When DNA Can’t: Ancient Proteins Reveal the Sex of 2-Million Year Old Paranthropus Specimen
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Study Conducts Paleoproteomic Investigation of Paranthropus Robustus Specimens
Early hominin species comprise a rich array of overlapping lineages that existed across the African Pliocene. Yet our knowledge of the diversity within any given lineage is limited. This is in part due to current ancient DNA technologies being unable to sequence genetic material older than ~0.02 million years. This means variations within a single lineage are difficult to attribute to a single cause, whether individuals are larger or smaller due to sexual dimorphism, population variation, or evolutionary change over time.
Yet recently, a breakthrough has provided insights into these questions for at least one ancient hominin group, namely Paranthropus robustus.
Using proteins preserved in the enamel (outer layer of your teeth), researchers Palesa Madupe and her colleagues were able to identify the sex of four P. robustus specimens, and what the sexes were, and how they help in our understanding of diversity and sexual dimorphism of P. robustus. Let us find out.
Paranthropus, Our Distant Relative
For the longest time, it was believed humans likely evolved in Eurasia. That is, until Raymond Dart made the controversial claim, based on the young skull of an Australopithecus africanus, the Taung Child, in 1925, that the origin of our species lay in Africa. This claim was supported by Scottish-born medical doctor and paleontologist Robert Broom (1866–1951), who had moved to South Africa in 1897.
From then on, until his death, Broom devoted much of his attention to human evolution. This led him to a particularly unique fossil jaw fragment in 1938. Initially presented with a few teeth by colleague George Barlow, who claimed they had come from the famous Sterkfontein site, Broom recognized these were not Au. africanus fossils and purchased the specimen for two pounds (despite being under strict orders not to purchase any fossils).
Knowing Barlow was not telling the truth, Broom pressed him for the true origins of the fossils, which he eventually admitted had actually been given to him by a local boy named Gert Terblanche, who had found them on a farm called Kromdraai. Broom set out to find Gert, but he was at school upon his arrival, and thus his sister led Broom to a hilltop where the specimen had been found. There, Broom collected more bones and teeth, which convinced him that the fossils belonged to a new species, P. robustus.
The name, Paranthropus, meant ‘beside man’ while robustus means ‘robust’ and refers to the large teeth and ridge at the top of the skull rather than the species’ overall size.
The large teeth and ridge at the top of the skull, which facilitated as chewing muscle attachments, allowed the species to crush and grind hard foods, such as nuts, seeds, roots, and tubers. Yet this was not the only diet of P. robustus; it is likely that it had a more generalist diet, also consuming soft fruits, possibly young leaves, insects, and meat.
Microscopic studies of bone fragments show that these early hominins probably used bones as tools to dig in termite mounds.
The Problem with Paranthropus
As more robust fossils were recovered, a debate began to emerge from the 40s into the 1970s, centered on whether these robust specimens simply represented the larger male individuals of Au. africanus. Eventually, three robust species, aethiopicus, boisei, and robustus, were recognized as representing their own separate genus, Paranthropus.
We now know that the genus Paranthropus first appeared in the fossil record around 2.8 million years ago (Ma) and persisted until 1 Ma, coexisting with various other hominins, including Australopithecus and Homo.
Most scientists think all Paranthropus species are monophyletic (descended from a single common ancestor), but physical similarities between certain Paranthropus and Australopithecus species suggest the possibility of paraphyly (evolving separately from different ancestors) or even interbreeding.
Additionally, studies of the enamel-dentine junction (the boundary between the outer and inner tooth layers) in Paranthropus have found significant variation, which has led to the debate on whether this variation was caused by the existence of distinct subgroups of P. robustus or whether these indicate significant sexual dimorphism similar to how gorilla males are significantly larger than gorilla females.
In Eurasian samples, this question can be solved by conducting aDNA analysis. However, aDNA has never successfully been recovered from African hominins older than ~0,02 million years (20,000 years) due to the continent’s relatively hot and humid climate.
However, proteins, even those millions of years old, preserve much better because they essentially stick to teeth and bones and are less affected by Africa’s warm weather.
Analyzing Four Fossil Teeth
It is thus that the researchers attempted to extract and analyze the protein preserved in four hominin fossils, SK 830, SK 835, SK 850, and SK 14132, originating from Swartkrans cave located around 40 km northwest of Johannesburg, in South Africa’s Cradle of Humankind World Heritage Site.
The site was discovered in 1948 by Robert Broom and John Robinson and was initially identified because it had deposits similar to those of the nearby hominin fossil site, Sterkfontein Caves. Shortly after its discovery, the site faced setbacks when lime mining operations in 1950 damaged its fossil deposits. Despite this, Robinson and geologist CK ‘Bob’ Brain continued to study the cave from 1953 to 1957, eventually leading to Brain overseeing excavations at the site between 1965 and 1986.
Archaeological investigations have revealed stone and bone tools used for butchering and digging edible roots. Additionally, the site is notable as one of the earliest places where our ancestors controlled and used fire, likely for cooking and defense.
All four teeth originated from the oldest deposits at Swartkrans, labeled Member 1 and dated to between 2.2 and 1.8 Ma. It has to date produced the largest collection of P. robustus specimens.
Analysis of the specimen revealed two female individuals and two males. More specifically, SK 850 and SK 835 were identified as clearly male due to the detection of AMELY-specific peptides, a unique protein fragment from the Y-chromosome amelogenin gene. The other two specimens did not have this male-specific peptide; however, the absence did not definitively prove they were female, as it was possible the signal was simply too weak to detect.
Thus, to address this, the researchers developed a method that estimates relative amounts using AMELX intensity thresholds and signal strength levels for a protein found in both sexes. In other words, the researchers measured the strength of the signal for a protein found in both sexes (AMELX). If the signal was strong in all samples, this would indicate the protein within is well preserved and thus should reflect AMELY (the male-specific protein) if it was ever present.
To ensure this method worked, they tested it on 11 modern human samples and were able to determine sex with 100% accuracy. They then turned to their fossil specimen and conducted the same test. It was found that both fossil specimens were female (SK 830 and SK 14132).
The researchers then compared their findings to traditional methods of sex estimation based on tooth size. Specimens SK 830 and SK 850 were correctly assigned as female and male, respectively, based on tooth size. However, SK 835, which was suggested to be female based on small tooth size, was actually male.
SK 14132 could not be compared because the tooth was incomplete, precluding sex estimation based on tooth size alone.
Putting it All Together
The identification of sex based on protein analysis first and foremost shows the limitations of using tooth size alone to determine sex. Additionally, the findings allowed the researchers to rule out sexual dimorphism as an explanation for the size variation observed in P. robustus, which has long been the primary explanation for size variation within P. robustus, making it similar to gorillas, where males are larger than females. Instead, it is more likely that size variation was due to size diversity within the species itself.
Interestingly, a single amino acid polymorphism, ENAM-137, suggests that specimen SK 835, the small-toothed male individual, might be more distantly related to the other three individuals. This could indicate that he belonged to a distinct Paranthropus group, which may explain his small size.
Additionally, a standout difference was observed in a gene which makes enamelin (enamel-forming protein); two of the specimens (SK 830 and 850) shared an amino acid similar to modern and early humans, gorillas, and chimpanzees, while the other two (SK 835 and SK 14132) had an amino acid that is so far completely unique to Paranthropus.
What was most intriguing was that SK 14132 specifically had both the distinct amino acids, indicating the first instance of heterozygosity (having two different versions of a gene). This variation could indicate genetic diversity within the P. robustus species, though the researchers caution that population sizes back then were likely different, so we can’t draw firm conclusions yet.
In a recent study, researchers used ancient proteins preserved in tooth enamel to determine the sex of four Paranthropus robustus individuals dating back nearly 2 million years. Despite the absence of aDNA, the researchers were able to determine the sex of four individuals: two females and two males.
One of the individuals was a small-toothed male, challenging the assumption that size variation in P. robustus was primarily due to sexual dimorphism. Instead, it is likely that variation in size was a reflection of size diversity within the species itself. The technique could be used on other fossil hominins across the African continent and may provide further insights into whether early hominins were more or less diverse than previously believed.
What do you think this technique might reveal about other early human relatives?
References
Madupe, P. P., Koenig, C., Patramanis, I., Rüther, P. L., Hlazo, N., Mackie, M., … & Cappellini, E. (2025). Enamel proteins reveal biological sex and genetic variability in southern African Paranthropus. Science, 388(6750), 969–973. https://doi.org/10.1126/science.adt9539
Wood, B., & Biggs, D. (2025). Birth of Paranthropus. Evolutionary Anthropology: Issues, News, and Reviews, 34(1), e70000. https://doi.org/10.1002/evan.70000
Originally Published On Medium
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