Souilmi, YassineWasef, SallyWilliams, Matthew P.Conroy, GabrielBar, IdoBover, PereDann, JacksonHeiniger, HollyLlamas, BastienOgbourne, StevenArcher, MichaelBallard, J. O.WilliamReed, ElizabethTobler, RaymondKoungoulos, LoukasWalshe, KerynWright, Joanne L.Balme, JaneO’Connor, SueCooper, AlanMitchell, Kieren J.2025-12-162025-12-160027-8424PubMed:38976766ORCID:/0000-0002-5550-9176/work/195963840ORCID:/0000-0002-4603-1473/work/195966093ORCID:/0000-0001-9381-078X/work/195968527https://hdl.handle.net/1885/733795111Dingoes are culturally and ecologically important free-living canids whose ancestors arrived in Australia over 3,000 B.P., likely transported by seafaring people. However, the early history of dingoes in Australia—including the number of founding populations and their routes of introduction—remains uncertain. This uncertainty arises partly from the complex and poorly understood relationship between modern dingoes and New Guinea singing dogs, and suspicions that post-Colonial hybridization has introduced recent domestic dog ancestry into the genomes of many wild dingo populations. In this study, we analyzed genome-wide data from nine ancient dingo specimens ranging in age from 400 to 2,746 y old, predating the introduction of domestic dogs to Australia by European colonists. We uncovered evidence that the continent-wide population structure observed in modern dingo populations had already emerged several thousand years ago. We also detected excess allele sharing between New Guinea singing dogs and ancient dingoes from coastal New South Wales (NSW) compared to ancient dingoes from southern Australia, irrespective of any post-Colonial hybrid ancestry in the genomes of modern individuals. Our results are consistent with several demographic scenarios, including a scenario where the ancestry of dingoes from the east coast of Australia results from at least two waves of migration from source populations with varying affinities to New Guinea singing dogs. We also contribute to the growing body of evidence that modern dingoes derive little genomic ancestry from post-Colonial hybridization with other domestic dog lineages, instead descending primarily from ancient canids introduced to Sahul thousands of years ago.We acknowledge the traditional custodians of the lands from which specimens used in this study have been collected and pay our respects to Elders, past, and present. Special thanks to Clem Lawrie, Senior Mirning Custodian, and Native Title Holder for facilitating access to dingo specimens from Koonalda Cave. We also acknowledge the La Perouse Local Land Council and Metropolitan Local Aboriginal Land Council for granting permission to radiocarbon date and genetically analyze dingo specimens from archaeological sites in NSW. We extend our sincere appreciation to the following individuals for their invaluable advice and assistance: Steve Johnson, Shing Kwong, Raphael Eisenhofer, Steven Bourne, Jessie Treloar, Mary-Anne Binnie, Michael Curry, Adara Curry, Brett Dalziel, Alan Treloar, Isabella Donato, Michael Westaway, Christian Huber, Pontus Skoglund, and Anders Bergström. The ARCHE at Griffith University and the ACAD at University of Adelaide provided access to ancient DNA laboratories. We extend gratitude to the staff at the Australian Museum—including Val Attenbrow, Allison Dejanovic, Niamh Formosa, Dale Higginson, Rebecca Jones, and Mariko Smith—for granting access to collections and museum study permits. We appreciate the South Australian Museum, QLD Museum, and Western Australian Museum for granting access to specimens in their collections. We thank QLD Parks and Wildlife Service and the Department of Environment and Science (QLD) for providing access to K’gari dingo specimens, and National Parks and Wildlife South Australia for assistance with field logistics and permitting. We are grateful to Australian Speleological Federation members—including Nicholas White, Susan White, Margaret James, Daryl Carr, Denis Marsh, Greg Leeder, Ian Curtis, and Steve Milner—who coordinated and conducted fieldwork on the Nullarbor. Additionally, we thank the AGRF, Garvan Institute of Medical Research, Macrogen, and Novogene Bioinformatics Technology Corporation Limited for providing high-throughput DNA sequencing services. Finally, we extend our gratitude to two anonymous reviewers, whose comments improved this manuscript. This work was supported by the Environmental Futures Research Institute Strategic Leverage Fund, Griffith University; the Australian Research Council Centre of Excellence for Australian Biodiversity and Heritage (ARC CE170100015); an Australian Research Council Laureate Fellowship (ARC FL140100260); and an Australian Research Council Discovery Project award (DP210101960). ER acknowledges support for Nullarbor fieldwork and radiocarbon dating from a Barbara Kidman Fellowship and funding from the Environment Institute, University of Adelaide. whose comments improved this manuscript. This work was supported by the Environmental Futures Research Institute Strategic Leverage Fund, Griffith University; the Australian Research Council Centre of Excellence for Australian Biodiversity and Heritage (ARC CE170100015); an Australian Research Council Laureate Fellowship (ARC FL140100260); and an Australian Research Council Discovery Project award (DP210101960). ER acknowledges support for Nullarbor fieldwork and radiocarbon dating from a Barbara Kidman Fellowship and funding from the Environment Institute, University of Adelaide.12en© 2024 the Author(s).ancient DNAdingodomesticationOceaniaPalaeogenomics2024-07-0810.1073/pnas.240758412185198252665