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Researchers excavating Estatuas cave in Spain found a long record of Neanderthal DNA in the sediments.

© JAVIER TRUEBA/MADRID SCIENTIFIC FILMS

DNA from cave dirt tells tale of how some Neanderthals disappeared

Estatuas cave in northern Spain was a hive of activity 105,000 years ago. Artifacts show its Neanderthal inhabitants hafted stone tools, butchered red deer, and may have made fires. They also shed, bled, and excreted subtler clues onto the cave floor: their own DNA. “You can imagine them sitting in the cave making tools, butchering animals. Maybe they cut themselves or their babies pooped,” says population geneticist Benjamin Vernot, a postdoc at the Max Planck Institute for Evolutionary Anthropology (MPI-EVA), whose perspective may have been colored by his own baby’s cries during a Zoom call. “All that DNA accumulates in the dirt floors.”

He and MPI-EVA geneticist Matthias Meyer report today in Science that dirt from Estatuas has yielded molecular treasure: the first nuclear DNA from an ancient human to be gleaned from sediments. Earlier studies reported shorter, more abundant human mitochondrial DNA (mtDNA) from cave floors, but nuclear DNA, previously available only from bones and teeth, can be far more informative. “Now, it seems that it is possible to extract nuclear DNA from dirt, and we have a lot of dirt in archaeological sites,” says archaeologist Marie Soressi of Leiden University.

“This is a beautiful paper,” agrees population geneticist Pontus Skoglund of the Francis Crick Institute. The sequences reveal the genetic identity and sex of ancient cave dwellers and show that one group of Neanderthals replaced another in the Spanish cave about 100,000 years ago, perhaps after a climate cooling. “They can see a shift in Neanderthal populations at the very same site, which is quite nice,” Skoglund says.

To date, paleogeneticists have managed to extract ancient DNA from the bones or teeth of just 23 archaic humans, including 18 Neanderthals from 14 sites across Eurasia. In search of more, Vernot and Meyer’s team sampled sediment from well-dated layers in three caves where ancient humans are known to have lived: the Denisova and Chagyrskaya caves in Siberia and Estatuas cave in Atapuerca, Spain.

In what Skoglund calls “an amazing technical demonstration,” they developed new genetic probes to fish out hominin DNA, allowing them to ignore the abundant sequences from plants, animals, and bacteria. Then, they used statistical methods to home in on DNA unique to Neanderthals and compare it with reference genomes from Neanderthals in a phylogenetic tree.

All three sites yielded Neanderthal nuclear and mtDNA, with the biggest surprise coming from the small amount of nuclear DNA from multiple Neanderthals in Estatuas cave. Nuclear DNA from a Neanderthal male in the deepest layer, dating to about 113,000 years ago, linked him to early Neanderthals who lived about 120,000 years ago in Denisova cave and in caves in Belgium and Germany.

But two female Neanderthals who lived in Estatuas cave later, about 100,000 years ago, had nuclear DNA more closely matching that of later, “classic” Neanderthals, including those who lived less than 70,000 years ago at Vindija cave in Croatia and 60,000 to 80,000 years ago at Chagyrskaya cave, says co-author and paleoanthropologist Juan Luis Arsuaga of the Complutense University of Madrid.

At the same time, the more plentiful mtDNA from Estatuas cave shows declining diversity. Neanderthals in the cave 113,000 years ago had at least three types of mtDNA. But the cave’s Neanderthals 80,000 and 107,000 years ago had only one type. Existing ancient DNA from Neanderthal bones and teeth had also pointed to a falloff in genetic diversity over the same period.

Arsuaga suggests Neanderthals thrived and diversified during the warm, moist interglacial period that started 130,000 years ago. But about 110,000 years ago, temperatures in Europe dipped suddenly as a new glacial period set in. Soon after, all but one lineage of Neanderthals disappeared. Members of the surviving lineage repopulated Europe during later, relatively warm spells, with some taking shelter in Estatuas cave.

Those survivors and their descendants include what Arsuaga calls the “famous” classic Neanderthals, such as skulls from Vindija and La Ferrassie in France. He notes they had bigger brains—up to 1750 cubic centimeters (cm3)—than earlier Neanderthals, whose cranial capacities were no larger than 1400 cm3. Arsuaga says this mirrors a similar pattern in modern humans in Africa, who also underwent a surge in brain size and multiple population replacements with the onset of the ice age.

"This pattern—dispersal over perhaps long distances and population replacement or admixture—is one that we find almost everywhere we look,” in humans or other mammals, says Beth Shapiro, a molecular biologist at the University of California, Santa Cruz.

Cave dirt DNA is likely to yield more clues. Paleogeneticist Viviane Slon, a co-author of the Science paper now at Tel Aviv University, says she and the MPI-EVA team are analyzing ancient DNA from sediments at dozens of sites worldwide. “Hopefully soon, we’ll start to get a very high-resolution, fine-scale view of ancient humans and who was where at what time,” she says.