Heritage Cases

Dental calculus is increasingly used as a source of information about the diets and health of extinct individuals (Henry and Piperno, 2008; Henry et al., 2011; Warinner et al., 2012; Adler et al., 2013), and has been successfully used on specimens as old as 1.9Ma, including the Australopithecus sediba individual MH1 from Malapa (Henry et al., 2012). We propose to sample the calculus from the roughly 110 hominin teeth recovered from the 'Rising Star' excavation of Empire Cave by Lee Berger and his team, and perform a series of analyses on these samples, including plant microremain analysis, organic residue analysis, and DNA and protein analysis. These three methods provide complementary information about plant foods and other contributions to the diet, as well as information about the health status of the individual. We wish to sample portions of the calculus from the teeth in South Africa, and export the calculus to Germany, and so are requesting a sampling permit and an export permit. Dental calculus is the result of the dense mineralization of the plaque that is formed by bacteria on the surface of the teeth. Saliva is supersaturated with calcium phosphate to prevent dissolution of the teeth by acidic foods. The calcium phosphate precipitates on the bacterial pellicle, or skin, that forms on the teeth, and over time, the bacteria and mineral form a thick, layered plaque, which continues to mineralize as the individual ages. As the plaque builds up, bacteria and food particles become trapped in the mineral matrix, which then preserves these important sources of dietary and health information. Plant microfossils, namely starch grains and phytoliths, are one such marker of diet, because their unique morphologies allow us to identify them to the plant taxa (family, genus and sometimes species) and/or plant organ (seed, fruit, or tuber) that produced them. By recovering microfossils from calculus, we can document the plant foods that may have made a significant addition to these individuals' diets. This method has been successfully used to document the use of a variety of C3 woody plant foods by the Au. sediba individual. Organic residue analysis of calculus can provide more information about the plant foods consumed by the individual, as demonstrated for Neanderthals (Hardy et al., 2012). The calculus is analyzed using a GC-MS system, which also allows the identification of lipids from animal sources (such as meat or fish), as well as the consumption of honey. Finally, calculus also preserves abundant proteins and DNA from the oral bacteria, which would allow us to record the health status of these individuals (Warinner et al., 2012). Markers of inflammation, oral disease, and even pneumatic disease have been recovered from calculus, which can provide unique information about the lifestyles and ultimately deaths of these individuals. The process of removing and sampling the calculus is straightforward and does not damage the surface of the tooth. The tooth is examined under a hand lens, in order to identify whether it has calculus. There are more teeth included in the sampling request than will actually be sampled. Whether a tooth is sampled depends on whether there is calculus visible on its surface. Once the calculus has been identified, a small part (generally less than 2 mm2) of the area of thickest calculus is chosen. Sampling the thickest calculus ensures there is enough volume of the sample while reducing the surface area of the enamel exposed by the removal. We collect only about half of the calculus that is preserved on any one tooth, and leave the remainder for future studies. The calculus is removed from the tooth using pressure applied with a dental scaler. Care is taken not to scratch the surface of the tooth, only the calculus itself. Our previous study comparing before (Figs 1and 3) and after (Figs 2 and 4) photographs and ESEM images clearly demonstrates that the enamel surface of the tooth is not in any way damaged by the removal of the calculus (Henry and Piperno, 2008). Though the tooth itself is not damaged, the calculus will be consumed during the processing. The small flakes of calculus are collected in foil, and placed in a centrifuge tube for transportation. In addition to the dental calculus samples from the hominins, we wish to collect several control samples, including sediment samples and surface washes of hominin long bones. Plant microremains, particularly phytoliths, from the environment are often preserved in sediments, and may be transfered to bone surfaces. Using a density-separation method, we will isolate the microfossils from the sediment. We will also collect surface washes (by placing a drop or two of distilled water on the cortical surface of the bone, and then collecting the water by pipette) of hominin long bones. By comparing the plant microremains found in the hominin calculus to those seen in the surrounding sediment and on the long bones, we will be able to identify and control for possible sources of contamination. We wish to perform the surface wash of five long bone fragments, and to collect 5g of sediment for analysis. These analyses will be performed by the Plant Foods in Hominin Dietary Ecology Research Group at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, and collaborators of that group. Graduate student Chelsea Leonard will collect the calculus, bone surface washes, and sediment specimens. Chelsea, along with the group leader Dr. Amanda Henry, and the group technicians will perform the plant microremain analysis. The Plants Working Group laboratories are ideally arranged for the analysis of ancient microfossils while minimizing any chance of contamination. Post-doctoral researcher Cynthianne Debono Spiteri will perform chemical extraction of subsamples of the calculus, and send the extractions to our collaborator, Dr. Claudia Birkemeyer of the University of Leipzig, to be run on her GC-MS system. There are no facilities in South Africa that can provide this kind of processing in a documented, clean-lab facility. Finally, further subsamples will be sent to another collaborator, Dr. Christina Warinner of the University of Oklahoma, for DNA and protein analysis.