For the first time ever, scientists have extracted ancient proteins from a 2-million-year-old tooth – and used them to map the sex, genetic variation and hidden relationships of the extinct human genus Paranthropus. This opens a rare window into how our distant relatives lived, how they were connected – and perhaps even to whom they were related.
It was early morning in Swartkrans Cave, just northwest of Johannesburg. The researchers were working beneath the steep rock walls, and dust from drill bits and brushes was thick in the air. Among fossilised animal bones and broken stone tools, a tooth emerged – a pale, smooth flap of ancient enamel.
It became immediately clear that the tooth belonged to Paranthropus robustus, an extinct human species that lived in the area nearly two million years ago alongside other hominins but without being our direct ancestor.
The species lived in what we now call the Cradle of Humankind – a network of caves, rock crevices and underground chambers about 50 km northwest of Johannesburg, South Africa. They walked upright but were still adapted to climbing. They ate coarse plant matter and possibly insects and lived in vivid interaction with other human species and predators.
Teeth like this have been known since the late 1940s, when palaeontologists first excavated Swartkrans and uncovered fossil skulls and jaw fragments. These discoveries opened the floodgates to a wave of new finds, and Swartkrans later became a centrepiece of the UNESCO-listed Cradle of Humankind – where scientists have been searching for traces of our origins for decades. Many of the teeth were catalogued and archived, but little more could be done with them at the time.
Back then, the technology did not exist to study anything beyond their appearance.
Now, after decades of waiting, an international team of researchers from South Africa and Denmark have done what no one thought was possible: extracted fossilised proteins and brought a prehistoric tooth back to life. They are the first in the world to isolate ancient proteins from the enamel of Paranthropus robustus teeth.
Forgotten proteins reveal hidden human variation
Although time and heat have long since erased these individuals’ DNA, the researchers gained unprecedented insight by analysing the proteins in the Paranthropus robustus tooth enamel. They used a new technique called palaeoproteomics – mapping ancient proteins preserved in fossils.
Co-author Claire Koenig, an postdoctoral researcher at the Novo Nordisk Foundation Center for Protein Research at the University of Copenhagen in Denmark, is thrilled that the team could determine the sex of four individuals and uncover genetic variation in their tooth proteins. The study shows that some individuals carried two versions of the same gene.
“We have never had access to this information in such ancient specimens. These are not just molecules – they are glimpses of real biological diversity in a long-extinct human lineage,” explains Claire Koenig.
Fossil teeth reveal sex
The team extracted protein fragments from the enamel and broke them into smaller chains. Each chain was then analysed using mass spectrometry – a technique that measures the weight and components of molecules to determine their structure. The method produced thousands of measurements per tooth – and many matches with known amino acid sequences. Thanks to these matches, the researchers could reconstruct parts of the proteins’ amino acid sequences – up to 780 positions in all – like rebuilding a sentence letter by letter.
The researchers found six enamel-specific proteins in all four samples, including amelogenin and enamelin. Enamel is especially valuable because it is so dense and well protected – and because it contains information about an individual’s sex and genetic traits. In two of the samples, the researchers found a version of the amelogenin protein only among males – a biological fingerprint that enables sex to be determined.
The other two lacked this protein. To rule out degradation, the researchers measured the intensity of a related protein, AMELX. Because its signal was strong, they could conclude with high confidence that the individuals were female.
In addition, one tooth showed that the individual had inherited two versions of the same gene – one from each parent. This genetic heterozygosity was found in a specific location in the enamelin protein: one version contained the amino acid glutamine and the other arginine. This offers direct evidence of genetic diversity in early human populations.
Hidden relationships buried in enamel
This is the first time such variation has been documented in fossils this old and so precisely, Claire Koenig notes.
“The discovery shows that we can now trace relationships and variation directly in fossil molecules from African hominins,” she says.
The findings also support a longstanding suspicion among researchers: that differences in tooth microstructure and genetic patterns might indicate multiple subgroups – or even distinct subspecies – within Paranthropus robustus.
When the researchers compared the protein data with anatomical measurements, one tooth stood out. It had arginine instead of glutamine in its enamelin protein, and its enamel–dentine junction showed structural traits more similar to individuals from the nearby Drimolen Palaeocave System than from Swartkrans. This deepens the hypothesis that several distinct Paranthropus groups may have coexisted in the region.
“Until now this was an informed guess – this is the first time we have biological data supporting the idea that there were multiple lineages, or even species, within Paranthropus,” adds Claire Koenig. She stresses that it is still too early to draw firm taxonomic conclusions – but the differences are significant enough to merit further investigation.
A new look at prehistoric life
For years, Paranthropus fossils have posed a mystery. Without the tools to extract molecular information, researchers could do little more than speculate. However, many of these remains clearly tell about a life marked by hardship. One example is the skeleton of a fully grown female, who was estimated to have been just 1 metre tall and weigh 27 kilograms.
Two deep marks in her skull matched the canine teeth of a leopard – evidence that she was likely killed by the leopard and dragged into the cave as prey. Nearby lay a nearly complete leopard skull.
Events like this are relatively straightforward to interpret. Mapping genetic variation and understanding what drove the emergence of subgroups are far more difficult.
Until now, it has not been clear whether such variation could be traced through skull shape or tooth size – or whether such features were more likely related to sex. The new results offer fresh biological evidence in debates that have shaped research on human origins for decades – and open a more nuanced view of both individuals and populations.
“This study is not just remarkable for the techniques it uses,” says Claire Koenig. “It also gives us access to family ties, social roles and life histories we have never been able to study before.”
She adds that combining palaeoproteomic and morphological analysis enables ancient hominins to be viewed in more detail – as individuals with genetic traits, social identities and biological diversity.
“These teeth, stored for decades in museum collections, hold more than fossilised traces. They reveal the diversity of life in a lineage that lived alongside us 2 million years ago – their sex, their genetic makeup and maybe even their relationships. And they open the door to new questions about our own beginnings.”
