Study of ancient human relative reveals unexpected absence of male markers

An international team of researchers extracted and analysed protein fragments for the first time from fossil teeth of Homo naledi, an extinct human relative found in the Cradle of Humankind World Heritage Site, South Africa. In a study published today in Cell, scientists from the University of Copenhagen, the National Geographic Society's Rising Star project and 13 collaborating global institutions found no signs of any biological males within the group of Homo naledi remains tested, providing new evidence that may shed light on behaviours that predate Homo sapiens.

The analysis examined 23 teeth representing at least 20 Homo naledi individuals. To determine the biological sex of each individual, researchers looked for Amelogenin-Y - a protein that is uniquely coded onto the male Y chromosome - within the tooth enamel. Thorough examination of ancient proteins from the enamel of 23 Homo naledi teeth revealed a complete absence of the marker, suggesting that all of the individuals analysed were biologically female. In mammals, females typically carry two X chromosomes and males one X and one Y; the presence of Amelogenin-Y is therefore used as a reliable indicator of biological sex.

_{A fossil mandible of Homo naledi photographed during palaeoanthropological research in the Cradle of Humankind. The specimen forms part of ongoing studies into the anatomy and evolution of the extinct hominin species. Photo by Mathew Berger.}

'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 South African-born scientist and lead study author Palesa Madupe. Madupe is a molecular scientist who was a postdoctoral researcher at the University of Copenhagen at the time of the study and currently a researcher at 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.'

As the largest extinct hominin population yet to be examined using ancient protein analysis, the findings raise new questions about the biology, evolution, culture and potential mortuary practices of Homo naledi.

When the team first discovered and excavated the Homo naledi remains in 2013, they observed an unexpected pattern: the adult fossils found in the Dinaledi Chamber were all very similar. The adult fossils had very little variation in size, shape and other physical traits. Typically, some variation is expected among hominins, especially among males and females, otherwise known as sexual dimorphism.

To investigate this anomaly, researchers applied a minimally destructive acid etching technique to carefully extract fragments of ancient proteins - called peptides - from the remains. The peptide samples were then analysed with a mass spectrometer to identify all of the proteins present in the remains, including amelogenin - a process called paleoproteomic analysis.

'It appears that the most likely explanation for the observed absence of an Amelogenin-Y marker in these individuals is that we are seeing a sex-bias in mortuary practice - a practice until now only observed in contemporary human cultures,' said National Geographic Explorer in Residence and corresponding author of the study Lee Berger. 'The chance of having sampled twenty individuals and they are all from one sex, is quite literally one in a million.'

First described by Berger and colleagues from the Dinaledi Chamber of the Rising Star cave system, Homo naledi is an extinct hominin species with a combination of human-like and pre-human-like features that lived between 335,000 and 241,000 years ago. In 2013, an all-female team of "underground astronauts" excavated more than 1,500 fossils and 150 hominin teeth - the largest ever discovered on the continent of Africa - which at the time represented at least 15 individuals. Later, this number grew as more fossils were found by the Rising Star team in this and other areas of the cave. The results of this study build upon previous insights from the Rising Star team about Homo naledi's purported behaviour. In 2023, the Rising Star team discovered evidence of possible Homo naledi fire, symbols and burials, and in 2025 the team published additional evidence to support their findings of intentional burial practices and the creation of rock engravings.

_{A panoramic photograph of Dinaledi Chamber within the Rising Star cave system. Photo by Robbie Shone/National Geographic.}

'There are many past human societies with sex-specific burial practices, but we've found very little hard evidence of this from the earliest burial sites of modern humans or Neanderthals,' said John Hawks, a co-author of the paper and member of the Rising Star team. 'These remains of Homonaledi are older than any known Neanderthal or modern human burial site, and it's remarkable to see that they may all be female.'

Still, the research team acknowledges in this work that there could be another reason for the absence of male markers in the tested Homo naledi remains: the Amelogenin-Y gene may have mutated or have been deleted over time.

'While the deletion of the entire AMELY gene has already been observed in extant male humans and even in the DNA of a Neanderthal male, it's very unlikely that this would be the case among even half of the 20 individuals we studied or for an entire population,' explained Enrico Cappellini, professor of paleoproteomics at the Globe Institute, University of Copenhagen, where the analytical work took place, an ERC grantee and senior and corresponding author of the study. '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.'

_{Enrico Cappellini preparing ancient samples for palaeoproteomic analysis in the ancient biomolecules clean laboratory at the Globe Institute, University of Copenhagen (Denmark). Photo by Meaghan Mackie, Globe Institute, University of Copenhagen (Denmark).}

This finding is particularly significant because Homo naledi is anatomically distinct from modern humans, possessing a brain size only slightly larger than that of a chimpanzee, the authors said.

Despite this smaller cranial capacity, a growing body of research suggests the species appears to have engaged in practices once considered uniquely human.

'Seeking to understand where we come from is truly core to the human experience. These new findings are amazing because they're challenging us to rethink what we thought we knew about our place in human history,' said Ian Miller, chief science and innovation officer at the National Geographic Society. 'What I love about the Rising Star team is their restless curiosity. They keep pushing the boundaries of paleoanthropology, and reminding us that the biggest discoveries are still yet to be made.'

The paper 'Proteomic analysis of dental enamel from 20 Homo naledi individuals shows no male markers' was published June 24, 2026 in the journal Cell.