New Insights on Ancient Human Relative Revealed by 2-Million-Year-Old Tooth Enamel

For nearly a century, scientists have been puzzling over fossils from a strange and robust-looking distant relative of early humans: Paranthropus robustus. It walked upright, and was built for heavy chewing with relatively massive jaws, and huge teeth with thick dental enamel. It’s thought to have lived between 2.25 million and 1.7 million years ago.

Humans today have a diverse array of hominin distant relatives and ancestors from millions of years ago. The South African fossil record ranges from early hominins such as Australopithecus prometheus, A. africanus (Taung child), A. sediba and P. robustus, to early members of the genus Homo (H. erectus/ergaster, H. habilis), to later hominins such as H. naledi and Homo sapiens (humans).

Fossils show how these early relatives evolved from as far back as A. africanus, 3.67 million years ago. They also document milestones in evolution, including the transition to walking on two legs, tool making and increased brain development. Ultimately, our species – Homo sapiens – appeared in South Africa 153,000 years ago.

Fossils of P. robustus were first discovered in South Africa in 1938. But crucial questions remained. How much variation was there within the species? Were the size differences related to sex, or did they reflect the presence of multiple species? How was P. robustus related to the other hominins and early Homo? And what, genetically, made it distinct?

Until now, answers to these questions have been elusive. As a team of African and European molecular science, chemistry and palaeoanthropology researchers, we wanted to find answers but we couldn’t use ancient DNA to help us. Ancient DNA has been a game-changer in studying later hominins like Neanderthals and Denisovans but it doesn’t survive well in Africa’s climate because of its simple structure.

We experienced a breakthrough when we decided to use palaeoproteomics – the analysis of ancient proteins. We extracted these from the enamel of the 2-million-year-old teeth of four P. robustus fossils from Swartkrans Cave in South Africa’s Cradle of Humankind.

Luckily, proteins that are millions of years old preserve well because they stick to teeth and bones and are not affected by the warm weather. One of these proteins tells us the biological sex of the fossils. This is how we found that two of the individuals were male and two were female.

These findings open a new window into human evolution – one that could reshape how we interpret diversity in our early ancestors by providing some of the oldest human genetic data from Africa. From there, we can understand more about the relationships between the individuals and potentially even whether the fossils come from different species.

The protein sequences also revealed other subtle but potentially significant genetic differences. One standout difference was found in a gene which makes enamelin, a critical enamel-forming protein. We found that two of the individuals shared an amino acid with modern and early humans, chimpanzees and gorillas. The other two had an amino acid that among African great apes is, so far, unique to Paranthropus.

What’s even more interesting is that one of the individuals had both the distinct amino acids. This is the first documented time we can show heterozygosity (a state of having two different versions of a gene) in proteins that are 2 million years old.

When studying proteins, specific mutations are thought to indicate different species. We were quite surprised to discover that what we initially thought was a mutation unique to Paranthropus robustus was actually variable within that group – some individuals had it while others did not. Again, this was the first time anyone had observed a protein mutation in ancient proteins (these mutations are usually observed in ancient DNA).

We realised that instead of seeing a single, variable species, we might be looking at a complex evolutionary puzzle of individuals with different ancestries. This shows that combining analyses of morphology (the study of the form and structure of organisms) and the study of ancient proteins, we can create a clearer evolutionary picture of the relationships among these early hominin individuals.

However, to confirm that P. robustus fossils have different ancestry, we will need to take samples of tooth enamel protein from more of their teeth. To do this, we plan to sustainably sample more P. robustus from other sites in South Africa where they’ve been found.

Our team was careful to balance scientific innovation with the need to protect irreplaceable heritage. Fossils were sampled minimally, and all work followed South African regulations. We also involved local laboratories in the analysis. Many of the authors were from the African continent. They were instrumental in guiding the research agenda and approach from the early stages of the project.

By combining data on molecules and morphology, our study offers a blueprint for future research – one that could clarify whether early hominins were more or less diverse than we’ve known.

For now, the Paranthropus puzzle just got a little more complex – and a lot more exciting. As palaeoproteomic techniques improve and more fossils are analysed, we can expect more surprises from our ancient relatives.

(Jesper V. Olsen, Rebecca R. Ackermann and Enrico Cappellini were also the principal investigators on this project.)