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Palaeoproteomic analyses of faunal bones from South Africa: Klipdrift Shelter and Blombos Cave

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Case Type: 

ProposalDescription: 

Our research aims to explore the potential of taxonomically identifying fragmentary bone assemblages from the Middle Stone Age (MSA) sites Klipdrift Shelter (KDS) and Blombos Cave (BBC), via the use of Zooarchaeology by Mass Spectrometry (ZooMS).

Expanded_Motivation: 

We want to analyse 201 bone fragments (less than 2cm each) to test if sufficient amounts of collagen survives in the bone from these sites and whether the collagen’s state of preservation is suitable for genus/species identification by ZooMS. Ultimately, we want to know whether ZooMS can be used in combination with traditional zooarchaeological techniques for better understanding bone assemblages from South African MSA sites, both for better characterising the faunal record, as well as for searching for hominin bone remains. The application of this method in Europe and Asia has led to important new discoveries, both of zooarchaeological and of paleoanthropological significance. We hope that the application of ZooMS to Pleistocene-age material from South Africa will lead to equally important results. I.Introduction Most Palaeolithic sites, in Africa and elsewhere, contain large numbers of bones that, due to human and carnivore processing and post-depositional influences, are either too small or lack the diagnostic features required for taxonomic identification. Because of these limitations, as well as time and funding constrains, often only a small portion of a bone assemblage is studied, usually the larger and more complete bones; the rest is set to one side and is not studied. However, this undiagnostic faunal fraction may contain taxa not previously found at the site or region, and in some cases, hominin bone fragments may also be present.Over the last decade, a new biomolecular technique (ZooMS: Zooarchaeology by Mass spectrometry) was developed for the taxonomic identification of bone fragments (Buckley et al. 2009, 2018) based on the analysis of collagen, the most abundant protein in modern and ancient bones. Collagen acts as a barcode for providing taxonomic identification, in a similar way that DNA is used to speciate biological material. Unlike DNA, however, ZooMS analyses are fast, cheap and can be done in a highthroughput format, so that several hundred samples can be analysed in a week by a single user. ZooMS, therefore, has the potential to increase the number of identified taxa and/or improve assessment of proportional abundance of species in an assemblage, especially when supplementing morphological identification, and offers a cost- and time-efficient solution compared to aDNA analyses. With this pilot study, we want to assess if the amount of collagen present in bone fragments from the MSA levels at KDS (Henshilwood et al. 2014) and BBC (Henshilwood et al. 2001), are suitable for the application of ZooMS and for genus/species identification. II. Archaeological background The southern Cape of South Africa contains some of the richest and most important record of archaeological sites containing MSA material dating to 120ka onwards. Faunal assemblages offer important insights into past environment and their composition, and may be used as a proxy of palaeoenvironmental reconstruction and animal territorial ranges, as well as for understanding human subsistence patterns, adaptation and resilience to changing climatic conditions (Thompson and Henshilwood 2011). Our hypothesis is that the unidentifiable faunal assemblages of MSA sites may contain important environmental indicators in the form of animal taxa not macroscopically identified, as well as hominin fossils fragmented beyond macroscopic identification. The application of ZooMS to sites in Eurasia, e.g. Vindija Cave, Croatia (Deviese et al. 2018), Grotte du Renne, France (Welker et al. 2016) and Denisova Cave, Russia (Brown et al. 2016; Douka et al. 2019) led to the discovery of several hominin fragments dating to ~200-40 ka and belonging both to Neanderthals and Denisovans. The aims of this study are twofold. i. We want to determine if there are sufficient amounts of collagen present in the bone assemblages from two MSA sites for ZooMS analyses. ii. If enough collagen is preserved, we will perform taxonomic identification of this material based on its collagen profile. Depending on the success rate of this study, ultimately, we want to establish ZooMS as a method available to South African researchers to supplement morphological identifications and applicable to all time periods and various research questions. Materials & Methods a. ZooMS Our research proposal revolves around the application of a method known as Zooarchaeology by Mass Spectrometry (ZooMS) (Buckley et al. 2009). This is a biomolecular technique that uses differences in the composition of collagen (type I), the major organic component found in bones and teeth of animals, to taxonomically identify them. Each family, genus, or, in some instances, species contains a unique combination of peptides present within their collagen, creating a unique “collagen fingerprint”. The method has been tested on large mammal assemblages from archaeological sites for all periods, but its application on Pleistocene-age sites over the last 5 years has provided exceptional results (e.g. Brown et al. 2016, Welker et al. 2016). We aim to use ZooMS as a high-throughput, time and costefficient biomolecular methodology for species identification of otherwise unidentifiable bones. b. Analytical protocols About 20mg of bone is demineralised in HCl solution which results in the release of acid soluble and insoluble collagen from the bone mineral. The supernatant is collected and incubated at 65oC for 1h where the collagen is converted to gelatine. The gelatine undergoes enzymatic digestion with trypsin and purification with C18 Zip Tips. The extract is then spotted on a target plate and analysed in a MALDI-ToF-Mass Spectrometer (Bruker Maldi Autoflex Speed LRF). The resulting spectra depicting the peptide “fingerprints” are analysed for their mass- to-charge (m/z) ratios and relative signal intensities, and are compared to a reference library compiled from specimens of known taxonomy. c. Materials requested for study We want to study 115 bone fragments from KDS and 86 from BBC (total: 201). A table with all samples and their provenance is shown at the end of the document (Table 1). All the Blombos bones are from the Still Bay layers (M1 and Upper M2); these are layers that have been analysed and recorded from a zooarchaeological perspective. All the Klipdrift Shelter bones are from the Howiesons Poort layers. All samples have been photographed and are included in the current permit application. We will regularly report about the progress of the project, including positive as well as negative results, to the responsible curators and researchers. We will publish the results together with all curators and researchers involved in the study. The materials provided and extracts made from them will be used for this study only, and no material will be released to any third party without explicit written permission. d. Need for sample exportation The successful application of ZooMS requires specialised laboratories with low levels of protein contamination, as well as the use of a MALDI-ToF mass spectrometer. Presently there is no equivalent expertise of this methodology in South Africa neither such facility, specialising on archaeological material. We propose to export and analyse the bones at the Department of Archaeology, Max Planck Institute for the Science of Human History, Jena, Germany, under the guidance of Dr Katerina Douka. The department has its own specialized MALDI instrument (~€0.5M) exclusively used on archaeological material. The analyses require 3 months and the material will be returned to South Africa immediately after. V. Significance Following the pilot study in this proposal, and with expertise and interest building in the country, it may become possible that the first part of the pretreatment (collagen extraction and purification) will take place in South Africa, so that all bone materials stay in the country and only collagen extract is exported. If successful, this study could produce valuable insights into African prehistory, all while preserving precious skeletal material or, in some cases, even generating data from archaeological sites that have yet to yield hominin fossils. It is hoped that a first successful round of results will facilitate the establishment and wider application of the method to bone material from all time periods from South Africa.

ApplicationDate: 

Wednesday, September 18, 2019 - 10:21

CaseID: 

14337

OtherReferences: 

ReferenceList: 

CitationReferenceType
Brown, S., Higham, T., Slon, V., Paabo, S., Meyer, M., Douka, K., Brock, F., Comeskey, D., Procopio, N., Shunkov, M., Derevianko, A., Buckley, M. 2016. Identification of a new hominin bone from Denisova Cave, Siberia using collagen fingerprinting and mitochondrial DNA analysis. Scientific Reports 6: 23559.
Buckley, M., Collins, M., Thomas‐Oates, J., & Wilson, J. C. 2009. Species identification by analysis of bone collagen using matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. Rapid communications in mass spectrometry 23(23), 3843-3854
Buckley, M. 2018. Zooarchaeology by Mass Spectrometry (ZooMS) Collagen Fingerprinting for the Species Identification of Archaeological Bone Fragments. In Giovas, C.M. & LeFebvre, M.J.: Zooarchaeology in Practice. Zooarchaeology in Practice: Case Studies in Methodology and Interpretation in Archaeofaunal Analysis. Springer International Publishing, pp. 227- 247.
Devièse, T., Karavanić, I., Comeskey, D., Kubiak, C., Korlević, P., Hajdinjak, M., Radović, S. et al. 2017. Direct Dating of Neanderthal Remains from the Site of Vindija Cave and Implications for the Middle to Upper Paleolithic Transition. Proceedings of the National Academy of Sciences of the United States of America 114 (40): 10606–11.
Douka, K., Slon, V., Jacobs, J., Bronk Ramsey, C., Shunkov, M.V., Derevianko, A.P., Mafessoni, F., Kozlikin, M.B. Li, Bo, Grun, R., Comeskey, D. Devièse, T., Brown, S., Viola, B., Kinsley, L., Buckley, M., Meyer, M., Roberts, R.G., Paabo, S., Kelso, J., Higham, T. 2019. Age estimates for hominin fossils and the onset of the Upper Palaeolithic at Denisova Cave. Nature 565(7741), 640-644
Henshilwood, C.S., Sealy, J.C., Yates, R., Cruz- Uribe, K., Goldberg, P., Grine, F.E., Klein, R.G., Poggenpoel, C., van Niekerk, K., Watts, I., 2001. Blombos Cave, Southern Cape, South Africa: Preliminary Report on the 1992e1999 Excavations of the Middle Stone Age Levels. Journal of Archaeological Science 28, 421-448.
Henshilwood, C.S., van Niekerk, K.L., Wurz, S., Delagnes, A., Armitage, S., Rifkin, R., Douze, K., Keene, P., Haaland, M., Reynard, J., Discamps, E., Mienies, S., 2014. Klipdrift Shelter, southern Cape, South Africa: preliminary report on the Howiesons Poort levels. Journal of Archaeological Science 45, 284-303.
Thompson, J.C., Henshilwood, C.S., 2011. Taphonomic analysis of the Middle Stone Age larger mammal faunal assemblage from Blombos Cave, southern Cape, South Africa. Journal of Human Evolution 60, 746-767.
Welker, F., Hajdinjak, M., Talamo, S., Jaouen, K., Dannemann, M., David, F., Julien, M., Meyer, M., Kelso, J., Barnes, I., Brace, S., Kamminga, P., Fischer, R., Kessler, B.M., Stewart, J.R., Pa€€abo, S., Collins, M.J., Hublin, J.-J., 2016. Palaeoproteomic evidence identifies archaic hominins associated with the Châtelperronian at the Grotte du Renne. Proceedings of the National Academy of Sciences of the United States of America 113, 11162-11167.
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