Understanding the changes in technology and population that went on during the Palaeolithic is the key to understanding much of the history of our species. Unfortunately, Palaeolithic deposits tend to be highly condensed, so that objects and remains found alongside one-another may be separated by hundreds or even thousands and years, making it impossible to connect artefacts to remains unless they were clearly directly buried together.
Recent advances in the recovery of DNA from ancient sediments provide us with the possibility of establish connections between artefacts and specific Human populations, although this would require the recovery of DNA directly from the objects in question. Potentially, artefacts made from tooth and bone have the best chance of preserving ancient DNA, as they are porous and capable of absorbing body fluids, and because they contain hydroxyapatite, which is known to both absorb and preserve DNA. Thus ancient pieces of tooth or bone may potentially preserve DNA not only from the Animal from which they came, but also DNA from other organisms that came into contact with them after that Animal's death, For the most part, this tends to be the DNA of Microorganisms which colonised the decaying body of the original Animal, but could also potentially include Humans which handled these pieces, or made things from them.
However, Palaeolithic artefacts made from tooth and bone are extremely rare, and therefore of great value to science, something particularly true of pendants or other objects likely to have been worn close to the skin for long periods of time, and therefore the most likely to have absorbed ancient Human DNA. The conservation of such objects is therefore considered a high priority, and archaeologists are reluctant to expose them to the destructive methods used to extract DNA, or even to soak them in buffers which might absorb DNA passively, as these can alter specimens irreparably.
In a paper published in the journal Nature on 3 May 2023, a team of scientists led by Elena Essel of the Max Planck Institute for Evolutionary Anthropology, Elena Zavala, also of the Max Planck Institute for Evolutionary Anthropology, and of the Department of Biology at San Francisco State University, and the Department of Molecular and Cell Biology at the University of California, Berkeley, Ellen Schulz-Kornas of the Department of Cariology, Endodontology
and Periodontology at the University of Leipzig, Marie Soressi of the Faculty of Archaeology at Leiden University, and Matthias Meyer, again of the Max Planck Institute for Evolutionary Anthropology, describe development of a method for non-destructively recovering DNA from ancient bone and tooth artefacts, and the first results obtained using this technique.
In order to test potential reagents for DNA extraction from ancient artefacts, Essel et al. first obtained ten pieces of faunal remains from the Quinçay and Les Cottés Palaeolithic sites in France, none of which are thought to have been deliberately modified by Human activity, but all of which were similar in size and shape to common bone and tooth artefacts. These were immersed in ten different potential reagents, including a
guanidinium thiocyanate-containing reagent previously suggested for
non-destructive DNA extraction, an ethylenediaminetetraacetate
(EDTA) solution, which is a decalcifier commonly used in ancient DNA
extraction, a sodium hypochlorite (bleach) solution, which is
an oxidizing reagent used to remove surface-exposed contaminant
DNA, and a sodium phosphate buffer supplemented with detergent, which has been recently shown to enable temperature-controlled
DNA release from powdered bone samples.
The microtopography of the surface of these artefacts was mapped using quantitative 3D surface
texture analysis prior to immersion, and then again once the samples were removed, in order to detect any changes caused by the reagents. The guanidinium thiocyanate-containing reagent and ethylenediaminetetraacetate solution were both found to cause significant alteration to the samples, whereas all of the other samples caused much smaller and more sporadic changes (none of the reagents caused zero alterations), possibly due to the removal of small particles of soil and other substances from the sample surfaces.
Faunal remains used in reagent testing. Photographs
of samples taken before and after treatment with various reagents used in
ancient DNA extraction (GuSCN, guanidine thiocyanate reagent; EDTA, ethylenediaminetetraacetate solution; Phosphate, sodium phosphate buffer
with detergent; Bleach, sodium hypochlorite solution). The black bar
represents 1 cm. Note that colours are not directly comparable, as the
photographs were taken at slightly different angles, with different light
settings and camera adjustments. Essel et al. (2023).
Based upon this, Essel et al. were able to develop a step-wise method for the extraction of DNA from ancient bone and tooth artefacts. using serial incubations
in sodium phosphate buffer at 21, 37, 60 and 90 °C, with three incubations per temperature.
Workflow of the gradual, non-destructive DNA extraction method using
sodium phosphate buffer at elevated temperatures. Essel et al. (2023).
Next Essel et al. applied this to eleven bone and tooth objects from Châtelperronian layers of Quinçay Cave in France, all of which are believed to have potentially been used as tools between 35 000 and 45 000 years ago. One of these objects (Q10), identified as a piece of Reindeer bone, yielded 1828 fragments of Cervid mitochondrial DNA, which showed elevated
frequencies of cytosine-to-thymine substitutions at their ends, which is consistent with the deamination of cytosine seen in other ancient DNA samples, and which has been used to date such DNA. Another object (Q15), identified as a piece of ivory, yielded 2004 fragments of Elephantid mitochondrial DNA, again showing cytosine-to-thymine substitutions. All eleven samples also yielded Hominid and Suid mitochondrial DNA, none of which showed cytosine-to-thymine substitutions, and all of which is therefore is considered to be the result of modern contamination. Since all of these samples were found several decades ago, and were neither collected or stored under sterile conditions, this was not surprising.
Artefacts before and after DNA extraction.
Photographs of samples taken before and after phosphate-based,
non-destructive DNA extraction. The black bar represents 1 cm. Note that
colors are not directly comparable, as the photographs were taken at slightly
different angles, with different light settings and camera adjustments. Essel et al. (2023).
Given that contamination by modern DNA appeared to be ubiquitous in ancient bone and tooth objects that had been handled by hand, Essel et al. decided to directly obtain objects from ongoing excavations at two Palaeolithic sites currently still under investigation; Bacho Kiro Cave
in Bulgaria, and Denisova Cave in the Altai Republic of southern Siberia. Three tooth pendants (‘BKP1–BKP3) were obtained from Bacho Kiro, and one (DCP1) from Denisova Cave, all by archaeologists wearing gloves and facemasks to prevent contamination.
Photograph of DCP1 as it became exposed during excavation. The photograph was taken shortly before the pendant was removed and
placed into a plastic bag using gloves. Essel et al. (2023).
Large and visible chunks of soil were removed from these artifacts by (gloved) hand, and they were washed in water three times before being subjected to the phosphate buffer DNA extraction technique. All four pendants produced ancient Mammalian mitochondrial DNA, with BKP1 producing Bovid DNA, BKP2 and BKP3 producing Ursid DNA, and DCP1 producing Cervid DNA. Human mitochondrial DNA was recovered at much lower levels than from the Quinçay material, and very few pieces of Suid DNA suggesting that the modified excavation method had helped prevent the contamination of these samples. Notably, the DCP1 Human mitochondrial DNA showed signs of significant cytosine deamination, suggesting that this was indeed ancient Human DNA.
Photographs of DCP1 before and after
cleaning and non-destructive DNA extraction. Essel et al. (2023).
Very little ancient Human mitochondrial DNA was recovered from the Bacho Kiro Cave material, with the largest sample, 29 deaminated fragments, coming from a soil particle attached to BKP3. In contrast, DCP1 produced significant amounts of ancient Human mitochondrial DNA, enabling the construction of a near-complete consensus sequence, which could be used to place the material within a phylogenetic analysis. This DNA seemed to mostly (but not exclusively) originate from a single individual, assigned to mitochondrial haplogroup U (because mitochondrial DNA is found in the mitochondria, organelles outside the cell nucleus, it is passed directly from mother to child without being sexually recombined each generation, enabling precise estimations of when individuals shared common ancestors, at least through the female line, forming a mitochondrial haplogroup).
Essel et al. estimate that this Human mitochondrial DNA came from an individual lived about 18 500 years ago, and falls within a group of Ancient North Eurasians who otherwise are known from further east within Siberia, including the 24 000-year-old Mal’ta
1 individual, and the 17 000-year-old Afontova Gora 3 individuals. All of these samples are more closely related to one-another than to modern North Eurasians, and show affinities to other ancient Siberians and Native Americans. As well as the mitochondrial DNA, sufficient chromosomal DNA was recovered to establish that the individual was female.
Essel et al.'s work establishes that it is possible to recover ancient Human DNA from bone and tooth artefacts, providing a previously unexplored source of information about the makers and users of these ancient objects. The amount of ancient DNA recovered from DCP1 was comparable to that obtained from well-preserved Pleistocene Human remains, and the recovery of both Human and Cervid DNA from the same object enabled two separate cytosine deamination dates from the same artefact.
Future work will determine how frequently such ancient Human DNA is preserved within bone and took artefacts. Essel et al. recommend that all archaeologists working with such material adopt the practice of wearing gloves and masks while extracting bone and tooth sample at archaeological sites, and express the hope that in future it might be possible to establish a dataset which can connect specific late Pleistocene technologies to specific ancient Human populations.
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