An international, interdisciplinary research team, led by the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, has developed a novel method for DNA isolation from bones and teeth. Although they are generally rarer than stone tools, the scientists focused specifically on artifacts made from skeletal elements—an ancient deer tooth pendant in this case—because these are more porous and are therefore more likely to retain DNA present in skin cells, sweat, and other body fluids.

Their research paper, “Ancient human DNA recovered from a Paleolithic pendant,” appears in Nature.

“Here we report the development of a non-destructive method for the gradual release of DNA trapped in ancient bone and tooth artifacts,” wrote the investigators. “Application of the method to an Upper Paleolithic deer tooth pendant from Denisova Cave, Russia, resulted in the recovery of ancient human and deer mitochondrial genomes, which allowed us to estimate the age of the pendant at approximately 19,000–25,000 years.

“Nuclear DNA analysis identifies the presumed maker or wearer of the pendant as a female individual with strong genetic affinities to a group of Ancient North Eurasian individuals who lived around the same time but were previously found only further east in Siberia. Our work redefines how cultural and genetic records can be linked in prehistoric archaeology.”

Ancient DNA studies also track changes in human biology over time

While the team’s goal was to directly link cultural objects to specific individuals and thus gain deeper insights into Paleolithic societies, “comparing past genomes also allows scientists to determine how different human groups are interconnected, and how migrations happened at different times in history,” according to Mateja Hajdinjak, PhD, a visiting scientist at the Francis Crick institute in London.

“Migrations allow people to mix and reproduce with new groups, which changes human biology over time,” she told Horizon last September.

The team tested the influence of various chemicals on the surface structure of archaeological bone and tooth pieces and developed a non-destructive phosphate-based method for DNA extraction. “One could say we have created a washing machine for ancient artifacts within our clean laboratory,” explained Elena Essel, the lead author of the study and a PhD student who developed the method. “By washing the artifacts at temperatures of up to 90°C, we are able to extract DNA from the wash waters, while keeping the artifacts intact.”

Lead author Elena Essel working in the clean laboratory on the pierced deer tooth discovered from Denisova Cave [
Lead author Elena Essel working in the clean laboratory on the pierced deer tooth discovered from Denisova Cave. [© MPI f. Evolutionary Anthropology]

The researchers first applied the method to a set of artifacts from the French cave Quinçay excavated back in the 1970s to 1990s. Although in some cases it was possible to identify DNA from the animals from which the artifacts were made, the vast majority of the DNA obtained came from the people who had handled the artifacts during or after excavation. This made it difficult to identify ancient human DNA.

To overcome the problem of modern human contamination, the researchers then focused on material that had been freshly excavated using gloves and face masks and put into clean plastic bags with sediment still attached. Three tooth pendants from Bacho Kiro Cave in Bulgaria, home to the oldest securely dated modern humans in Europe, showed significantly lower levels of modern DNA contamination; however, no ancient human DNA could be identified in these samples.

Pendant from Denisova Cave

The breakthrough was finally enabled by Maxim Kozlikin, PhD, and Michael Shunkov, PhD, archaeologists excavating the Denisova Cave in Russia. Kozlikin is a senior researcher and Shunkov is a professor at the Institute of Archaeology and Ethnography (Siberian Branch of the Russian Academy of Sciences).

In 2019, unaware of the new method being developed in Leipzig, they cleanly excavated and set aside an Upper Paleolithic deer tooth pendant. From this, the geneticists in Leipzig isolated not only the DNA from the animal itself, a wapiti deer, but also large quantities of ancient human DNA. “The amount of human DNA we recovered from the pendant was extraordinary,” said Essel, “almost as if we had sampled a human tooth.”

Excavations in the South Chamber of Denisova Cave in 2019.
Excavations in the South Chamber of Denisova Cave in 2019. [© Sergey Zelensky]

Based on the analysis of mitochondrial DNA, the researchers concluded that most of the DNA likely originated from a single human individual. Using the wapiti and human mitochondrial genomes they were able to estimate the age of the pendant at 19,000 to 25,000 years, without sampling the precious object for C14 dating.

In addition to mitochondrial DNA, the researchers also recovered a substantial fraction of the nuclear genome of its human owner. Based on the number of X chromosomes they determined that the pendant was made, used, or worn by a woman. They also found that this woman was genetically closely related to contemporaneous ancient individuals from further east in Siberia, the so-called “Ancient North Eurasians” for whom skeletal remains have previously been analyzed. “Forensic scientists will not be surprised that human DNA can be isolated from an object that has been handled a lot,” said Matthias Meyer, group leader, advanced DNA sequencing techniques group, Max Planck Institute for Evolutionary Anthropology, “but it is amazing that this is still possible after 20,000 years.”

The scientists now hope to apply their method to many other objects made from bone and teeth in the Stone Age to learn more about the genetic ancestry and sex of the individuals who made, used, or wore them.

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