Heritage Cases

The project plans to sample fragmentary, non-identifiable faunal remains (<3 cm; non-human) from the Middle Stone Age site of Sibhudu for paleoproteomic analyses. Tübingen University has recently built-up a cutting-edge paleoprotemics laboratory run by Dr. Samantha Brown who will conduct all analyses. Studies with these methods and laboratory setup require the bones to be exported to Tübingen (see e.g., Brown et al. 2021; Janzen et al. 2021). The paleoproteomics facilities at Tübingen are part of the zooarchaeological department which is directed by Prof. N. Conard, current permit holder of the Sibhudu excavations. The key objectives of this pilot study are threefold: 1) find out whether ancient proteins have survived in the faunal assemblages over the last 40.000-80.000 years before today; 2) examine the differential preservation of ancient proteins across different archaeological layers and taphonomic regimes of the site; 3) identify the fragmentary faunal remains by genus and species, which is impossible from these remains by classic macroscopic zooarchaeological methods. This will be the first time that the ZooMS (see below) will be applied to Middle Stone Age faunal remains and the site of Sibhudu, providing not just novel scientific insights but also potential new ways of analyses for Stone Age remains across southern Africa if successful. In terms of materials and methods, the pilot study would be based on n=500 fragments which will be sampled from the entire culture-stratigraphic sequence of Sibhudu (n=100 each from: Sibudan, HP, SB, “pre-SB Wadley”, “pre-SB Conard”). Only non-identifiable bone fragments <3 cm will be used, which have already been weighted, counted and entered in the Sibhudu database, but have no further intrinsic scientific value (i.e., they cannot be identified to species/genus by classic zooarchaeological methods). All of these bone fragments are currently stored in bulk by excavated bucket (incl. information on stratigraphic unit and weight) in the KwaZulu-Natal Museum in Pietermaritzburg. The 500 bone fragments will be hand-carried to Tübingen (Germany) by Dr. Manuel Will (Co-PI of the current research grant to excavate Sibhudu) and will be returned to South Africa after the analyses are finished (i.e., in February 2023). These bones will be analysed using a paleoproteomic technique called ZooMS (Zooarchaeology by Mass Spectrometry). ZooMS allows for the taxonomic identification of bone owing to the small differences in the amino acid sequences of collagen between species (e.g. Brown et al. 2021; Janzen et al. 2021). 20mg subsamples will be taken from each of the fragmented bones for analysis. Subsamples are taken using a microdrill with care taken to avoid any diagnostic features on the bone. Preferably, a small bone chip is removed rather than powdering the sample as powdering can sometimes lead to collagen loss. Drilling is kept as minimal as possible however a small hole/mark will be present on the bone after drilling is complete (damaging but not destroying the bone fragment in the process). Collagen is extracted from the subsample by first demineralising the bone and then using heat to gelatinise the collagen. Once extracted, the collagen chains will be enzymatically digested into their smaller peptides and these will be measured using a MALDI-TOF MS (matrix-assisted laser desorption/ionisation time-of-flight mass spectrometer). We then match each of the collagen peptides identified in a sample to a reference library which allows for the taxonomic identification of the collagen sample. The method is highly time and cost efficient allowing us to run fairly large assemblages of bone fragments within a relatively short time period, making this the ideal technique to assess biomolecular preservation at Sibhudu and to taxonomically identify the highly fragmented assemblage.