Ancient teeth suggest Homo naledi fossils in a cave may all be female

New protein analysis adds to the mystery of how these bones ended up deep underground

To the point

• Minimally invasive technique: Ancient proteins were extracted from the teeth using a minimally invasive technique, which is well suited to rare fossils.
• Assemblage may represent females only: Analysing 23 fossilised teeth from at least 20 individuals of the extinct human relative Homo naledi, scientists found no ancient protein markers for males. This suggests that the assemblage may represent females only.
• New clues towards solving the mystery: The fossils were discovered in a chamber deep within a cave system in South Africa. How they got there is still unresolved, but these results could provide new clues towards solving this puzzle.
• Possible explanations: The unusual skew may mean that only the remains of certain individuals ended up in this part of the cave. Alternatively, Homo naledi may have lacked the gene that produces the male marker.

An international research team has conducted the first analysis of ancient proteins from Homo naledi, an extinct human relative that was discovered in the Rising Star cave system at the Cradle of Humankind World Heritage Site in South Africa.

The study was led by Palesa Madupe, a molecular scientist and the study's lead author, and Enrico Cappellini, a professor of palaeoproteomics and the study's senior author. The team collaborated with researchers from the National Geographic Society’s Rising Star project and its partner institutions. Led by paleoanthropologist Lee Berger, the Rising Star project first brought Homo naledi to global attention following the discovery and description of fossils in a cave system.

The new analysis examined enamel proteins from 23 fossilised teeth, representing at least 20 individuals. The researchers searched for Amelogenin-Y, a protein encoded by the AMELY gene on the Y chromosome and normally present only in biological males. However, no Amelogenin-Y was detected in any of the teeth tested.

Ancient proteins preserved in tooth enamel

To conduct the study, the researchers used a minimally destructive acid-etching technique to extract peptides, which are fragments of ancient proteins, from tooth enamel. These were then analysed using mass spectrometry.

“Unlike those found in other remains like bone fragments, proteins in tooth enamel are preserved because dental enamel – the hardest tissue in the human body – shields proteins from environmental contamination even for millions of years. This makes them ideal carriers of genetic information from deep time,” said Palesa Madupe, formerly a postdoctoral researcher at the University of Copenhagen and now at the Max Planck Institute for Evolutionary Anthropology in Germany. “Our study helps resolve the long-standing mystery of why Homo naledi lacked significant variation; it’s probably because they could have all belonged to one sex.”

Adult Homo naledi fossils from the Dinaledi Chamber have long puzzled researchers because they show unusually little variation in size, shape, and other physical traits. In many hominin groups, some of this variation reflects differences between males and females. The new protein evidence therefore adds important data to discussions about the biology of Homo naledi and how its remains came to be deposited in the cave system.

Cappellini said the findings also highlight the need for further research: “Intriguingly, these results raise profound new questions, chief among them: if the H. naledi individuals in the Rising Star cave system are all females, then where are the males? We need to deploy a new generation of palaeoproteomic tools to address this and other fascinating questions.”

If the individuals tested were female, the findings could point to sex-specific burial or deposition practices in Homo naledi. However, the researchers stress that another biological explanation remains possible: deletion of the AMELY gene. Such deletions have been observed in some living humans and in ancient DNA from a Neandertal male. If Homo naledi males had a similar deletion, they could appear female in enamel-protein analysis even if they were biologically male.

“Either scenario, namely the absence of H. naledi males in the Rising Star cave system or a systematic deletion of their AMELY gene, is fascinating and would have deep implications for a better understanding of the biology and evolution of this species,” said Enrico Cappellini, professor of paleoproteomics at the Globe Institute, University of Copenhagen, where the analytical work took place.

“What I find most exciting is that our results further demonstrate that protein analysis of Pleistocene hominins is not just possible, in some cases, it can be done in a minimally destructive way,” said Madupe, emphasising the broader methodological importance of the study. “This means potentially opening the door to a whole new way of sustainably investigating the differences between sexes in groups of extinct hominins and other animals without causing visible damage to these priceless fossils.”

A new tool for studying ancient human relatives

Homo naledi lived approximately 335,000 to 241,000 years ago and combined human-like and more primitive anatomical features, including a brain only slightly larger than that of a chimpanzee. The Rising Star fossils represent one of the largest known assemblages of an extinct hominin species.

The fossils were first excavated in 2013 by the Rising Star team, which included an all-female group of cavers and scientists known as the “underground astronauts”, as part of a project led by Berger and colleagues. More than 1,500 fossils and 150 hominin teeth were recovered from the Dinaledi Chamber, with additional material later discovered elsewhere in the cave system.

The recovery of ancient proteins from Homo naledi tooth enamel opens new possibilities for studying extinct human relatives in contexts where DNA preservation is limited. While the findings bring researchers closer to understanding the mystery of Homo naledi, they also emphasise that much remains to be discovered about this enigmatic species.