Extraction of ancient human DNA from artefacts

The Denisova cave in southern Siberia is now famous for the evidence that it has provided for Neanderthals and Denisovans and their interbreeding based on DNA recovered from their bones, even a tiny finger bone of the latter. Indeed we would not know of the former existence of Denisovans without such a clue. Scientists at the Max Planck Institute for Evolutionary Anthropology in Leipzig, responsible for both breakthroughs, also pioneered the extraction of hominin DNA from soil in the cave. Now they have refined the intricate extraction of genetic material to such an extent that detailed hominin DNA sequences can be analysed from ornaments worn by ancient people, in much the same manner as applied in forensic studies of crime scenes (Essel, E. and 22 others 2023. Ancient human DNA recovered from a Palaeolithic pendant. Nature, early release 3 May 2023; DOI: 10.1038/s41586-023-06035-2).

Elk-tooth pendant found at Denisova cave, before cleaning and DNA extraction (top) and after the ‘washing’ procedure (bottom). Credit: Essel et al., Fig 1.

Russian archaeologists who continue to work at Denisova cave found a pierced pendant made from the tooth of a Siberian elk or wapiti during the 2019 field season. It was sent to Leipzig, where the palaeogenetics team had been trying to extract the DNA of whoever had worn personal artefacts found in French and Bulgarian caves. Their efforts had been unsuccessful, but such an object from Denisova clearly spurred them on. When someone wears next to the skin objects made of porous materials their sweat and the DNA that it carries seeps into the pores. If the materials decay very slowly, as do bone and especially teeth, genetic material can, in principle be extracted. But crushing up important ancient objects is not an option: for such rarities the extraction has to be non-destructive. It can only be done by ‘washing’ it in reagents that do not themselves break down DNA. Elena Essel and her many colleagues experimented with many ‘brews’ of reagents and repeated immersion at steadily rising temperature (up to 90°C). This releases genetic material in a stepwise fashion, allowing separation of contaminants in the host sediment from that which had penetrated into the tooth’s pores from whoever made the pendant and the wearer, and the animal from which it came

 Analysis of the recovered material yielded elk mtDNA, which was compared with that from four other ancient elks of known ages. This suggested that the elk had lived between 19 and 25 ka ago, thereby indirectly dating the time when the pendant was made and worn. A surprisingly large amount human DNA showed that the wearer was a female who was genetically allied with ancient anatomically modern humans who lived further east in Siberia at about that time.

Obviously this astonishing result opens up a wide vista for archaeology, though not from Palaeolithic burials, which are extremely rare. But artefacts of various kinds are much more common that actual human remains. Because the technique is non-destructive museums may be more willing to make objects in their collections available for analysis. Maybe the approach will be restricted to porous bone or tooth ornaments worn for long periods by individuals. Yet stone tools that were handled continually could be a more important target, depending on the rock from which they were made and its porosity.

See also: Lesté-Lasserre, C.. DNA from 25,000-year-old tooth pendant reveals woman who wore it. New Scientist, 3 May 2023.

A stratigraphic timeline for the Denisova Cave

Denisova Cave was named to commemorate an 18th century hermit called Denis, who used it as his refuge. The culmination of more than four decades of excavation, which followed the discovery there of Mousterian and Levallois tools there, has been the explosion onto the palaeoanthropological scene of Denisovan genomics, beginning in 2010 with sequenced DNA from a child’s finger bone. The same layer yielded Neanderthal DNA from a toe bone in 2013. Another layer yielded similar evidence in 2018 of an individual who had a Neanderthal father and a Denisovan mother. Application of the new technique of peptide mass fingerprinting, or zooarchaeology by mass spectrometry (ZooMS), to small, unidentifiable bone fragments from the cave sediments revealed further signs of Denisovan occupation and the first trace of anatomically modern humans (AMH). So far the tally is 4 Denisovans (two female children and two adult males), a Neanderthal woman and the astonishing hybrid. Analyses of the sediments themselves showed traces of both Neanderthal and Denisovan mtDNA from deeper in the stratigraphy than levels in which human fossils had been found, but which contained artefacts. The discovery of the first Denisovan DNA revealed that AMH migrants from Africa who reached the West Pacific islands about 65 ka ago carried fragments of that genome. As well as hybridising with Neanderthals some of the people who left Africa had interbred with Denisovans sufficiently often for genetic traces to have survived. Yet, until now, the ages of the analysed samples from the cave remained unknown.

That is no surprise for two reasons: cave sediments are complex, having been reworked over millennia to scramble their true stratigraphy; most of the organic remains defied 14C dating, being older than its maximum limit of determination. However, using alternative approaches has resulted in two papers in the latest issue of Nature. The first reports results from two methods that rely on the luminescence of grains of quartz and feldspar when stimulated, which measures the time since they were last exposed to light (Jacobs, Z. and 10 others 2019. Timing of archaic hominin occupation of Denisova Cave in southern Siberia. Nature, v. 565, p. 594-599; DOI: 0.1038/s41586-018-0843-2). Over 280 thousand grains in 103 sediment samples from different depths and various parts of the cave system have yielded a range of ages from 300 to 20 ka that span 3 glacial-interglacial cycles except for a few gaps, giving rough estimates of the timing of hominin occupation shown by fossils and soil layers that contain DNA. The youngest evidence for Denisovans is shown to be roughly 50 ka; a time when AMH was present elsewhere in Siberia. They lived at a time halfway between the 130 ka interglacial and the last glacial maximum. Two Neanderthals, a Denisovan and the hybrid occupied the site during the 130 ka interglacial. Soils from the previous warm episode from 250 to 200 ka contain both Neanderthal and Denisovan DNA traces. The oldest occupancy, marked by the presence of a Denisovan bone sample, was 300 ka ago, once again midway between an interglacial and a glacial maximum.

All the hominin remains found in Denisova Cave: Note the common scale. (Credit: Douka et al. 2019; extended data Figure 1)

The second paper (Douka, K. and 21 others 2019. Age estimates for hominin fossils and the onset of the Upper Palaeolithic at Denisova Cave. Nature, v. 565, p. 640–644; DOI: 10.1038/s41586-018-0870-z) focused on direct dating of the hominin fossils themselves – and thus their DNA content, important in trying to piece together timings of genetic mixing. In the absence of radiocarbon dates from the bones themselves because of most specimens’ >50 ka ages, except in the case of the youngest whose 14C age is at the 50 ka limit. They resorted to a hybrid technique based on a means of modelling fossils’ ages from differences in mtDNA between the specimens and that in the youngest hominin, which, luckily, was dateable by radiocarbon means. Weighted by dating of the actual sediments that contain them, the differences should become greater for successively older fossils because of random mutations: a variant of the ‘molecular clock’ approach. It’s complicated and depends on assuming that mitochondrial mutation rate was the same as that in modern humans. Unsurprisingly the results are imprecise, but sufficient to match the hominin fossil occurrences with different environmental conditions

Pollen grains and vertebrate fossils from various levels in the cave system demonstrate the wide climatic and ecological conditions in which the various hominins lived. The warmest episodes supported broad-leafed forest, offering maximum resources for hominin survival. Those between interglacial and full glacial conditions were much less benign, with alternating dry and wet cold conditions that supported open steppe ecosystems. The oldest Denisovan occupation was at the close of a period of moderately warm and humid conditions that supported mixed conifer and broad-leafed trees that gave way to reduced tree cover.

As well as the presence of stone tools sporadically through the sedimentary sequence, in the youngest levels there are bone rings and pendants made from deer teeth; clearly ornamental items.  Did the late Denisovans make them or do they signify anatomically modern human activity? Radiocarbon ages do not give a concrete answer, one of the pendants is about 45 ka old with an error that puts it just within the range of age variation of the oldest Denisovan fossil. No AMH remains have been found in Denisova Cave, but remains of a modern human male have been found at Ust’-Ishim, in NW Siberia. At 45 ka, he represents the earliest arrival of AMH in northern Asia. So it may have been members of this new population that left ornaments in Denisova, but, for the moment, artistic Denisovans are a possibility.

Further deployment of rapid screening for hominin bone fragments using the ZooMS method and analyses for traces of DNA in soils is likely to expand the geographic and time ranges of Denisovans and other close human relatives. Denisova Cave formed in Silurian limestones of the Altai Range, and there are other caves in those hills …

Related article: Dennel, R. 2019. Dating of hominin discoveries at Denisova. Nature, v. 565, p. 571-572; DOI: 10.1038/d41586-019-00264-0)

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