Heritage Cases

Introduction and background Kruger Cave is a Later Stone Age rock shelter overlooking the Hex River in the eastern fringe of the Magaliesberg. The site preserves two Oakhurst expressions dating from 10480-5400 BP, and a Wilton assemblage 1390-1020 BP (Mason 1988). Along with other hunter-gatherer sites in the Magaliesberg valley, Kruger Cave was abandoned between AD 300-600 when the first Bantu-speaking farmers infiltrated the landscape. Indeed, Kruger Cave preserves the earliest record (in the form of pot sherds) of Iron Age occupation of the Magaliesberg (Mason 1962), although there is no evidence for farmer occupation of the cave. As with other sites in the region, Kruger Cave was reoccupied from AD 600-1300, after which the region was permanently abandoned when the farmers returned. The first millennium AD occupation was restricted to the outer section of the shelter, near the drip line (Mason 1988), and the artefacts from this period resemble contemporaneous assemblages from other sites in the area, such as Munro Cave and Oliebooomspoort (Mason 1988). Kruger Cave is one of only seven rock art sites in the Magaliesberg. Pager (1987) recorded 57 paintings on the walls of the shelter, the overwhelming majority of which are depictions of humans. Pager noted that, although the paintings follow the San stylistic conventions, the human depictions at Kruger Cave are on average larger than similar depictions at other sites in the area. The paintings likely date to the first millennium AD occupations, that is, the contact period (Mason 1988). The Kruger Cave deposit is extremely rich in terms of organic preservation. One of the remarkable organic finds from this site includes a bladder cap with human hair preserved (Figure 1). Most of the organic finds were recovered in the rear of the shelter, except the bladder cap with human hair, which was recovered from an undated deposit in the fore of the shelter. Bladders and pegs were used as ornaments among the Nguni, though not the Tswana, leading Mason to conclude that this artefact might date to the second quarter of the 19th century when Mzilikazi’s Ndebele were marauding through the area (Steele 1987; Mason 1988). It is, however, possible, considering no other 19th century material items were recovered from the site, that the hair is much older – dating to the period of contact: 1st millennium AD. Objectives and rationale The objectives of this study are to properly establish the age of the hair and bladder cap, and to ascertain whether it belongs to a person of Bantu-speaking or San extraction. Analysis of the DNA will also allow us to ascertain whether and to what degree intermixture existed at this time between the two populations. The extent of admixture between early Iron Age farmers and San hunter-gatherers and/or Khoekhoe herders is an open question and a focus of ongoing research (e.g., Schlebusch et al. 2012, 2017; Breton et al. 2014). Modern and aDNA studies assist in providing scientific evidence for past admixture events and the extent to which populations across southern Africa migrated and interacted with one another (e.g., Li et al. 2014; Schlebusch et al. 2012, 2013a, b). These studies are also able to provide relative timings for admixture events and offer insights that transcend traditional oral and historiographical sources. It therefore adds important aspects to the population histories of individuals and groups. Our study will contribute to the ongoing aDNA studies on early Iron Age remains. Contact between two ethnically distinct groups may take various forms, with material culture items migrating from one group to the other via direct or indirect exchange (Mazel 1989; Wadley 1996; Sadr 2002; Van Der Ryst 1998; van Doornum, 2007; Hall et al. 2013). The Magaliesberg region is of interest owing to the close proximity in which farmers and foragers lived to one another (see Mason 1981; Wadley 1996). Kruger Cave, although in close proximity to farmer sites like Oliefantspoort and Broederstroom, contains little in the way of contact material, save some pot sherds and the bladder cap with human hair. Should the hair sample turn out to be of 19th century Nguni origin, the relevance of this project is not diminished, as the aDNA will still provide an insight into the degree of population admixture at this time. The dating and aDNA data generated through this study will contribute our understanding of contact in the western Magaliesberg, and to the provincial and national database on Iron Age human remains. The results of this study will be disseminated via scientific and popular platforms, including The South African Archaeological Bulletin, South African Journal of Science, The Conversation, the Heritage Portal. Information will also be disseminated locally as part of the larger, NRF-sponsored Kruger Cave Rehabilitation Project. Sampling procedure The clump of human hair is preserved adhering to a bladder cap. Individual strands of hair can be removed from the clump using a tweezers. Sampling will take place at the Origins collections room under the supervision of the curator of the collection. Three tufts of hair will be removed and placed in sterile plastic vials for transport to Sweden. All samples will be clearly marked with its unique identifying number, and a note will be placed in the repository box to indicate that sampling has taken place, for what purpose and by whom. Sampling of the bladder material for AMS dating will take place at the same time in the Origins collection room. Approximately 20 mg of tissue will be cut from one of the edges of the bladder by the dating technician. The sample will be removed using a scissor or scalpel depending on how brittle the bladder is. Laboratory procedure and analysis AMS The processing of tissue such as the bladder is routine for AMS radiocarbon dating. The sample will be subject to the standard acid-base-acid (ABA) pretreatment for organic materials (the protocol that is followed is outlined in Brock et al. 2010). This ensures that no surface contamination or soluble organics remain in the aliquot that will be processed. Thereafter the sample is combusted to form CO2 and then reduced over iron to form graphite (Lowe 1984). Measurement of the 14C concentration of the graphite will be done on the Tandem accelerator at iThemba LABS in Johannesburg (on the WITS campus) (Mbele et al. 2017). Calculations of the radiocarbon age will follow the protocol of Zoppi (2010). aDNA It has been shown previously that hair (both from modern-day individuals and ancient material) is resistant to contamination and/or can be easily decontaminated, making hair a good source for endogenous DNA (Gilbert et al., 2006). As hair grows, hair shaft cells become keratinized, providing a physical barrier between the endogenous DNA in the keratinized cells and the outside environment. Hair is also hydrophobic, which prevents damage of the endogenous DNA (Gilbert et al., 2006). We will make use of established protocols to extract DNA from hair (Gilbert et al., 2004; Vilstrup et al., 2013), which have recently been implemented successfully in our laboratory on hair originating from the southern Cape coast of South Africa (Coutinho et al, unpublished - manuscript in preparation – available on request). We will construct DNA sequencing libraries from the DNA extracts using specific techniques, that are tailored to very challenging materials, such as the ‘blunt-end’ (Meyer and Kircher 2010) and ‘single-strand’ (Gansauge and Meyer 2013) methods. We will also apply enrichment techniques if needed, in which specific DNA sequences of interest are captured from the DNA extracts through hybridization of biotinylated oligos to streptavidine coated beads and multiplied prior to sequencing (Enk et al. 2014; Maricic et al. 2010). We, and others, have applied successfully the above methods to retrieve sequence data from Neolithic and Mesolithic humans from Scandinavia and Europe (Skoglund et al. 2012, 2014), from 300 000-year-old hominids and cave bears from Spain (Dabney et al. 2013, Meyer et al. 2014), from a 600 000-year-old horse from Canada (Orlando et al. 2013), from a 30 000 to 50 000-year-old Denisovan from Siberia (Meyer et al. 2012) and recently our team was successful with extracting and reconstructing full ancient genome data from both inland and coastal regions of South Africa (Schlebusch et al. 2017). The DNA libraries will be sequenced at the Uppsala SNP&SEQ platform, which has worked closely together with the Human Evolution group in order to fine-tune the sequencing of ancient DNA libraries. We will generate genomic data from the autosomes, the X-chromosome, the mitochondria and the Y-chromosome if the sample contains any ever-so-small amounts of human sequence data. All the labwork (DNA extraction, library construction and amplification) will be conducted at the dedicated ancient DNA facility at the Human Evolution Program at Uppsala University. Digital DNA sequence data will be deposited and made available for download through the European Nucleotide Archive (EBI-ENA) once the paper is published (expected 1-3 years after the sampling has been conducted). This publicly available data can then be used by all aDNA research teams as comparative data. Four sub-Sharan African genomic ancient DNA studies are available at the moment (Gallego Llorente et al 2014, Skoglund et al, 2017, Schlebusch et al., 2017, Prendergast 2019) but many more are ongoing, Releasing digital data will permit other ancient DNA teams to make use of the data generated by this study and compare their own data to. Since the aDNA approach of this team is to sequence full genomes of ancient remains (i.e. full information of all chromosomes, including autosomes, X-chromosome, Y-chromosome and mitochondrial DNA is obtained) – no further studies on ancient remains is necessary after a successful aDNA extraction and sequencing effort (see for example Schlebusch et al., 2017 where full genomes were sequenced). Other ancient DNA teams are using “capture” approaches (Skoglund et al., 2017), this approach only captures parts of the genome that is variable in human groups today. A big disadvantage of the capture method is that it does not give full genomic information and it only “captures” variation that is present in present-day groups. Variation that is unique to groups that are not living today or perhaps were not represented when they designed the capture panel will thus be missed. The variation targeted on the capture panels is also biased towards non-African populations and therefore the approach misses a large amount of African genetic variation. Since a successful full genomic study will produce the full genetic sequence of an individual, it encapsulates the variation that will be produced by a capture approach and aDNA labs using capture approaches will also be able to make use of the data generated by a full genomic study. However, for capture approaches, full genomic information of individuals is not obtained and to get fuller information on the ancient genomes, further ancient DNA studies might be needed in the future on the same individual. Processes, ethics and permissions Upon permission of the relevant curator an application for destructive sampling and export will be submitted to SAHRA (the South African Resources Agency) for permission. The researchers undertake to treat all human remains with respect. We also undertake to abide by the rules set out in the Wits Collections and South African Resources Agency policies. Copies of all results and publications thereof will be provided to the Museum and heritage authorities for archival purposes.