Heritage Cases

Methodology (short): 15N analyses of Member 4 fossil tooth enamel: We will analyze nitrogen isotope ratios of tooth enamel powder of the diverse fauna from the Sterkfontein Member 4 (herbivores, omnivores and carnivores; n=42) to establish a baseline 15N record for interpreting early hominin meat consumption. We will develop a complex picture of the unprecedented dataset of the food chain with trophic levels for diet, habitat preferences and water balance in animals. Ultimately, seven early hominin teeth fragments will be analyzed and the amount of Australopithecus’ consumption of meat resources (and with it the determination of their trophic level) will be quantified for the very first time. Note that we will only use tooth fragments which we will use for sampling, and only a very small amount of tooth enamel (5 mg) will be taken, the rest of the tooth fragment will remain at the ESI. The organic matter bound within the mineral structure of the fossil liberated after the dissolution of the mineral is oxidized to nitrate (see e.g., Ren et al., 2009, Martinez-Garcia et al., 2014). The resulting nitrate is converted into nitrous oxide using the denitrifier method (Sigman et al., 2001). The 15N of the N2O produced by denitrifying bacteria is measured using an automated extraction system coupled to a Thermo Plus 253 isotope ratio mass spectrometer (Weigand et al., 2016; Sigman, 2016). Around 1 to 5 mg (depending on the N content) of sample powder will derived from the fossil with a handheld diamond-tipped dental drill (1 mm diameter). This sampling will take place under a microscope at the ESI and only already destructed/fragmented fossils will be used. The enamel powder will be taken to Germany, where, in the laboratories of the MPIC, it will be prepared for 15N determination. High-resolution photographs will be taken before and after sampling of each specimen. Statement why this study cannot be done in South Africa: 15N analyzes on fossil teeth older than 100,000 years can only be archived with the new method described above. There are only two laboratories in the world that have suitable equipment, one is the USA (Princeton University), the other at the MPIC in Germany, where the Sterkfontein samples will be analyzed by the PI of this project, T. Lüdecke. The method proposed is now routinely applied to determine 15N values in different types of fossils including corals, diatoms and foraminifera. The method consists of several steps that collectively permit the analysis of much smaller nitrogen concentrations when compared to conventional extraction methods. It shows that organic material is still preserved in old fossil teeth in quantities high enough for accurate analysis (i.e. around 7 nmols/mg). Results of Phase 1: (SAHRA Permit ID 2898) Extensive lab work has been performed at the facilities of the MPIC in April/May 2019, for proof of concept for this new method and the establishment of a (preliminary) baseline δ15N dataset for the Sterkfontein Member 4 fauna (see. Fig. 1 & Tab. 1). First, we established a redox-cleaning method for raw enamel powder which removes all non-enamel bound nitrogen. We then analyzed δ15N isotope ratios in the enamel of modern rodents which were fed plant, insect, or meat-based diets with known δ15N values under controlled conditions. In the following weeks, we analyzed tooth enamel from modern African fauna including herbivores (elephants, bovids, equids), omnivores (baboons, suids), and carnivores (lions, hyenas) from different habitats. This modern dataset allowed us to characterize trophic and habitat-driven variation in the δ15N values of modern taxa. We also developed a new in-house standard (AG-Lox) using the tooth enamel of a modern herbivore. In the final step of phase 1 of this project, we measured preliminaryδ15N ratios in fossil tooth enamel from Sterkfontein Member 4, which I sampled in March at ESI. This process is still ongoing, and all sampled teeth (n = 40) are expected to be analyzed by the end of July. In this step, we establish the needed baseline for the Sterkfontein Member 4 fossil assemblage and reconstruct geochemical differences according to the tropic variation in this mammalian assemblage. 2nd phase of the project: Trophic level and meat consumption of Australopithecus After establishing baseline δ15N values for a diversity of modern and fossil fauna in the first phase of this project, nitrogen isotope ratios for primate and hominin teeth will be analyzed to evaluate their tropic behavior, (i.e. estimate the importance of animal resources in their diets). We plan to measure tooth enamel from eight non-hominin primates (Cercopithecoides and Papio) and seven hominins (Australopithecus) to produce the first nitrogen isotope dataset for early Pleistocene primates. This new analytical method for quantifying enamel-bound nitrogen isotopes requires only enamel fragments from already broken teeth. Thus this method represents a novel approach for reconstructing trophic behavior and animal consumption in early hominins while minimizing destruction to precious hominin samples. Timeline for phase 2: - August 2019: Sampling primate tooth enamel at ESI - September/October 2019: Measuring and evaluation δ15N values of these primates and hominins - January 2020: Publish first data in high-impact journals Description of the laboratory work: δ15N analyses of Member 4 fossil tooth enamel: Approximately 5-10 mg of sample powder will be drilled from the fossil teeth with a handheld diamond-tipped dental drill (1 mm diameter). This sampling will take place in the collection of the ESI. I will take the enamel powder to Germany, where, in the laboratories of the MPIC, it will be prepared for δ15N determination. The organic matter bound within the mineral structure of the fossil enamel is cleaned and liberated via dissolution of the mineral matrix and then oxidized to nitrate (Ren et al., 2009, Martinez-Garcia et al., 2014). The resulting nitrate is converted into nitrous oxide via bacterial conversion(Sigman et al., 2001). The δ15N of the N2O produced by denitrifying bacteria is then measured using an automated extraction system coupled to a Thermo Plus 253 isotope ratio mass spectrometer (Weigand et al., 2016). This project is funded by the German Science Foundation (DFG grand LU 2199/1-2).