Heritage Cases

South African archaeogenomics: The KhoiSan presence prior to the Bantu migration into South Africa Project description The Khoi and San people carry the oldest lineages of humankind (Behar et al. 2008). They inhabited modern day South Africa for approximately 9,000 years (Pfeiffer and Sealy 2006), prior to any interaction with the incoming Bantu groups approximately 1,000 years ago. For the sake of brevity, the term KhoiSan will be used throughout to collectively refer to both groups. Contemporary KhoiSan and Bantu genomes have been made available in the work of Schuster et al. 2010. The contemporary genomic data can be used as a window into past events and processes such as admixture and gene flow. Additionally, there have been numerous studies focusing on the genetic diversity, population structure, population dynamics and interactions between contemporary KhoiSan and Bantu populations (Barbieri et al. 2013; Batini et al. 2011; Behar et al. 2008; Marks et al. 2015; Montinaro et al. 2016; Pickrell et al. 2012; Sikora et al. 2011; Tishkoff et al. 2007, 2009). In contrast, prehistoric genetic information for the KhoiSan population groups in South Africa is sparse. At present, only one genome is publicly available which was obtained from archaeological human remains (Morris et al. 2014). Furthermore, to date no study using the genetic analysis of isotope dated human remains has been conducted in Africa. Such a study may reveal a unique and highly accurate understanding of the past events (similar to for example Pedersen et al. 2016; Rasmussen et al. 2014, 2015; Seguin-Orlando et al. 2014; Sikora et al. 2014). This study will analyse ancient KhoiSan remains found in South Africa by using massively parallel sequencing (MPS) in order to produce complete endogenous genomes and expand current genomic knowledge of these groups. Learning more about the KhoiSan genomes will further biogeographical analysis between ancient population groups and contribute to our understanding of ancient migrations and human evolution (similar to for example Raghavan et al. 2014; Rasmussen et al. 2014; Skoglund et al. 2014). Unfortunately, the samples collected from the Albany museum will be analysed in Copenhagen as there are no facilities in Africa let alone South Africa and thus a export permit will be required. Specific objectives The main aim of the project is to employ genomic practices to study ancient human remains excavated from South Africa (between 9,000 and 500 years old). The data obtained from the human remains will be compared with contemporary genomic data to understand the temporal dynamics of the KhoiSan populations. Methods and materials Pre-analysis The methods employed to investigate prehistoric population groups are mostly destructive processes. Therefore, before any destructive methods are employed, scans and photos will be taken so that if necessary, 3D replicates can be printed. This will ensure that if further studies are required, a digital and/or physical specimen will be available for analysis. Genetic analysis All genetic analysis will be performed in a highly specialized ancient DNA laboratory at the University of Copenhagen’s (KU’s) Geogenetics Centre in Denmark. The useable genetic molecules in bones and teeth will be preserved by the protective effects of crystalline hydroxyapatite. To extract the specific endogenous DNA molecules, they will first be separated from exogenous molecules originating from modern human and bacterial contaminants as in Allentoft et al. 2015. This separation is done by chemical and mechanical eradication of contaminants on the exterior surfaces. Following the separation, the “clean” bone/teeth sections will be pulverised to aid with metal chelation which removes the hydroxyapatite and releases the DNA into solution. The extracted DNA will be concentrated with spin columns or solid phase reversible immobilization beads. The quality of the extracted DNA will be checked on a Bioanalyser (Agilent technologies, CA, USA) to evaluate the origins of the DNA. If Ancient DNA is present it will appear as short fragments (40 – 100 bp) and contaminants of modern DNA will appear as longer fragments (> 200 bp). The small (ancient) DNA fragments will subsequently be prepared for MPS using the HiSeq platform (Illumina, San Diego, USA). After sequencing, the genomes will be assembled and compared to the plethora of published ancient genomes to infer ancestry, population migration and insights into human evolution. To investigate these questions, we will perform several analyses to examine the differences in nucleotide compositions and the differences between groups or individuals. To do this we will employ analysis such as Bayesian evolutionary analysis by sampling trees (BEAST), principal component analysis (PCA), Admixture analysis and STRUCTURE. Isotyping analysis Highly stable Strontium isotopes are found in geological structures and are naturally occurring. These isotopes are passed through the earth and into the water system and therefore also into the food chain of inhabitants of the area. The humans tend to cover a large geographical area to obtain food and therefore they will provide a geographic average of the Strontium isotopes for their locale. Once assimilated, Strontium isotopes are permanently fixed into bone and teeth structures. The ratio between the radiogenic (87Sr) and non-radiogenic (86Sr) can then be used to infer an average local geographical location (see review by Price, Burton, and Bentley 2002). The calculus of teeth contains Strontium assimilated during childhood and there is little change during an individual’s lifetime. Unlike teeth, bones are constantly growing and reabsorbed and therefore elemental proportions are in constant flux throughout an individual’s lifetime. Therefore, to determine the childhood and adult geographic origins Strontium typing of teeth and bones respectively, can be done. In a similar fashion, Carbon, Nitrogen and Sulphur isotypes in nails and hair can be used to infer the diet of an individual. During consumption of foods the elemental compounds are assimilated into the body and these signals can be used to infer diet (Lehn, Mützel, and Rossmann 2011; Schoeninger 2014). The above methods have been carried out at the Geogenetics Centre at KU. A total of eight skeletal remains (ranging between 6,800 and 2,000 years old) originating from middens, dunes, rock shelters and hot subtropical regions within South Africa were tested. The results were excellent with a total of 40% endogenous DNA being recovered, resulting in seven complete genomes. These preliminary results are profound as they demonstrate that ancient DNA methodology in leading labs is now sufficiently advanced to support investigation of samples thousands of years old from hot environments that were not previously possible. The initial analysis to be conducted on the remains from the Albany museum will be carried out by Assistant Professor Lasse Vinner and Peter Damsgaard from the KU, and Peter Ristow from the KU and University of the Western Cape. Benefits of the project The Albany museum will benefit from this project by way of research output in the highest impact journals (e.g. Nature and Science). The results of this project will also attract media attention due to the potential for new findings, leading to further exposure for the Albany museum. This project will generate local knowledge of complex laboratory techniques not facilitated by any institution on the African continent. The transfer of these highly specialised and scarce skills to South African researchers can be used locally to further investigate problems/questions which cannot be answered with contemporary populations. The results of this study will further a more complete understanding of the populations who occupied prehistoric South Africa.