Demarchi, BeatriceStiller, JosefinGrealy, AliciaMackie, MeaghanDeng, YuanGilbert, TomClarke, JuliaLegendre, LucasBoano, RosaSicheritz-Ponten, TMagee, John2025-04-082025-04-080027-8424https://hdl.handle.net/1885/733747440The realization that ancient biomolecules are preserved in "fossil" samples has revolutionized archaeological science. Protein sequences survive longer than DNA, but their phylogenetic resolution is inferior; therefore, careful assessment of the research questions is required. Here, we show the potential of ancient proteins preserved in Pleistocene eggshell in addressing a longstanding controversy in human and animal evolution: the identity of the extinct bird that laid large eggs which were exploited by Australia's indigenous people. The eggs had been originally attributed to the iconic extinct flightless bird Genyornis newtoni (†Dromornithidae, Galloanseres) and were subsequently dated to before 50 ± 5 ka by Miller et al. [Nat. Commun. 7, 10496 (2016)]. This was taken to represent the likely extinction date for this endemic megafaunal species and thus implied a role of humans in its demise. A contrasting hypothesis, according to which the eggs were laid by a large mound-builder megapode (Megapodiidae, Galliformes), would therefore acquit humans of their responsibility in the extinction of Genyornis. Ancient protein sequences were reconstructed and used to assess the evolutionary proximity of the undetermined eggshell to extant birds, rejecting the megapode hypothesis. Authentic ancient DNA could not be confirmed from these highly degraded samples, but morphometric data also support the attribution of the eggshell to Genyornis. When used in triangulation to address well-defined hypotheses, paleoproteomics is a powerful tool for reconstructing the evolutionary history in ancient samples. In addition to the clarification of phylogenetic placement, these data provide a more nuanced understanding of the modes of interactions between humans and their environment.We thank two anonymous reviewers for their comments, which have helped us improve the manuscript. We also thank Dr. Jorune Sakalauskaite and Prof. Kirsty Penkman for support and useful discussion. B.D. was supported by the Italian “Ministero dell'Università e della Ricerca” (Young Researchers Program—“Rita Levi Montalcini”) and M.J.C. by the Carlsberg Foundation Semper Ardens grant CF18-1110. G.M. was funded by US-NSF grant 0914821. M.B. received support from an Australian Research Council Discovery grant. J.M. was funded by the Australian Research Council and the Australian National University. M.M. is supported by Danish National Research Foundation Award PROTEIOS (“Understanding and exploiting the survival of ancient proteins”) (DNRF128). We are grateful to the Science Faculty at the University of Copenhagen for free access to Computerome 2.0. We thank Prof. Jesper Velgaard Olsen at the Novo Nordisk Center for Protein Research for providing access and resources, which were also funded in part by a donation from the Novo Nordisk Foundation (Grant No. NNF14CC0001). The Pearcey Supercomputing Cluster at Commonwealth Scientific and Industrial Research Organisation was used to carry out aDNA phylogenetic analysis.application/pdfen-AU©2022 The authorshttp://creativecommons.org/licenses/ by-nc-nd/4.0/202210.1073/pnas.21093261192023-12-17Creative Commons Attribution licence