Artefacts made out of stones but also made out of bones and teeth are frequently found in archaeological excavations. They provide us with invaluable insights into the behaviors, technologies, and survival strategies of our ancestors. However, due to the rarity of burials during the Palaeolithic, it has been extremely challenging to link these artefacts to specific individuals, and to make inferences about social structures or division of labor. Therefore, we set up to develop a non-destructive DNA extraction method which enables us to isolate the genetic material from bone and tooth artefacts - allowing for the direct identification of the individual (or individuals) who handled the object. This technique has the potential to deepen to our understanding of ancient societies and provide a new lens for examining the social and cultural dynamics of our distant past.
Have you ever admired an ancient artefact in a museum exhibition and marveled at its intricate details, the grooves of a bone tool or the smoothness of a pendant made out of a massive animal tooth? The first time I have ever held a Paleolithic bone tool in my hands (wearing gloves, of course!), I felt transported back in time, imagining the human hands that had created and used it thousands of years ago. As I looked at the object, a flood of questions came to mind: who was the person who made it? Was this tool passed down from one generation to the next, from a mother to a daughter or from a father to a son?
When Prof. Marie Soressi, a renowned professor of Hominin Diversity and Archaeology at the University of Leiden and a true expert on Palaeolithic artefacts, visited the Max Planck Institute for Evolutionary Anthropology in 2018 and brought with her a variety of ancient tools and jewelry made from bones, teeth, and even antlers. With each artefact she showed, my curiosity grew. I couldn't help but bombard her with my questions: How were they made? How were they used? And, perhaps, most interestingly - who were the people behind these items? Fortunately, Marie was more than willing to listen and answer all my questions. She explained that Palaeolithic graves are indeed very rare, making it nearly impossible to attribute any of these artefacts to specific individuals. But despite this challenge, Marie's enthusiasm for the subject was contagious, and I couldn't help but be to inspired by her passion for uncovering the mysteries of our ancient past.
Figure 1. Personal ornaments from layer 11 in the South Chamber of Denisova Cave: 1-4 – pendants made of mammalian teeth; 5 – ornamented bone pendant; 6 - bone tubular bead; 7 – flat bead made of bone; 8 - shell bead; 9 - chrysotile pendant. © Alexandr Fedorchenko.
The working group she visited in Leipzig, which I am fortunate enough to be a part of, was the one of Dr. Matthias Meyer, a team of scientists with a knack for developing cutting-edge methods tailored to ancient DNA (aDNA) research. The aim of our group is to both develop and refine aDNA methods, with the ultimate goal of pushing methodological advances beyond their limits, which then allows us the the exploration of hominin genetic history beyond what is currently thought possible and with deeper resolution. The year before, Matthias' team managed to isolate ancient hominin DNA from archaeological sediments, which initially sounded like a crazy idea. There was a bit of a gold-rush atmosphere in the team: on the one hand, to investigate exciting questions about human history using this new tool, and on the other hand, to believe that maybe – just maybe – some of the other crazy ideas that were slumbering in our drawers might, actually, work!
One of these, previously labeled ‘crazy’, ideas was to try to isolate authentic ancient DNA directly from the artefacts. Since we learned that DNA can bind to minerals and preserve in archeological sediments, our first attempts were trying to isolate ancient DNA from stone tools. Not much unlike the modern forensic investigations, the basic idea was to try to isolate the DNA that a person left on an object while they were handling it, - allowing the identification of the said person via genetic profiling. First, we had to figure out which artefacts to target. Stone tools seemed like the logical choice since they are abundant and durable. Ironically, the ancient DNA in the sediments these objects were buried in for the thousands of years was our biggest challenge! Even after a thorough washing, it was near to impossible to tell if the DNA we extracted came from the tool itself or just the surrounding sediment. So we turned our attention to skeletal artefacts – – which is just a fancy name to say the artefacts are made out of bits of bones and teeth (most often animal). As they are quite porous (you can think of them as a sponge), they could have potentially absorbed some of the DNA other than its own over the years.
Figure 2. Schematic of the temperature-controlled phosphate-based DNA extraction method that preserves the surface and integrity of osseous artefacts. ©Merlin Szymanski
Now, you may wonder, “why not work with bone and tooth artefacts from the beginning?” It turns out that extracting DNA from these types of artefacts presents special challenges. Our traditional ancient DNA extraction methods risk damaging the delicate surface structure and overall integrity of such objects. So how do you extract the DNA without damaging these rare and precious artefacts?
This is where Dr. Ellen Schulz-Kornas, an expert in surface structure measurements at the University of Leipzig, comes in. She worked with us in trying to develop a non-destructive DNA extraction method that does not damage the surface structure and does preserve the integrity of bone and tooth artefacts. We studied the effects of different extraction reagents on the surface structure of ancient faunal bones and teeth, which were to material typically used for artefact production. With Ellen's help, we developed a temperature-controlled phosphate-based DNA extraction method that preserves the surface structure and integrity of the objects it is used upon– just what we wanted!
Figure 3. Matthias Meyer at work in the clean laboratory at the Max Planck Institute for Evolutionary Anthropology © Max Planck Institute for Evolutionary Anthropology.
With this new tool in our toolbox, we could start working on some real artefacts. Thus, Marie went carefully through the collection of artefacts excavated back in the 1970s-1990s from the French cave, Quinçay, and selected the ones that looked most suitable for our first attempts of trying to get that precious ancient DNA. She came to Leipzig to join us in the eight–hours long extraction process in the clean lab of the Max-Planck-Institute for Evolutionary Anthropology (let me take this opportunity to say: I have a lot respect for Marie’s endurance and patience during the timely process under the somewhat challenging working conditions in the clean lab.).
Several weeks passed from when the extracts were converted into sequencing format, enriched for human DNA – the material we are looking for after all – and sequenced. We had been working on this first dataset for weeks, pouring all of our energy into it. We were excited to finally see the results of our work, but also nervous about what we might find. I told myself several times while starring at the white letters and numbers running through the black computer screen, that finding ancient human DNA on the artefacts was a long shot, but I couldn't help but hope. However, the moment the results came in, my heart sank. There was no ancient human DNA to be found. Instead, there was tons and tons of modern human DNA, that dreadful contamination every ancient DNA person fears the most. This contamination was mostly likely introduced during the excavation and subsequent studying of the artefacts. It was disappointing. But when looking deeper into the data coming from some of these artefacts, we found ancient mammalian DNA of the animal the artefact was made from. This was the proof that we needed that our approach worked in principle, but the massive amounts of modern human DNA hampered any possibility of the detecting traces of ancient human DNA, if they were there at all.
Therefore, we decided that we needed to shift our focus again, this time to artefacts that were excavated using measures to prevent the introduction of modern contamination. We were grateful when Tsenka Tsanova, one of the leading figures behind the team excavating Bacho Kiro cave (Bulgaria), contacted us asking if we were interested in working on three artefacts excavated under clean conditions (wearing gloves and face masks). We were enthusiastic and excited to work on these freshly excavated artefacts, and equally disappointed when we got back the sequencing results: again no trace of ancient human DNA. However, these artifacts came out with substantially less modern human contamination and loads of DNA from the animal of which the artefacts were made. We were on the right track after all! This raised our hopes that with more samples excavated cleanly there would be one that not only showed ancient animal DNA, but also ancient human DNA.
Figure 4. The pierced deer tooth discovered from Denisova Cave after DNA extraction © Max Planck Institute for Evolutionary Anthropology
In 2019, Maxim Kozlikin arrived at the MPI EVA with a small but promising sample in his bag - a pierced wapiti tooth still embedded in sediment and that had been excavated with gloves and face masks and set aside for us. We were particularly excited since Denisova Cave has been a treasure trove for ancient DNA researchers in the past, offering up breakthrough after breakthrough. Through years of close collaboration between the local excavation team and the MPI-EVA it was possible to sequence the first high coverage Neandertal genome, reveal the existence of a new ancient hominin group - the Denisovans - and even uncover the first sequenced hybrid offspring from Neandertals and Denisovans. We wasted no time in bringing the wapiti tooth to our clean lab and getting to work.
Though we processed the sample on the fast lane, the wait for the sequencing data felt like an eternity. The team tried to temper their expectations, reminding each other not to expect too much. But when we finally looked at the data together, we were stunned. There it was - ancient human DNA, and lots of it, in the highest temperature fractions recovered in the sequential extraction approach! It almost seemed too good to be true, so the team generated more data, ran more tests and did sanity checks to confirm the results. Fortunately, the results held up and it was time to call upon population genetics experts. Elena Zavala, back then a PhD student in Matthias’ group and Mateja Hajdinjak, back then PostDoc at the Francis Crick Institute, combined their skills. Through their expertise in analyzing challenging ancient DNA datasets, they were able to determine that the individual whose DNA was on the pendant was genetically related to contemporaneous ancient individuals from Siberia's east, known as the 'Ancient North Eurasians.' Moreover, they estimated the pendant's age to be between 19,000 and 25,000 years old, rendering it unnecessary to destroy the precious object for C14 dating. Benjamin Vernot, specialized on analyzing challenging data from sediment samples, applied methods highly specialized for data that are mixtures of different DNA sources. He was able to determine that the DNA we found on the pendant comes from a female individual, despite the challenge of retrieving mixed DNA from the wapiti and the human.
For the first time we were able to directly link the genetic trace of an ancient individual to an object handled by that person. It finally allowed us to answer some of the very questions that came into my mind when I looked at Paleolithic artefacts for the first time. With more samples being excavated using gloves and facemasks, thereby limiting the introduction of modern DNA, the doors have been opened to the possibility to someday systematically study the DNA of the makers or users of ancient artefacts that were made from bones and teeth.
Figure 5. Elena Essel during working on the pierced deer tooth discovered from Denisova Cave in the clean laboratory © Max Planck Institute for Evolutionary Anthropology
Working late in the evening in the clean lab is always a slightly eerie experience given the human remains stored there. We often talk about their spirits lingering around, playing tricks on us. So, on one such late night, during the initial testing of our novel DNA extraction approach, I set up a Thermo-Shaker at 90°C and carried on with my focused-pipetting. So focused that I paid no mind to the tubes that were under increasing pressure due to the heat. The sudden popping of all the tubes sounded like a ghostly chorus of angry spirits - it gave me such a fright that I jumped so high out of my chair, probably setting a new world record for lab-gymnastics. Fortunately, as it turned out, the spirits must have been pleased with our non-destructive DNA extraction approach, as it ended up being a resounding success. Who knew that the spirits could be such excellent project reviewers?
If you want to read in more detail how we recovered ancient human DNA from a Paleolithic pendant and what were able to find out about the person handling it, you can delve into our paper out today in Nature: www.nature.com/articles/s41586-023-06035-2
I am grateful to Louisa Jáuregui and Mateja Hajdinjak for their feedback on this blogpost, Alexandr Fedorchenko and the Max Planck Institute for Evolutionary Anthropology for providing photos, and Merlin Szymanski for the wonderful illustrations.
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