Heritage Cases

Case for sampling Australopithecus sediba Well studied and documented Australopithecus sediba was discovered in 2008 and first described in 2010. To date Au. sediba has more than 2100 citations in the literature and is thus one of the most studied early hominid species. In addition, sampling strategy in both the lab and at the site have ensured that substantial numbers of hominid specimens remain either unprepared or remain in-situ for future sampling methods. This means that well thought through decisions can be made about destructive sampling of material from this species and this site. Furthermore, the sample from Au. sediba has a number of parts preserved from the right and left sides of body, and this means that parts are replicated. Additionally, the advent of high-resolution scanning means that we have good records of this material for both gross and micro-anatomical studies available. High likelihood of proteins being present In 2013 aDNA sampling was attempted at the Max Planck Institute. While we were unsuccessful in recovering aDNA, the sampling procedure recognised proteins in the sample. While palaeoproteomics was not advanced at that time, the recognition of proteins in those samples indicated the high probability of success for a modern attempt at extracting useful samples for palaeoproteomics analysis and making the chance of failure in this species lower than in untested samples from other sites. Samples We have selected two dental samples for palaeoproteomics. UW block 10 left mandibular ramus with M2 exposed. Part of MH-1. This specimen is part of the Block 10 accumulation which recently travelled to the Perot Museum for the 2019 – 2020 Origins exhibit. The specimen has been chosen as it is well studied, is a mirror of the right tooth and has both dentine and enamel exposed on the broken edge. It is thus ideal for sampling with minimal impact on future scientific study. 3d scans, micro-CT scans and a full image catalogue will be stored with the Curator of collections and uploaded to Morphosource.org before sampling and after sampling. Lingual side of MH2’s third mandibular molar This specimen has been selected for two reasons. The first is that there is already damage to the lingual side of the specimen where sampling can occur, and the third molar is the least diagnostic of mandibular teeth. Documentation protocols will follow that for other specimens. Case for sampling Homo naledi Homo naledi was discovered in 2013 and described in 2015. The sample is represented by over 2500 individual fossils including several hundred teeth. In sheer numbers of preserved specimens, it is the best represented extinct hominin species in the African fossil record. It is also among the best described and work on the species already has over 1800 citations. The sample is extremely homogenous in its morphology. This homogeneity and the abundance of material make this species ideal for sampling for palaeoproteomics. Given the young age of Homo naledi, there is a high probability of success in recovering ancient proteins. This alone is a reason for sampling the specimens. In addition, however, the phylogenetic position of Homo naledi remains unresolved and understanding its relationships, if any, with other species of ancient hominins and perhaps living humans is of the utmost importance. We are proposing to sample four specimens. Two from the Dinaledi Chamber, one from the Hill Antechamber and one from the Lesedi Chamber. Protocols for documentation will follow that for the specimens of Au. sediba selected for the same study. For the Dinaledi Chamber samples, an additional criterion has guided our selection. This fossil sample contains the commingled remains of several individuals and we have elected to select teeth that have a high confidence of coming from two different individuals. Samples U.W. 101-809 left mandibular first molar This tooth is relatively complete. It has been chosen because it is the likely antimere of the right first molar in the U.W. 101-377 mandible, which has been attributed to the DH7 individual. This would be a high priority for future sectioning to understand development and exposure to rare isotopes. This tooth is fully published and described with microCT data in hand. U.W. 101-886 right mandibular canine This tooth is a complete crown with partially complete root. It is the antimere of U.W. 101-1126, and is associated with a complete set of mandibular incisors, giving it the possibility of representing an individual that is represented by several teeth within the sample. This individual must be different from DH7 represented by the U.W. 101-377 mandible, because that mandible already has a right canine. U.W. 101-020 left maxillary first molar This tooth was the first recovered from the Hill Antechamber and unlike other material from this chamber is fully published and described with micro-CT data. The base of the crown is broken with no roots attached, and enamel may be sampled from the natural breaks. U.W. 102b-511 left mandibular canine The 102b locality within the Lesedi Chamber has a mandibular fragment and five isolated teeth from a juvenile H. naledi individual. This canine tooth has no root and enamel may be sampled from the mesial face or possibly via the base of the tooth. Sample collection for dentine and dental enamel palaeoproteomics analysis All of the sample preparation procedures for mass spectrometric analysis of ancient proteins will be conducted in laboratories dedicated to the analysis of ancient DNA and ancient proteins, in clean laboratory rooms fitted with filtered ventilation and positive pressure. Sample collection and preparation for mass spectrometry will be executed by experienced staff at the Globe Institute, with extensive and demonstrated experience in performing these operations. The operator will wear facemasks, nitrile gloves, hairnets, and bodysuits all the time while in the clean lab. After the specimens will arrive at the University of Copenhagen (Denmark), 150 mg of dentine plus dental enamel or 100 mg of dental enamel will be sampled from each ancient hominin tooth fragment using a sterilized drill and crushed to a rough powder, Fig. 1. An aliquot of 10-20 mg of dentine and/or dental enamel will be prepared for mass spectrometric analysis to test biomolecular preservation and preliminary recovery of genetic information. A mock ‘extraction blank’, containing no starting material, will be prepared, processed and analysed together with each batch of ancient samples to monitor possible contaminations. Sample preparation for palaeoproteomics analysis Enamel and the dentine samples will be demineralized in 10% trifluoracetic acid (TFA) at 3 °C for 24 h, Fig. 2. Subsequently, solubilized protein residues will cleaned, concentrated and immobilized on C18 Stage-Tips. The C18 Stage-Tips will be transferred to the Novo Nordisk Foundation Centre for Protein Research, at the University of Copenhagen, for mass spectrometric analysis. Statement why this study cannot be done in South Africa: There are no suitable laboratories capable of conducting this research in Africa, nor the expertise. The experimental process was pioneered by the Danish lab we are partnering with.