Heritage Cases

The Terminal Holocene in southern Africa is characterized by the transition of human subsistence from relying exclusively on foraging to food production. Given the absence of wild domestic prototypes in the region, researchers agree that domesticate animals (cattle and sheep/goats) were introduced from farther north following one or more several routes. The modalities of herding practices’ introduction into the region remain debated and several models explaining the introduction and movement of livestock into southern Africa have been proposed. One model, which is relatively well supported by genetic and linguistic studies, suggests that the introduction of domesticate caprines is consistent with significant migrations of pastoralist groups moving into the region (Gifford-Gonzalez & Hanotte 2011; Smith 1990, 1992, 1998, 2008, 2009). Archaeologically, however, early herding practices are only documented in the form of a few sheep bones at Later Stone Age (LSA) sites, dated to ca. 2000 BP. To account for these “invisible herders,” another model proposes diffusion/infiltration through trade and the slow transformation of local foragers into hunter-herders (Sadr 2003, 2008, 2015). In southern Africa, a key issue regarding faunal data from this period relates to the secure taxonomic identifications of bovid remains. One major limitation for archaeozoologists is the great degree of morphological overlap between bones of wild bovids and domesticate caprines. Hence, the secure identification of sheep/goat bones in the archaeological record is problematic. Palaeogeneticists have questioned the taxonomic attribution of bones based on morphology alone (e.g. Horsburgh et al. 2016a). There have been some discrepancies between morphological and biomolecular identifications of potential sheep and cattle bones from sites in southern Africa. For example, using palaeoproteomics, Le Meillour et al. (2020) have reattributed teeth from Leopard Cave in Namibia initially identified as caprines by Pleurdeau et al. (2012) to springbok. Similarly, bones once identified as of cattle and sheep/goat from Sehonghong in Lesotho and Blydefontein Rock Shelter in South Africa were reclassified as wild animals using DNA (Horsburgh et al. 2016b; Horsburgh & Moreno-Mayar 2015 respectively). There is still controversy regarding the sheep/wild bovid bones from Blydefontein, where classical osteology and genetics do not concur (see Horsburgh & Moreno-Mayar 2015 and Scott & Plug 2016). The specimens selected from Diepkloof Rock Shelter and Faraoskop Rock Shelter are good examples of such bones. Based on their morphology only, it is impossible to identify them beyond family level (i.e. Bovidae). The chronology of the deposits of interest is, respectively between 1590 ± 45 and 390 ± 30 BP for Diepkloof (uncalibrated dates from the Bedding unit; Parkington & Poggenpoel 1987) and between 2510 ± 60 and 2000 ± 70 BP for Faraoskop (uncalibrated dates from units MAC and MAC(C) in Layer 2; Manhire 1993). Previous analyses identified the presence of sheep in the LSA faunal assemblage from Diepkloof Rock Shelter (Steele & Klein 2013). This is confirmed by the ongoing taxonomic reappraisal conducted by Humphrey Nyambiya, under the supervision of Louisa Hutten and myself. The bulk of the ungulate assemblage, however, cannot be identified to either sheep or wild antelopes of similar size and morphology (e.g. springbok, grey rhebuck) and falls within the category of “bovids class II” or “medium bovids”, following the size class system commonly used by zooarchaeologists working in South Africa (Brain 1974). The use of palaeoproteomics to the non-identifiable bovid remains will help estimating more accurately the proportion wild antelopes and sheep occupied in the diet of the shelter’s LSA inhabitants. Based on published data and ongoing zooarchaeological study of the material, we hypothesize that sheep were central to the subsistence of the LSA groups occupying Diepkloof in the terminal Holocene. Combined with the palaeoproteomic results, we will provide a comprehensive view of how herding practices were integrated into foragers’ lifeways in the Western Cape. I have attached the PhD research proposal of Humphrey Nyambiya that includes a detailed description of the research background, aims and methodological approach of the PhD project in the appropriate section of the permit application. At Faraoskop, the preliminary analysis of the faunal assemblage conducted by Liora Horwitz (in Manhire 1993) underlined the predominance of bovids, but did not firmly establish the presence of sheep. Manhire highlights that “due to the very poor preservation of the mammalian remains very few of the bones could be identified as to species” (1993: p.15). The bovid material was assigned to class sizes; small and medium –“sheep size”- bovids are the most abundant. The faunal material from Faraoskop selected for palaeoproteomics is stratigraphically associated with the remains of 12 human skeletons. Several lines of evidence, including new and not yet published chronological, stratigraphic, isotopic, genetic and physical anthropological data, concur to support the hypothesis of a single-event leading to the death and disposal of the 12 individuals at the back of the cave (Manhire, 1993, Parkington et al., in prep. and a series of research articles for a special issue planned for 2023 in the journal Archaeological and Anthropological Sciences). One possible scenario involves a violent encounter between sheep thieves and sheep owners at the rock shelter. Based on bone morphology only, it is not possible to confirm or infirm the presence of sheep in the deposits. Palaeoproteomics will help assigning the medium bovid bones to either sheep or wild antelopes. This will contribute to shed light on the circumstances that led to the death and quick internment of the 12 individuals recovered at Faraoskop. This is particularly relevant due to the chronology of the event, which is consistent with the earliest traces of herding practices in the Western Cape . Palaeoproteomic analytical methods From the LSA faunal assemblages of Faraoskop and Diepkloof, we have selected, respectively, 47 and 100 specimens to be sampled for palaeoproteomics. The Faraoskop material is curated at the Department of Archaeology of the University of Cape Town (UCT). A letter of consent from the curator, Louisa Hutten, is enclosed with this application. This letter also concerns the specimens from Diepkloof curated at UCT. Additional material from Diepkloof is curated in the Archaeological Collections at the Iziko Museums of South Africa in Cape Town, and we provide in this application the associated letter of consent from the curator there, Wilhelmina Seconna. We have selected 10 specimens curated at UCT and 90 specimens curated in Iziko. All specimens consist of non-identifiable bovid size II. The specimens have been photographed and described, i.e. measured (length, width and cortical thickness) and, as much as possible, anatomically identified (skeletal element, skeletal portion, side). When possible, we have assigned an age group (adult, juvenile, or neonate) to the specimens based on fusion degree. The two Excel spreadsheets attached with this application contains this information. We have selected unburnt specimens because burning affects the internal composition of bone and decreases the potential for collagen preservation. Upon reception of the permits, we intend to courier the specimens to the Section for Molecular Ecology and Evolution at the Globe Institute in the University of Copenhagen (KU), Denmark. The sampling and analysis will be conducted by the PhD candidate (Humphrey Nyambiya), under the supervision of Dr. Louise Le Meillour, during a 3-month research stay from March to May 2023. Dr. Louise Le Meillour is a Fyssen Foundation and Marie Curie post-doctoral fellow and a specialist in palaeoproteomics applied to southern African bovids (e.g. Le Meillour et al. 2018, 2020). She has developed specific extraction protocols to both minimize sampling and expand protein recovery for remains in arid environments, where this is particulary challenging. She has also developed a specific comparison database of type I collagen sequences for more than 20 wild African bovids. Facilities to perform palaeoproteomic analyses are currently absent from the African continent and the Section for Molecular Ecology and Evolution at the Globe Institute in Copenhagen is one of the leading institutes worldwide for such analyses. Following ancient DNA guidelines, the Globe has developed a specifically dedicated lab for ancient proteins extraction and purification (dir. M. Collins & E. Cappellini) in order to avoid any type of contamination and expand ancient proteome recovery. The Globe is also associated with the Center for Protein Research (CPR), located in Copenhagen too and part of KU. CPR is a world leading center in terms of protein analyses in a variety of application (medical, fundamental study but also ancient proteins). The Globe has thus access to cutting-edge mass spectrometry equipments, including the most recent developed ExPloris 2 from Thermo. Humphrey Nyambiya will both be trained in ZooMS (Zooarchaeology by Mass Spectrometry), which used peptide-mass fingerprinting to assign remains to species, and in shotgun proteomics which allow, amongst others, the sequencing of proteins. Combined will Dr. Le Meillour’s expertise in both preparation of the samples from Southern Africa and the available bioinformatics tools, chances of success of this research are very high. In order to taxonomically identify the specimens selected from Diepkloof and Faraoskop to either sheep/goat or wild antelopes, we will perform both ZooMS and shotgun proteomics analyses. Palaeoproteomics allow the identification of otherwise unidentifiable faunal remains because of unique sequencing of amino acids in bone collagen of species that result in characteristic peptide profiles. Palaeoproteomics have been used to identify and distinguish morphologically similar faunal remains such as sheep (Ovis aries) and goat (Capra hircus) that are difficult to identify using a modern comparative reference (see for instance Buckley et al. 2010). Recently, both shotgun proteomics and ZooMS have also been successively applied to distinguish between caprines and wild African antelope remains (Coutu et al., 2021; Janzen et al., 2021; Le Meillour et al., 2020). ZooMS uses matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize profiles of peptides from bone collagen when shotgun proteomics rely on the use of ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). In this research, a drill will be used to obtain bone powder from the specimens. Palaeoproteomics workflow will be performed as follows: gentle decalcification of bone powder, extraction and solubilisation of preserved proteins, hydrolysis and purification prior to mass spectrometry analyses. This method will be useful in increasing the sample of securely identified domestic bones and to distinguish them from wild bovid remains. This method is relatively cost-effective and less destructive than other biomolecular methods such as aDNA. For each bone, as little as 20 mg of bone powder will carefully be extracted for the analysis. The extraction will not affect the general morphology and dimensions of the specimens. The specimens will be returned to South Africa after completion of sampling and laboratory analysis. We expect a high percentage of ancient protein recovery and we plan on publishing the obtained results in high impact journals, both benefiting Humphrey Nyambiya PhD outcomes and South African parties that will be associated in the publication process.