Source: Institut of Evolutionary Biology (IBE, CSIC-UPF)

Published in the prestigious journal Science, the research has achieved a milestone in the study of human evolution through the recovery of ancient protein sequences from four approximately 2-million-year-old teeth from Swartkrans, South Africa. The fossil remains have been attributed to the extinct hominin species Paranthropus robustus. Considered a side branch of our evolutionary tree, Paranthropus emerged and evolved in Africa between 2.8 and 1.2 million years ago, coexisting with early Homo species and possibly interacting with them. Paranthropus was bipedal but also exhibited adaptations in the upper limbs for climbing and had a broad dietary niche, based on various types of plants and possibly insects.

Led by the University of Copenhagen and the University of Cape Town, with participation from the Institut Català de Paleontologia Miquel Crusafont (ICP) and the Institute of Evolutionary Biology (IBE)—a joint center of the Spanish National Research Council (CSIC) and Pompeu Fabra University (UPF)—the team extracted protein fragments from the dental enamel of these ancient human relatives and used cutting-edge mass spectrometry techniques to partially reconstruct their sequences.

Ancient Proteins Recovered in Africa from Deep Time

The pioneering approach employed by the team made it possible to determine the biological sex of individual hominins and revealed crucial genetic variability that had not previously been detected in Paranthropus robustus. These findings not only challenge assumptions based solely on skeletal morphology but also open new pathways for understanding the complex evolutionary history of these ancient relatives. The successful application of paleoproteomics to determine sex and genetic variation in Paranthropus represents a potential shift in paleoanthropology, overcoming the limitations of morphological analysis and the challenges of preserving ancient DNA (aDNA), the recovery of which in Africa does not currently extend beyond approximately 20,000 years ago.

The four Paranthropus specimens were recovered from consolidated cave sediments, which may have contributed to the preservation of proteins within the fossil teeth. However, time also significantly affects the recovery of proteins from fossil samples, increasingly shortening peptides, which can hinder their detection through mass spectrometry.

“Due to poor preservation, we used peptide fractionation—a method widely used in modern proteomics—and state-of-the-art mass spectrometers to maximize protein coverage in these rare samples, thereby increasing the chances of identifying key phylogenetic information,” explains Jesper Olsen, senior author of the article and Executive Director of the Protein Research Center at the University of Copenhagen.

This advancement suggests that a wide range of African hominin fossils, previously considered beyond the reach of genetic analysis, could provide crucial biomolecular data, with the potential to transform our understanding of the diversity and variation that existed among African hominins millions of years ago.

Evolutionary Perspectives

The analysis of ancient proteins revealed several key findings. By analyzing the enamel of the teeth, the team was able to identify the biological sex of the specimens through the detection of specific variants of a protein called amelogenin. The detection of a male-specific variant allowed researchers to confidently identify two Paranthropus specimens as males, while a novel quantitative approach enabled them to infer that the other two specimens were females. The ability to accurately determine the biological sex of fossilized remains is a crucial advancement, allowing for a more precise understanding of sex-based differences in land use, distribution, and social behaviors among our long-extinct relatives.

“Determining the biological sex of these individuals who lived two million years ago is truly remarkable. This opens the door to understanding the biology of other populations within the human lineage,” says Esther Lizano, Junior Distinguished Researcher at the Institut Català de Paleontologia Miquel Crusafont (ICP), who participated in the study.

The team also discovered that the mutation found in the polishin protein, initially identified as a unique genetic variant of the species, exhibited variation among the four Paranthropus individuals analyzed.

The team also conducted morphological analyses of the teeth, which identified a possible substructure within Paranthropus. The results suggest that the considerable morphological variation observed in Paranthropus may reflect more complex population dynamics, microevolution, or even the presence of distinct groups or taxa.

Most of the recovered Paranthropus protein sequences were identical to those of modern humans, with only two differing positions between the groups. As a result, these specimens appear to be more closely related to the Homo lineage (including ourselves and our genetic cousins, the Neanderthals and Denisovans) than to any other extant species, although they remain slightly distinct. However, the age of the samples affects total protein recovery, reducing the likelihood of identifying crucial phylogenetic information that would enable precise comparisons between closely related groups.

“The study highlights both the possibilities and the limitations of our current technology. It demonstrates the complementary power of morphological and molecular analyses in the study of human evolution, but it's also important to understand that molecular diversity is limited and not always informative,” concludes Tomàs Marqués, principal investigator at the Institute of Evolutionary Biology (IBE), head of the Paleogenomics and Paleoproteomics research group at the Institut Català de Paleontologia Miquel Crusafont (ICP), and ICREA research professor at Pompeu Fabra University (UPF), who participated in the study.

The study was funded by the European Union’s Marie Skłodowska-Curie Training Network “PUSHH”, under the Horizon 2020 research and innovation program (grant no. 861389, E.C.); by the European Research Council (ERC) Advanced Grant “BACKWARD” (grant agreement no. 101021361), also under the Horizon 2020 program (E.C.); and by the ERC Proof of Concept Grant “SSPIN” (grant agreement no. 101138962), under the Horizon Europe program (E.C.).

Main image: SK 48, the skull of an adult Paranthropus robustus recovered at Swartkrans. Credit: Dr. Bernhard Zipfel (with permission from Dr. Lazarus Kgasi, Assistant Curator of Plio-Pleistocene Paleontology at the Ditsong National Museum of Natural History).

Original research article: 

• Madupe, P. P., Koenig, C., Patramanis, I., Rüther, P. L., Hlazo, N., Mackie, M., Tawane, M., Krueger, J., Taurozzi, A. J., Troché, G., Kibii, J., Pickering, R., Dickinson, M. R., Sahle, Y., Kgotleng, D., Musiba, C., Manthi, F., Bell, L., DuPlessis, M., Gilbert, C., Zipfel, B., Kuderna, L. F. K., Lizano, E., Welker, F., Kyriakidou, P., Cox, J., Mollereau, C., Tokarski, C., Blackburn, J., Ramos-Madrigal, J., Marques-Bonet, T., Penkman, K., Zanolli, C., Schroeder, L., Racimo, F., Olsen, J. V., Ackermann, R. R., & Cappellini, E. (2025). Enamel Proteins Reveal Biological Sex and Genetic Variability in Southern African Paranthropus. Science, 388, adt9539. DOI:10.1126/science.adt9539.