After the unearthing of a Neanderthal-Denisovan fossil, UK scientists are using groundbreaking techniques to learn more of the species’ complex bonds with humans

Of all the ancient peoples that have been studied by scientists, none has set puzzles quite so profound as those left behind by the Denisovans. Only a few tiny pieces of bone and teeth have ever been found of this long extinct species – fragmentary remains that would all fit snugly inside a cigarette packet.

Yet these fossil scraps suggest that Denisovans had a considerable influence on people today. Up to 6% of the genes now found in modern New Guineans and 3-5% of the DNA of aboriginal Australians is made up of Denisovan DNA, scientists have discovered. The gene that allows Tibetan people to survive high altitudes is also believed to have been inherited from them. This information tells us one thing: tens of thousands of years ago, modern humans encountered Denisovans – and had sex with them. It is a startling discovery that raises many basic questions. Just who were the Denisovans? What did they look like? And what were their relations with the Neanderthals, their closest evolutionary cousins? Did they have tools and art like the Neanderthals?

At present, researchers have few answers to these questions, such is the paucity of the Denisovan fossil record. But a new project, Finder – Fossil Fingerprinting and Identification of New Denisovan Remains from Pleistocene Asia – which has been just been launched with backing from the European Research Council, aims to put that right and transform our knowledge of the Denisovans and their relations with both Homo sapiens and the Neanderthals. All three species interbred, we now know, and a key aim of the study is to help understand these complex bondings: Denisovans will be a special focus, however.

“We aim to find out where they lived, when they came into contact with modern humans – and why they went extinct,” says project leader Katerina Douka, of the Max Planck Institute in Jena, Germany and a visitor at Oxford University.

Denisovan research faces a basic problem, however – paucity of fossils. Denisova Cave in Siberia – where their eponymous remains were first found in 2010 – is our sole source, and only a handful of fossils have ever been dug up there (along with several Neanderthal pieces).

“It is a wonderful site,” says Tom Higham, deputy director of Oxford University’s Radiocarbon Accelerator Unit and an adviser to Finder. “It is cool inside, so DNA in bones does not disintegrate too badly. However, nearly all the bones there have been chewed up by hyenas and other carnivores.” As a result, Denisova’s cave floor is littered with tiny, unidentifiable bone fragments.

Facebook Twitter Pinterest A molar tooth found at Denisova Cave. Photograph: Robert Clark

“You cannot tell whether a piece comes from a mammoth or a sheep – or a man or woman,” adds Higham. “Only a very few will be human, though they are certainly worth finding – they could tell us so much.”

Current techniques for identifying bone fragments involve the time-consuming process of extracting and sequencing DNA. “That takes far too long to be practical,” says Higham. “There are tens of thousands of bits of bones here.”

However, Douka and Higham will use a new technology called Zooarchaeology by mass spectrometry – ZooMs. Developed by Mike Buckley, at Manchester University, the technique, which is derived from food science research, exploits the fact that collagen, a protein fibre found in bone, can survive for hundreds of thousands of years. Every major mammal group has a distinctive type of collagen, and ZooMs can read its structure like a molecular barcode, identifying which animal was the originator of a particular bone. This makes it ideal for differentiating human and animal remains.

“We asked Anatoly Derevyanko and Mikhail Shunkov, who direct the cave’s excavations, for samples – and they gave us a big bag full of bits of bones. All the fragments were considered to be unidentifiable,” says Douka.

The team began to prepare the bones. A 20mg slice was cut from each and placed in a test tube and given an identification code. After three months, 150 samples had been prepared. “That was nowhere near enough,” says Higham.

So Douka and Higham asked their postgraduate students for a volunteer to work on the project. “No one came forward,” says Higham. “A week went by, then a fortnight. I was beginning to get worried. Then an Australian student, Samantha Brown, knocked on my door and volunteered to work the bones as part of her master’s dissertation. She saved the day.”

Over the next few weeks, Brown undertook the laborious task of cutting and labelling tiny slices from each bone fragment. “I eventually prepared about 700 samples,” she recalls. These were then taken to Manchester for analysis in Buckley’s laboratory. “The results showed we had a lot of cow bones, a few other animals but no humans,” says Brown. “It was very disappointing.”

At that point, Brown could have left the project with honour. But she chose to go on. “Thank God she did,” says Higham. A further 1,500 Denisova bone samples were prepared by her and then taken to Manchester. This time, the results were spectacularly different. One bone, number 1,227, was identified as being from a human species.

“We could not believe that it had actually worked. It was wonderful,” says Douka. Brown was also overjoyed. “It was unbelievably exciting. We had not only shown the technique works but we had found a hominin. And I had been prepared for the worst.”

Facebook Twitter Pinterest Site of the Denisova Cave in Altai Mountains of southern Siberia. Photograph: AP

It was tremendous news for the team. But their discovery lacked a key piece of information. Yes, they had found a human of some kind, but which species? ZooMs can tell only if a bone comes from a member of the hominidae family, which includes great apes and humans, including Homo sapiens, Neanderthals and Denisovans. It cannot differentiate within this group. “There have never been great apes around Denisova so that meant we had to have found a piece of a human,” says Higham. “But which species?”

To find out, the sample was taken to Svante Pääbo at the Max Planck Institute for Evolutionary Anthropology in Leipzig, whose team had sequenced the first Denisovan genome in 2010. Initial analysis showed that the bone was more than 50,000 years old and from a person who had been 13 or older when they died.

Then the Leipzig team – led by Pääbo’s student Viviane Slon – began more detailed genetic analysis and made a startling discovery. Exactly half the sample consisted of Neanderthal DNA. The other half was made up of Denisovan DNA. At first, the researchers assumed that the sample was contaminated. “I thought they must have screwed up something,” says Pääbo.

But re-testing confirmed the finding: the Oxford team had discovered the 90,000-year-old remains of a hybrid daughter of a Neanderthal mother and a Denisovan father. She was nicknamed Denny. “If you had asked me beforehand, I would have said we will never find this, it is like finding a needle in a haystack,” Pääbo told Nature.

To discover a first-generation person of mixed ancestry was extraordinary. But there were more revelations to come. Further detailed studies of the genes of Denny’s Denisovan father were found to contain fragments of Neanderthal DNA. These indicated that interbreeding between the two species had also occurred at an earlier time.

At first sight, Denny’s remarkable ancestry suggests that Neanderthals and Denisovans must have mated with each other regularly. But Douka counsels caution. “The DNA of Neanderthals and Denisovans are distinct. We can easily tell them apart. That argues against frequent interbreeding. Otherwise they would have ended up with the same DNA.”

Past studies have provided clear evidence that Denisovans and modern humans interbred, and also that Neanderthals and modern humans mated with each. Now, thanks to ZooMs – which has since been used to pinpoint other ancient human remains – there is dramatic evidence of Denisovan and Neanderthal intermingling. But why at Denisova?

One suggestion is that the cave represents a border outpost for both species, one that was situated at the very eastern edge of the range of the Neanderthals, who were primarily a European species, and at the very western tip of the homelands of the Denisovans, who were an eastern species. Occasionally members from both groups would have reached the cave at the same time – with amorous consequences.

It is an idea borne out by detailed studies of Denny’s Neanderthal mother. Her genes show a particularly close affinity with Neanderthals who lived in Croatia, suggesting that the immediate predecessors of Denny’s mother may have been part of a group who slowly migrated east from Europe towards Denisova – where she encountered Denny’s father at the outer edges of each other’s homelands.

Facebook Twitter Pinterest Excavation at Denisova Cave. Photograph: Robert Clark

It is an intriguing picture, though much more information is required to confirm it. Scientists have no direct evidence that the Denisovans’ homeland range was primarily to the east of the cave, although the fact that their genes have been detected in the DNA of populations in Australia, New Guinea and other parts of Oceania, provides support for this idea and suggests future searches for sites should be focused on eastern Russia, China and south-east Asia.

A great deal more needs to be learned about the Denisovans, says Higham: “What was their distribution? What is the earliest evidence for their emergence from the common ancestor they shared with Neanderthals 500,000 years ago? If we could get a bone or two from other sites, that would be tremendously helpful.” One possible source of fossils could include remains of ancient humans that were placed in museums in Asia decades ago. These could be wrongly labelled, and could be Denisovans, researchers suggest. Unfortunately, it has proved difficult to get hold of these specimens for sampling.

For their part, Douka and Higham are planning on a number of approaches. One of these will be to collaborate with researchers in Chinese laboratories, teaching them how to use ZooMs, and how to use the technology to uncover more Denisovan sites.

“ZooMs is going to be crucial to this project,” says Douka. “We have shown that it is a powerful tool for pinpointing human fossils. That makes it ideal for tracking down Denisovans.”

The riches within Denisovan DNA

Knowledge of the Denisovan people emerged from studies of ancient genomes, which began 20 years ago when scientists first developed techniques for extracting DNA from fossils and for creating copies of that genetic material which allowed them to study it.

Scientists at the Max Planck Institute in Leipzig were studying bones and teeth found in Denisova cave in the Altai Mountains in Siberia, where Neanderthal fossils had been found. One bone and one tooth, they discovered, belonged to a previously unknown species of ancient human.

These few fossils proved to be spectacularly rich in genetic material, allowing scientists to sequence entire genomes. It is this detailed information about the Denisovan genome that has demonstrated they interbred with modern humans.

Descendants of these unions, carrying small amounts of Denisovan DNA, went on to settle in Melanesia and Australia thousands of years ago.

In this way, we can see Denisovan DNA in that of modern humans, though we have no idea what the Denisovans looked like or where, exactly, they lived.