IT WAS an astonishing discovery: a chamber deep underground, packed full of ancient human remains. The excavators who uncovered the fossils at South Africa’s Rising Star cave in 2013 described the experience as “breathtaking” and “emotional”. Then they took a proper look at the bones, and exhilaration gave way to bewilderment. This new species of ancient human, which the researchers called Homo naledi, had such an odd combination of primitive and modern features that it was impossible to know how it was related to other ancient humans and, ultimately, to us.
About 20 years ago, it looked like the human evolutionary tree was coming into focus. Then palaeontologists started finding ancient humans, like H. naledi, that are so strange, it is as if they had walked off the pages of a Tolkien fantasy. We can’t expect ancient DNA to help resolve their place in the human family tree because most of these misfit cousins were found in places too warm for genetic material to survive. The trail seemed to have gone cold.
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In the past few years, however, we have learned to read the signals in other organic molecules that tend to survive longer than DNA and persist even in warm environments. Researchers have already analysed samples of proteins extracted from ancient bones and teeth to reveal relationships between ancient mammals. Now, some think they could reveal how archaic humans like H. naledi evolved and interacted. “I’m confident that it will be possible to put some of these very unusual hominins on the [family] tree,” says at the University of Copenhagen in Denmark.
Human hybrids
It is no exaggeration to say ancient DNA has transformed our understanding of human evolution. It confirmed that our ancestors interbred with Neanderthals between about 100,000 and 50,000 years ago. It revealed the existence of a distinct group of Stone Age humans we had never recognised before – the Denisovans of east Asia – and showed that our ancestors interbred with them too, between about 50,000 and 15,000 years ago. More recently, ancient DNA studies have even begun to find evidence that Denisovans once interbred with a far more ancient group of humans, perhaps a species called Homo erectus that appeared almost 2 million years ago and vanished about 100,000 years ago.
In other words, ancient DNA has revealed that the Stone Age world was populated by many distinct human groups that, despite their genetic differences, were more than willing to interact and interbreed when their paths crossed.
Ancient genes can’t tell us everything, though. All of the revelations came from analysis of DNA from living people and samples from a handful of ancient humans who lived in cooler parts of Eurasia within the past 50,000 years. We could learn a lot more by analysing even older genetic material, but DNA tends to fall apart over such time spans. That means we are missing vital genetic information from most of human evolutionary history, which arguably began around the time that H. erectus evolved and began spreading across Africa and Eurasia. Furthermore, DNA is completely silent on our earlier, more ape-like hominin ancestors that lived in Africa between about 7 million and 2 million years ago.
This is where proteins can help. Large and complex molecules, they are built from smaller components, amino acids, that occur in sequences according to instructions encoded in genes, so they contain the same sort of information as DNA. We have known for 65 years that proteins, or at least bits of them, might survive in the fossil record. The problem was that studying them was always too fiddly and difficult.
Things changed at the beginning of the 21st century with the development of new techniques. They involved adding electrically charged ions to the ancient and fragile protein fragments, which means the molecules can be run through a machine called a mass spectrometer to quickly identify their amino acid sequence. Ancient protein research took a huge leap forward. “Anyone can do this,” says Collins.
And they have every reason to. Analysing fragments of protein in this way can offer insights into ancient human behaviour, including what sort of foods people ate and even clues about their sex lives (see “Extracting insights into ancient lives“). Extract larger chunks of ancient protein, however, and you might be able to work out where our strange cousins belong in the hominin family tree.
Biologists build evolutionary trees by examining similarities and differences between species, whether in terms of their physical appearance or their molecular make-up. By and large, the more similarities two species share, the more likely they are to be closely related. Proteins are suitable for this sort of evolutionary analysis because animals typically produce equivalent versions of the same proteins – collagen, keratin, haemoglobin and so on – and because the sequence of amino acids within these proteins can differ slightly between species. This means that if you extract large chunks of particular proteins from extinct hominins and read their amino acid sequences, you can use that to work out how they relate to one another, and to living humans.
“We are missing vital genetic information from most of human evolutionary history”
There are some caveats. Although the human body contains tens of thousands of distinct proteins, surprisingly few of these are found in the tissues that readily become fossilised. Teeth are a good example. Tooth enamel preserves ancient proteins very well, but even in a living human it contains just 10 or so different proteins, says Frido Welker, also at the University of Copenhagen. Each protein is generally 50 to 2000 amino acids long, so even if all the proteins found in enamel – the “enamel proteome” – are recovered from a fossil tooth, there might be a combined sequence of about 20,000 amino acids at most. For comparison, a complete ancient human genome contains a genetic sequence billions of base pairs long. The question then is: do ancient proteomes contain enough information to build a reliable evolutionary tree?
Recent work suggests they do. Over the past five years, this approach has been used to construct evolutionary trees for various ancient mammals. A 2019 analysis of sloths, for instance, looked so different to conventional evolutionary trees for this group of animals that it was viewed suspiciously by some people in the field. But in a second study, geneticists independently analysed sloth relations using a tried-and-trusted DNA analysis and it gave essentially the same result as the protein study.
The field of palaeoproteomics, as it is known, has now moved into the realm of primates. Last year, Welker and Enrico Cappellini, also at the University of Copenhagen, led an analysis of an ancient enamel proteome taken from the largest of extinct apes, Gigantopithecus. The information it contained suggested that the ancient primate, which lived in South-East Asia until about 300,000 years ago, was related to living orangutans – an idea that matched expectations.
What made the result particularly intriguing was that the Gigantopithecus tooth the researchers sampled is 1.9 million years old and came from a subtropical cave in southern China. That is exactly the sort of place in which H. erectus and enigmatic species like H. naledi have lived over the past 1.9 million years. The implication is that the fossils they left behind might contain enough protein to work out where they fit in the hominin evolutionary tree. “The Gigantopithecus study definitely pushed the boundaries of what we know about protein preservation,” says Jessica Hendy at the University of York, UK.
By the time the Gigantopithecus paper came out, the researchers had already moved on from apes to hominins. A few months earlier, Welker and his team had published work describing the dentine proteome from a 160,000-year-old hominin jaw. The fossil, they concluded, may have belonged to a Denisovan. It was a conclusion with profound implications, because the jawbone had been found 3280 metres above sea level on the Tibetan plain. Perhaps, they suggested, Denisovans adapted to life at such altitudes.
Earlier this year, Welker and Cappellini notched up another success. Their team reconstructed an enamel proteome from a tooth that was potentially 950,000 years old and that belonged to another poorly understood early human, Homo antecessor, that once lived in Spain. The information within the proteome firmed up the idea that H. antecessor was closely related to the common ancestor our species shared with Neanderthals and Denisovans. It was spectacular proof that ancient proteins really can shine a light on the murkier early chapters of our evolutionary history.
Now thoughts are turning to the contentious hominins that walked Earth more recently, particularly the Indonesian “hobbit” (Homo floresiensis), discovered in 2003, and H. naledi. Both were alive when our species, Homo sapiens, evolved about 300,000 years ago, but both show a truly bizarre mix of features. H. floresiensis had a chimpanzee-sized brain inside a miniature H. erectus-like skull, while its shoulders, wrists and feet are reminiscent of the ape-like hominins, including the famous Lucy, that lived in Africa more than 2 million years ago. H. naledi, meanwhile, had a brain only marginally larger than a chimpanzee’s, hands that looked a little like Lucy’s and feet very similar to those of living humans.
We still have no idea where either H. floresiensis or H. naledi sits on the human evolutionary tree. We don’t even know whether they really are humans that ultimately descended from a species like H. erectus, or whether they group with the ape-like hominins.
“We still have no idea where Homo naledi sits on the human evolutionary tree”
Fresh clues
Both scenarios are plausible – and proteins should help us figure out which is right, says Welker. This is because the amino acid sequences within proteins change at a relatively constant rate, like a clock. It isn’t a very accurate clock, he says, but by comparing the ancient protein sequences with similar sequences from living people, it should be possible to determine whether H. naledi and H. floresiensis branched off our family tree a few hundred thousand years ago – making them human – or more than 2 million years ago, when the Lucy-like hominins had their heyday.
Collins says he is involved in ongoing discussions about putting an H. naledi specimen under the drill to extract proteins. There is no official word on when – or if – the work will go ahead. However, Lee Berger at the University of the Witwatersrand, South Africa, who leads the H. naledi research, says he was due to take fossil samples to Europe several months ago, only for the coronavirus pandemic to thwart the plans.
Within a few years, then, proteins might help some of our more inscrutable relatives find a place in the human family tree. Whether or not the scientists who study hominin fossils will accept the evidence from protein analysis is another matter. “I’m confident that the new data would be taken very seriously by most,” says William Jungers at Stony Brook University, New York, but he adds that it probably wouldn’t be seen to override all of the evidence gleaned from the shape of the fossil bones themselves.
That’s probably a good thing as molecular information isn’t infallible, says John Hawks at the University of Wisconsin-Madison. For instance, he recalls that when geneticists first looked at segments of Neanderthal DNA in the late 1990s, they concluded that our species didn’t interbreed with Neanderthals, which we now know is incorrect.
But although some caution is required, ancient proteins are almost certain to be one of the next big things in human evolutionary studies. Assuming the work on H. naledi goes ahead successfully, there is potential to recover proteins from far older fossils. Already, Welker and his colleagues have extracted them from a 1.9-million-year-old H. erectus tooth. They were too degraded to be useful, although Welker says that might be because the tooth in question was damaged, meaning some of the proteins it originally contained might have leached out.
Collins says it could even be possible to extract proteins from the ape-like hominins that came before humans, and use the information to work out how some of them relate to each other and to us. That might seem far-fetched – but in 2016, Collins was involved in a study that successfully extracted proteins from ostrich eggshell fragments collected at Laetoli, a 3.8-million-year-old site in Tanzania world famous for preserved footprints left by a Lucy-like hominin.
“We have such an interesting family tree with some curious critters in there,” says Collins. “But I’m pretty sure the protein work will be able to put some of them on the right evolutionary branch.”
Extracting insights into ancient lives
As we get better at reading the information they contain, ancient protein fragments are telling us about how our ancient ancestors behaved. For instance, Neanderthals who lived in southern France during the Stone Age typically hunted reindeer. But earlier this year, an ancient protein analysis by Naomi Martisius at the University of California, Davis, and her colleagues revealed that the ancient humans chose to make their bone tools from the ribs of aurochs and bison, although these animals were less common.
Studying ancient proteins can also throw new light on recent human evolution. Earlier this year, Shevan Wilkin at the Max Planck Institute for the Science of Human History in Germany and her colleagues examined food proteins trapped in ancient dental plaque. They concluded that people on the eastern Eurasian steppe began consuming dairy produce at least 5000 years ago. This means there is an equally long history of dairy consumption in Europe and on the Eurasian steppe. And yet, while many Europeans now carry a genetic adaptation that makes digesting milk easier for adults, few people on the Eurasian steppe do. “These two regions seem to take these different trajectories when it comes to evolutionary selection for dairy consumption, and it’s so interesting to ask why,” says Jessica Hendy at the University of York, UK, a co-leader of the study. It is a question she is now eager to answer.
Ancient proteins can even guide us to a better understanding of prehistoric sex lives. One of the biggest science stories of 2018 was the discovery that a 90,000-year-old bone fragment belonged to a teenage girl – nicknamed Denny – who had a Neanderthal mother and Denisovan father. Although it was DNA analysis that revealed Denny’s remarkable parentage, geneticists might not have chosen to study the bone fragment if not for the fact that ancient proteins had already shown it belonged to an ancient human.
