Heritage Cases

Introduction Numerous palaeontological studies have been unsuccessful in finding definite metrics for sex and palaeodietary constraints (e.g. palaeotrophic level, palaeodietary range) of different extinct tetrapod groups. Sex determination in dinosaurs is beneficial to avoid misclassification of taxa and may even solve long-standing debates on taxonomy such as the debate on Triceratops and Torosaurus possibly being two separate dinosaur lineages or the same dinosaur lineage but differ due to evolution (Longrich and Field, 2012). It may also provide a better understanding of sexual anatomical differences within different tetrapod groups and their population dynamics within groups. Currently, only vague anatomical proxies such as horn size are used to suggest sex (Petrie et al., 1991). Understanding palaeotrophic levels and palaeodietary range are also crucial for past ecosystem characterisation, especially in species-rich domains such as the Karoo where many different taxa, potentially with varying dietary habitats, as they are stratigraphically well-constrained (Jaouen et al., 2012). Developing metrics that can contribute to understanding population dynamics and dietary behaviour is therefore a critical endeavour for advancing palaeontological analysis of past ecosystems. Together with a French team of isotope specialists, we have developed Ca and Sr isotopic tools in the Wits Isotope Geoscience Laboratory (WIGL; Wits University) that have been shown to trace sex and palaeodietary factors in tooth enamel of extant and fossilised species. In this heritage application, we request permission to sample exceptionally small amounts of tooth enamel and bone (<1 milligrams) from a selection of Karoo-aged co-existing taxa, with an emphasis but not exclusive focus on dinosaurs from the Elliot Formation, using tested micro-drilling techniques, ion-exchange geochemistry and subsequent cutting-edge mass spectrometry to produce requisite Ca and Sr isotopic ratios to that may indicate sex, palaeotrophic levels and palaeodietary range. Background and Rationale: Calcium is an alkaline metal that is found in the living organisms (Martin et al., 2017). Previous studies have shown that male and female mammals fractionate calcium differently as females undergo lactation (Tacail et al., 2017). Strontium isotopes, also found in bones and teeth, represent a traditional isotope system that may provide more information about diet and trophic levels of humans and animals as shown in Knudson et al., 2010. The stable strontium isotope ratio, 88Sr/86Sr, can be used in palaeotrophic-level analysis, as mass dependant fractionation of 88Sr and 86Sr occurs through the trophic spectrum (Martin et al., 2017). The radiogenic strontium isotope ratio, 87Sr/86Sr, found in animal tissue, reflects the same 87Sr/86Sr ratio of the habitat (e.g. geological substrate, food and water source) in which the organism was living and provides a proxy of palaeodietary range (Knudson et al., 2010). 87Sr/86Sr analysis has been successfully used to track the movement of hominids (Schweissing and Grupe, 2003; Knudson et al., 2010). In the study by Balter et al., 2012, strontium and calcium isotopes were shown to provide further information on diet and range. Past studies have shown that there is variation in calcium isotopes (δ44Ca) in humans and other animals based on sex differences and breeding behaviour (Tacail et al., 2017). Other traditional and non-traditional isotope systems fractionating in the body such as strontium (δ88Sr and δ87Sr), copper (δ65Cu) and zinc (δ66Zn) isotopes have shown variation in plants, animals and humans when studying diet and trophic levels (Jaouen et al., 2016). Specifically, previous studies on humans have shown that calcium isotopes can aid in sex determination, while strontium, copper and zinc isotopic analysis can provide further understanding of sex, diet and trophic levels. Our testing shows they may be extendable to dinosaurs and other extinct organisms. This study will focus on Karoo-aged dinosaurs and extinct taxa that once co-existed in the Elliot Formation, because of the large potential sample size, as well as the high level of generic diversity (Barret, 2004). In particular, in well-known ecosystems of the upper Elliot Formation, there are a variety of individuals that co-existed at the same time period, however little independent evidence exists identifying where they may be placed on the trophic spectrum. This provides an excellent opportunity to apply the palaeodietary isotopic tools to a well-constrained scientific problem within vertebrate palaeontology. The fossil specimens requested (see Table 1) will first be used to determine sex, palaeotrophic levels and palaeodietary range. If we can see a difference between the presumed herbivore and carnivore specimens, I will then expand into looking at the ontogenetic differences in diets. Among presumed herbivores, such as Massospondylus, it has been hypothesised that juveniles differed from adults in terms of their foraging habits. Also, there is uncertainty to what roles the different sexes may have in population dynamics and what sexual attributes classify the different sexes. These non-traditional isotopic systems allows us to test these hypotheses for the first time using state-of-the-art isotopic techniques. I have already started to test this in living crocodilians with promising results, and based on this, I wish to extend the method to a selection of Karoo-aged specimens that co-existed in possibly similar ecologies. Isotopic analyses of bone and teeth could provide a proxy for sex determination in extinct tetrapods as it has been shown to differentiate between the sexes, diet and trophic levels of extant animals (Tacail et al., 2017; Jaouen et al., 2012). Proposed Fossil Sampling Process A major advantage of these non-traditional isotope studies (e.g. Ca and Sr) is that the mass of enamel/bone required is extremely small, on the order of <500 μg. Specialist microdrills and non-contaminating bits (Fig. 1) are used to ensure that sampling is as minimally destructive as possible. Creating the requisite enamel powder (<500 μg) produces exceptionally small spots on the surface of the bone, on the order of ~500 μm in diameter (Figure 2 a, b). Under normal lighting conditions, these marks can be barely be seen. Highly magnified images shown in Fig 2 and 3 illustrate the minimal damage that is created on the sampling process. A maximum of 2 drill spots will need to be created on any tooth or bone fragment. I have been trained in these micro-drilling techniques during a research visit to our collaborating team at the ENS Lab in Lyon (France) and we have successfully tested these drilling techniques in the ultra-clean WIGL at Wits University (Fig 2). Given this success, all articulated fossils and teeth or bone fragments will be sampled in this laboratory. Sampling of tooth enamel will form the majority of the sample material for this project as enamel is effectively resistant diagenetic alteration that would normally alter isotopic ratios in dentin and/or bone material (Fisher, 1981). This is because tooth enamel has small pore spaces and larger apatite crystals making it more resistant to physical and chemical alteration during fossilisation (Botha et al., 2004). Sampling of the specimens and the ion-chromatography will be carried out in the WIGL. The detailed sampling protocol, in preparation for the ion-exchange chromatography and Ca-Sr separation from the bone/enamel, is as follows: 1. High-resolution, scaled photographs are taken of the teeth and/or bones before sampling begins. 2. When sampling, it will be vital to remain consistent in sampling the same spot i.e. enamel and cortical bone for all the teeth and bones for the various specimens. This is to avoid a variation in development of that part of the tooth or bone in the life in the specimen. 3. The sample spot is gently cleansed using minimal amounts of distilled water and methanol. 4. The sample is secured, and a tiny drill spot is created using tungsten bits that are thoroughly cleaned prior to sampling, producing a small amount of powder which is carefully collected on weighing paper surrounding the sample. 5. Samples will be re-photographed to document the sample spot. 6. Extensive cleaning of the drill and individual drill bits takes place in between each specimen to avoid cross-specimen contamination. Ion exchange chromatography in the Wits Isotope Geoscience Laboratory: Ion-exchange chromatography will be performed on the powdered tooth enamel to separate calcium and strontium as shown in figure 4 below (Martin et al., 2017). This will be carried out mainly at the Wits Isotope Geoscience Laboratory at the University of the Witwatersrand, where the separation methods for these ions from tooth material are well established. I have been trained at the ENS in ion-exchange chromatography and have successfully implemented the ion exchange separation techniques in the WIGL at Wits University. MC-ICPMS analysis using the Thermo-Finnigan Neptune-plus MC-ICPMS facilities at the Université de Lyon will be carried out as outline in Tacail et al., 2014. This novel study may lead to ground-breaking research which could open up many exciting research pathways in palaeosciences and geosciences. This research also strengthens research collaborations between two fundamental research thrusts in South Africa, namely palaeontology and geochemistry and is supported by the Centre of Excellence and the PAST fund. I will be able share my skill set and ideas through what I have learnt in this study to contribute to South African geochemistry and palaeontology techniques. This pioneering project may also be beneficial for tourism in our country as South Africa has a rich and diverse collection of fossils. It will also assist many palaeontological studies in unravelling how these extinct fossils may have lived and the different characteristics that separated females, males and carnivores, herbivores and omnivores. Through this research, I would like to encourage students to think of ways to carry out multidisciplinary studies, so we can create new and exciting scientific innovation.