, such as habitat fragmentation or the disappearance of more superficial and seasonal water bodies. However, the presence of swan eggs and other non-migratory birds suggests a continuity of the occurrence of more stable and deeper wetlands throughout the year and supports the possibility of the site being occupied by humans year-round, a crucial and much-debated requirement for the development of agricultural practices in the area. For the PPNA period, our data were not sufficient for an in-depth analysis of the environment, although the presence of anatids was recorded. In conclusion, this study contributes to the broader understanding of human-environment-bird interaction during the Pleistocene-Holocene transition in the Near East, and highlights subtle environmental changes over time. The paleoproteomic approach, despite some limitations, has proven effective for taxonomic identification, enabling environmental reconstruction based on ecological requirements refined to the genus level. Future technological advancements and improved reference databases will further refine our understanding of climate change and past societies dynamics.