It was an otherwise ordinary day in 2015 when Viviane Slon had her eureka moment. As she worked at her computer, the results revealed the sample she was examining contained human DNA. There was nothing so unusual about that in itself: at the time, the ancient DNA (aDNA) revolution was in full swing, and surprising new insights about our ancestors were being gradually unveiled. But Dz’s sample wasn’t from human remains – it was just dirt from a cave floor. That immediately told her she was onto something big.
Many archaeological sites yield tools and artefacts that tell us about human occupation, but few have provided the bones or teeth that could still harbour human aDNA. Even when such remains are present, the chances that genetic material survives within them is slim because DNA is damaged by heat, moisture and acidity. So finding another source of aDNA – the soil itself – was a game changer. “That opens up hundreds of prehistoric sites that we couldn’t work on before,” says Slon.
Besides, humble dirt can reveal a lot about our distant past. Whereas fossils provide snapshots of prehistory, sediment gives a DNA source that can, in theory, generate an unbroken narrative. Researchers can study hominins predating the practice of burial. They can work out which groups created particular tools and other artefacts, learning more about their cognitive and artistic capacities. And, because the hominin DNA comes with that of ancient plants, animals and microbes, analysis of sedimentary aDNA can reconstruct what life was like for prehistoric humans. “It’s a huge advance,” says Chris Stringer at the Natural History Museum in London.
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As with some of the best discoveries in science, was an accident. Now at Tel Aviv University in Israel, in 2015 she was one of the young researchers in lab at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, who were attempting to refine the methods for extracting and analysing aDNA. Fossilised remains of hominins are rare and probing for genetic material damages them. So, she thought, before tampering with a precious relic in an attempt to extract a nugget of DNA, it would be useful to have an indication of whether any had survived.
Slon got her inspiration from a group led by geneticist at the University of Copenhagen, Denmark, which in 2003 had shown that . Her initial idea was that if aDNA from other mammals had survived to be detected in the soil around hominin remains, perhaps those remains would be more likely to contain aDNA too. Tests showed she was right.
But the surprise came when she used the method on soil from a Spanish cave called El Sidrón where 13 Neanderthals were buried 49,000 years ago. In 2017, Slon and her team reported finding . This was a first for aDNA in sediment (sedaDNA), but it was just the beginning.
The team had identified mitochondrial DNA (mtDNA), a type of genetic material that passes from mother to child. Cells contain many mitochondria, so, in theory, mtDNA is more plentiful than DNA from cells’ nuclei and hence dirt samples will be richer in it. But the latter is more informative, because the nuclear genome is larger and contains contributions from both parents. Finding nuclear DNA in sediment would be the next challenge. In 2021, , then in Meyer’s lab but now with his own group at the Max Planck Institute for Evolutionary Anthropology, . This time, the results weren’t just proof of principle, they also told a story.
Neanderthal revelation
Vernot analysed soil from three caves – two in Siberia and a third in Spain called Galería de las Estatuas – and found ancient human nuclear DNA in all three. Since the two Siberian caves had previously yielded hominin bones, he could show that the sedaDNA in the dirt samples confirmed what was already known. But the Spanish cave is where the new method came into its own. Although it was already established that this site had been occupied by Neanderthals for 40,000 years, it had only given up one tiny Neanderthal bone. The sediment analysis revealed much more: it showed that two distinct populations of Neanderthals had occupied Estatuas, with one replacing the other around 100,000 years ago.
That’s not all. By comparing the sedaDNA with aDNA from other Neanderthal populations across Eurasia, Vernot was able to link these two populations to two Neanderthal range expansions. These occurred on either side of the date that one group replaced the other in Estatuas – perhaps during warmer periods. “It’s a change in the populations in that area of the Neanderthal world that we didn’t have any inkling of before,” says Slon. Suddenly, the great arc of human history was visible, ebbing and flowing across Eurasia.
The ancient DNA field is often seen as cutthroat due to the scarcity of bones
Given its potential for such revelations, it is hardly surprising that many palaeoanthropologists and geneticists are embracing sedaDNA analysis. The cherry on the cake is that there is a near-unlimited supply of source material. That is a relief to some in the aDNA field, which has a reputation for being cutthroat due to the scarcity of bones. “There’s enough dirt in the world to go around,” says at Yale University, who was also previously in Meyer’s lab. The challenge is finding the DNA in all that dirt – and distinguishing hominin aDNA from that of the other species lurking there.
If you are lucky, hominin aDNA constitutes 0.001 per cent of sedaDNA, says Massilani. This makes sense, because in the deep past humans were massively outnumbered by other animals – even megafauna such as mammoths, bison and horses – as well as plants and microbes. But it also complicates matters. “It’s like looking for a needle in a haystack,” he says.
So far, the best sites for yielding sedaDNA have been dry caves. In these cases, geneticists work closely with archaeologists, taking sediment cores during active excavations where possible and correlating their findings with the stratigraphy – the geologically distinguishable layers.
More recent disturbance of these layers, for example by burrowing animals or leaching water, can complicate matters, but archaeologists are able to recognise and account for this when working out the chronological sequence. They can also date the layers using artefacts within them that are amenable to radiocarbon dating, along with clues from pollen and bone fragments from extinct species. The assumption is that any sedaDNA will be the same age as the layer it is found in. However, if enough is present, it can even date itself, by virtue of the mutations the DNA has clocked up over evolutionary time compared with known older or younger samples of the same species.
In a clean room, the geneticists extract the DNA from their sediment cores and “enrich” it for hominin DNA. This entails adding probes or bait comprising fragments of DNA found only in the archaic human group of interest that bind to their counterparts in the mix, so that these can be fished out. The hominin aDNA is then sequenced, which gives much finer-grained information about the group concerned, and also allows researchers to verify that it is ancient. They do this by discerning the chemical changes that have accrued in it over time, and that set it apart from potentially contaminating modern DNA.
“It’s the combination of the genetic work and the really detailed archaeological work that makes the technique powerful,” says Vernot. And, as Massilani told at the European Molecular Biology Laboratory in Heidelberg, Germany, it has already earned its stripes.
If you are lucky, hominin DNA constitutes 0.001 per cent of the ancient DNA in sediment
Take research done in Denisova cave, Siberia, which was occupied by a variety of human groups for more than 200,000 years. A team led by Elena Zavala at the University of Copenhagen, who is yet another Meyer lab alumna, used sedaDNA to . In another first, the researchers were also able to work out which groups created which of the thousands of artefacts that have been discovered in various layers of the cave floor.
Their analysis of mtDNA from 175 sediment samples revealed that the first hominins to occupy Denisova, starting around 250,000 years ago, were the eponymous Denisovans. They probably fashioned the oldest stone tools found there. At least two different populations of Denisovans and another of Neanderthals then alternated intermittently until about 45,000 years ago, when modern humans appeared in its cavernous chambers.
“In the upper layers, people have found these beautiful artefacts, bracelets and pendants that are usually associated with Homo sapiens,” says Zavala, “but there was no evidence that sapiens had been in the cave.” Archaeologists had wondered if Denisovans or Neanderthals made the objects, which would have been remarkable if true. Zavala’s discovery of genetic material from H. sapiens in the cave increases the odds that they were responsible for creating them.
The researchers also found animal DNA preserved in hundreds of other sediment samples, leading them to suggest that archaic humans had probably followed the mammals they preyed on into the Altai mountains where Denisova cave is located. Yaks and horses, for example, are known to have migrated there from South-East Asia via the foothills of the Himalayas, when the climate permitted.
We know that Denisovans and Neanderthals interbred at times, and that at least one individual found in Denisova cave had mixed Neanderthal-Denisovan parentage. Unfortunately, it is hard to tell from the sedaDNA whether the different lineages of Denisovans, Neanderthals and humans met in the cave: one of its limitations is that DNA traces found in the same layer can’t be differentiated in time. Nevertheless, with sedaDNA we can start to test predictions about the ranges and possible interactions of these hominins.
For years, Denisovans could only be definitively tied to Denisova cave. One or two other remains had come to light elsewhere, but they were so sparse that together they would fit inside a large envelope. Then, in 2020, a team led by at Lanzhou University, China, found DNA in cave sediments on the Tibetan plateau, which showed that were there 100,000, 60,000 and possibly 45,000 years ago. This fits with a recent prediction, based on evidence from climate science and palaeobiology, that these human ancestors and that, at times, their territories would have overlapped with that of Neanderthals, who preferred more temperate climes.
“With cave sediment DNA, we can really start to get a fix on the ranges of Neanderthals and Denisovans,” says Stringer. We might also learn more about how the areas they occupied overlapped with early modern humans. For example, Massilani has teamed up with archaeologists from Mongolia and the US to try to identify the hominin group that 45,000 years ago. “We suspect that these are the cultural remains of the first Homo sapiens living in the region, who would have met with Denisovans while expanding eastward,” says at the University of California, Davis.
Clues from dog DNA
Undoubtedly, sedaDNA has the potential to reveal a lot more about human evolution. But there are still some big unanswered questions, including where exactly any hominin DNA in sediment comes from. Corpses are obvious candidates, but such remains could have wound up there naturally or through burial, or may even been excreted by a predator. Fossilised hominin faeces and blood are other potential sources. Nor can geneticists know, at first glance, if they are dealing with one or more individuals. However, an ingenious experiment by Vernot is helping to narrow the possibilities. He asked his students to search for dog DNA throughout the Max Planck Institute of Evolutionary Anthropology building. Dogs, which are allowed inside, must pass through lifts and corridors to get to their owners’ offices. However, the students only detected canine DNA in the offices themselves. In other words, it requires a prolonged presence or concentrated deposition to produce DNA in detectable quantities – even when not accounting for a time lag of thousands of years.
Over millennia, DNA is gradually lost, and extracting informative quantities of the hominin variety is another problem. Massilani, for one, has been working on ways to optimise the return, using soil archives that have been hoarded by some archaeologists over the decades. Sampling blocks of sediment from 13 prehistoric sites across five continents, he found that . The clusters were often associated with bone fragments or fossilised faeces, giving a clue to the source of the DNA in soil. Then, by analysing the blocks containing concentrated amounts of hominin aDNA at much finer resolution, he was able to obtain far more information about the individuals who shed the DNA, down to their sex.
As the pioneers of this field improve their method, one of its most exciting applications could be in parts of the world that have yielded little or no aDNA to date, notably places where heat and high humidity are common. Massilani is currently testing his approach at cave sites in his native Gabon, and Slon has already up to 70,000 years old in an Israeli cave.
Other opportunities abound. Lake sediments could tell us about the world when sea levels were lower. Sites like the Tibetan cave that produced Denisovan sedaDNA could reveal how humans adapted to life at high altitude. From the assemblies of species that occur with archaic humans, researchers hope to track the domestication of plants and animals, as well as changes in human diet and health and the associated genetic adaptations. Within prehistoric settlements, they might be able to discern the segregation of people of different sexes or ancestry. Massilani can even envisage a future where it is possible to detect the presence of ancient people where no human sedaDNA has been found, just from changes in the associated microbial community.
The potential, if not as limitless as the supply of dirt, is huge – and still mostly unexplored. As Slon says: “We’re far from having exploited the possibility to its maximum.”
