Enamel proteins reveal biological sex and genetic variability in southern African Paranthropus

Abstract

Europe PMC Funders Author Manuscripts Paranthropus robustus is a morphologically well-documented Early Pleistocene hominin species from southern Africa with no genetic evidence reported so far. We describe the mass spectrometric sequencing of enamel peptides from four ca. 2 million year old dental specimens attributed morphologically to P. robustus, from the site of Swartkrans in South Africa. The identification of AMELY-specific peptides enabled us to assign two specimens to male individuals while semiquantitative mass spectrometric data analysis attributed the other two to females. A single amino acid polymorphism and the enamel-dentine junction shape variation indicate potential subgroups present within southern African Paranthropus. This study demonstrates how palaeoproteomics can help distinguish sexual dimorphism from other sources of variation in African Early Pleistocene hominins. Europe PMC Funders Author Manuscripts While our understanding of the evolution of Middle to Late Pleistocene hominins is becoming increasingly clear, in large part due to ancient DNA (aDNA) sequencing data (1, 2), the biological and behavioral variation among earlier Plio-Pleistocene hominins remains poorly understood. The genus Paranthropus first appeared in the fossil record ca. 2.8 million years ago (Ma) and persisted until 1 Ma, coexisting in time and space with a number of other hominins, including Australopithecus species and members of the genus Homo. Most researchers consider Paranthropus to be monophyletic (3) however, morphological similarities between P. robustus and A. africanus (4, 5), and between P. aethiopicus and A. afarensis (6, 7), have raised the possibility of paraphyly or even admixture between species (8, 9). Furthermore, analyses of the enamel-dentine junction (EDJ) of southern African Paranthropus indicate significant variation, suggesting the possibility of detectable substructure within P. robustus (10), or even the presence of more than one species of this genus in southern Africa (11, 12). Other researchers have argued that the observed morphological differences stem from sexual dimorphism (13). Determining to what extent the variation within and between Plio-Pleistocene hominins is due to evolutionary diversification versus intraspecific variation, of which sexual dimorphism is likely a major contributor, is fundamental to interpreting their evolutionary history. Although genetic data from the African continent have provided insights of unprecedented resolution into human demography and evolution (14, 15), aDNA has never been successfully recovered from African hominin material older than ~ 0.02 Ma (15). As phylogenetically informative ancient protein sequences have been retrieved beyond the limits of aDNA preservation in Eurasia (16–18), we attempted to recover them to help investigate the causes of Plio-Pleistocene hominin variation in Africa. We used liquid chromatography coupled to high-resolution tandem mass spectrometry to reconstruct dental enamel protein sequences from four southern African hominin specimens assigned to P. robustus (Supplementary Materials). The four hominin fossils analyzed (SK 830, 835, 850, and 14132) originated from Swartkrans cave, located approximately 40 km northwest of Johannesburg, in South Africa’s Cradle of Humankind World Heritage Site (Fig. 1). The teeth are from the oldest deposits at Swartkrans, Member 1 (MB1), which is dated to between 1.8 and 2.2 Ma (19, 20). Although Swartkrans has produced the largest collection of specimens attributed to P. robustus, the Science. Author manuscript; available in PMC 2025 June 24. Madupe et al. Page 4 relationships between this material and the Paranthropus fossils from other southern African sites has been the subject of various interpretations, summarized by Martin et al. (21).