bioRxiv preprint doi: https://doi.org/10.1101/2020.06.11.145482; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Ancient DNA Insights into Aboriginal Australian Mortuary Practices 2 Sally Wasef1*, Joanne L Wright1, Shaun Adams1, Michael C Westaway1, 2, Clarence Flinders3, 3 Eske Willerslev 4, 5,6 and David Lambert1* 4 1Australian Research Centre for Human Evolution, Environmental Futures Research Institute, Griffith 5 University, 170 Kessels Road, Nathan 4111, Australia. 6 2 Archaeology, School of Social Science, University of Queensland, Sir Fred Schonell Drive, St Lucia 7 QLD 4072 8 3 Cape Melville, Flinders and Howick Islands Aboriginal Corporation, Cairns, Australia. 9 4 Department of Zoology, University of Cambridge, Cambridge, UK 10 5 Wellcome Trust Sanger Institute, Cambridge, UK 11 6 Lundbeck Foundation GeoGenetics Center, University of Copenhagen, Copenhagen, Denmark 12 * Correspondence: 13 David Lambert 14 [email protected] 15 Sally Wasef 16 [email protected] 17 Keywords: Aboriginal Australians 1, bioarchaeology 2, genomic enrichment 3, mitochondrial 18 DNA 4, paleogenetics 5, Ancient DNA6. (Min.5-Max. 8) 19 Abstract 20 Paleogenetics is a relatively new and promising field that has the potential to provide new information 21 about past Indigenous social systems, including insights into the complexity of burial practices. We 22 present results of the first ancient DNA (aDNA) investigation into traditional mortuary practices among 23 Australian Aboriginal people with a focus on North-East Australia. We recovered mitochondrial and 24 Y chromosome sequences from five ancestral Aboriginal Australian remains that were excavated from 25 the Flinders Island group in Cape York, Queensland. Two of these individuals were sampled from 26 disturbed beach burials, while the other three were from bundle burials located in rock shelters. 27 Genomic analyses showed that individuals from all three rock shelter burials and one of the two beach 28 burials had a close genealogical relationship to contemporary individuals from communities from Cape 29 York. In contrast the remaining male individual, found buried on the beach, had a mitochondrial DNA 30 sequence that suggested that he was not from this location but that he was closely related to people 31 from central Queensland or New South Wales. In addition, this individual was associated with a 32 distinctive burial practice to the other four people. It has been suggested that traditionally non-locals 33 or lower status individuals were buried on beaches. Our findings suggest that theories put forward 34 about beach burials being non-local, or less esteemed members of the community, can potentially be 35 resolved through analyses of uniparental genomic data. Generally, these results support the suggestion 36 often derived from ethnohistoric accounts that inequality in Indigenous Australian mortuary practices 37 might be based on the status, sex, and/or age of individuals and may instead relate to place of bioRxiv preprint doi: https://doi.org/10.1101/2020.06.11.145482; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 38 geographic origin. There is, however, some departure from the traditional ethnohistoric account in that 39 complex mortuary internments were also offered to female individuals of the community, with genomic 40 analyses helping to confirm that the gender of one of the rockshelter internments was that of a young 41 female. 42 1 Introduction 43 A better understanding of how people lived in the past can be revealed by an examination of their 44 skeletal remains, which can assist in reconstructing their life history. The examination of their burial 45 context reveals information on how they were treated after death. Traditionally this level of 46 understanding has been reconstructed using methods from biological anthropology. These methods can 47 include studies of craniometrics and biological traits that can provide insights into the genetic 48 relatedness of an individual Pardoe (1993). Moreover, the examination of the health status of an 49 individual which provides insights into how the individual may have been nursed or cared for by that 50 society during life (Tilley, 2015). Taphonomic investigations of burial sites and the arrangement of the 51 deceased in the grave provide important information on the status of an individual (Pearson, 1999). 52 However, the methods based on archaeological and anthropological assessments are sometimes 53 insufficient to establish the sex and biological kinship relationships particularly when ancestral remains 54 are heavily eroded or damaged and when comparative datasets simply do not exist for multivariate 55 analyses of metric and non-metric data. The uses of ancient DNA (aDNA) can provide more precise 56 information about the biological affinity among individuals in past populations to complement the 57 other bioarcheological findings. The field of ancient DNA has expanded rapidly since its inception in 58 1984 (Kutanan et al., 2017). It has now impacted a large number of different disciplines including 59 biological anthropology. Ancient DNA studies have the potential to provide new information about 60 cultural traditions and specifically burial practices. 61 Biparentally inherited genomic data has been successively used in population studies for repatriation 62 purposes (Heupink et al., 2016; Malaspinas et al., 2016). However, due to the highly fragmented nature 63 of aDNA, this whole-genome approach could be inefficient when studying the burial practices of 64 poorly preserved remains (Collard et al., 2019). It is typically more feasible to recover high copy 65 number uniparental mitochondrial DNA (mtDNA) from human remains (Wright et al., 2018). 66 The Flinders Island group is located in the tropical north-east of Queensland, off the eastern coast of 67 Cape York Peninsula. The group consists of seven continental Islands, sitting within the Princess 68 Charlotte Bay, west from Cape Melville (Figure 1). Over the past two centuries, Indigenous people 69 from these Islands have been regularly involved in maritime industries. Cultural changes that have 70 included increased European contact have also resulted in the removal and theft of many Indigenous 71 cave bundle burials (Horsfall, 1991 ). Not only were ancient remains affected by these activities, but 72 so too were the Indigenous people of Flinders themselves. Since the Second World War many 73 Indigenous people were forcefully removed from Flinders Island (Wurrima) to the mainland as a result 74 of government regulations at that time (Rigsby & Chase, 1998). 75 Princess Charlotte Bay was first recorded by British navigators in 1815 (Jack, 1921). Princess Charlotte 76 Bay was situated within the early shipping routes of the east coast of Australia, allowing early contact 77 between Europeans and Traditional Owners in the 19th Century (Coppinger, 1883; King, 1827; Roth, 78 1898). Although there has been extensive archaeological research conducted in the Flinders Group, no 79 research to date has extended to mortuary practices. Little attention has been paid to many of the burial 80 sites on the islands, or indeed throughout many parts of Cape York. Archaeological examinations 81 carried out by Beaton in 1985 revealed that there has been an Aboriginal occupation of the Flinders bioRxiv preprint doi: https://doi.org/10.1101/2020.06.11.145482; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 82 group for at least 2,300 years (Beaton, 1985). Beaton also suggested that the initial occupation of 83 Flinders Island occurred ~2500 years ago and was probably closely related to the introduction of 84 Papuan/Melanesian outrigger canoes (Beaton, 1985). This interpretation is now under question with 85 the discovery of archaeological evidence at Endean Shelter illustrating that Aboriginal occupation of 86 the islands extended back to 6,280 calBP (Collard et al., 2019). Ethnographic records provide detailed 87 information on mortuary protocols after initial contact, but little is known about how these customs 88 may have differed in pre-European contact. 89 Knowledge of Aboriginal mortuary practices in north-east Australia is limited to ethnographic accounts 90 by anthropologists and observers in the 19th and early 20th Centuries (Roth, 1898, 1907). According 91 to Roth, people of Cape York interpreted death as a result of spiritual intervention or human agency, 92 rather than natural phenomena. It was believed that spirits of the dead could harm the living (Roth, 93 1907). The deaths of prominent and/or powerful people were often avenged by their remains being 94 carried from camp to camp and defleshed before finally being buried or interred in trees or caves. While 95 old, less esteemed, or infirmed people were given simpler burials with minimal ceremony and often 96 buried within close proximity