Human DNA extracted from a Palaeolithic pendant.

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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Jaculus jaculus & Jaculus hirtipes: Are the African Hammada Jerboa and the Lesser Egyptian Jerboa really separate species?

As desert specialist rodent species, African Jerboas, Jaculus spp., have drawn the attention of researchers due to their broad distribution across the Saharan-Arabian region and their high phenotypic and genetic variation. Within the five recognized species in the genus, particular attention has been given to two putative sister cryptic species, the African Hammada Jerboa, Jaculus hirtipes, and the Lesser Egyptian Jerboa, Jaculus jaculus, sometimes considered as a single species due to incongruences between molecular and morphological studies. Cryptic species are species that closely resemble other species, and cannot be separated by simple, non-invasive examination. While taxonomists have known about cryptic species for a long time, it was not realised how numerous such species were until taxonomists began to use genetics to determine relationships between species, about twenty years ago. The presence of cryptic species can have a profound influence on conservation efforts, as ‘species’ thought to be wide ranging with large populations and broad environmental tolerances can turn out to be complexes of closely related but reproductively isolated species, each with a smaller population and distribution, and narrower range of environmental tolerances. 

In a paper published in the journal BMC Evolutionary Biology on 13 February 2020, Ana Filipa Moutinho of the Department of Evolutionary Genetics at the Max Planck Institute for Evolutionary Biology, and the Research Centre in Biodiversity and Genetic Resources and the Department of Biology at the University of Porto, Nina Serén, also of the Research Centre in Biodiversity and Genetic Resources and the Department of Biology at the University of Porto, and of the Division of Ecology and Evolutionary Biology at the University of Jyväskylä, Joana Paupério, again of the  Research Centre in Biodiversity and Genetic Resources  at the University of Porto, Teresa Luísa Silva, again of the Research Centre in Biodiversity and Genetic Resources  at the University of Porto, and of the Life and Health Sciences Research Institute at the University of Minho, Fernando Martínez-Freiría, Graciela Sotelo, and Rui Faria, again of the Research Centre in Biodiversity and Genetic Resources  at the University of Porto, Tapio Mappes, also of the Division of Ecology and Evolutionary Biology at the University of Jyväskylä, Paulo Célio Alves and José Carlos Brito, again of the Research Centre in Biodiversity and Genetic Resources and the Department of Biology at the University of Porto, and Zbyszek Boratyński, once again of the Research Centre in Biodiversity and Genetic Resources  at the University of Porto, present the results of a genetic study based upon mitochondrial DNA designed to resolve the status of the two species. 

Northern Africa holds a great biogeographical interest owing to the strong species interactions (such as the competition for limited and ephemeral resources), the wide diversity of habitats and heterogeneous landscapes, and the complex palaeoclimatic and geological history. Available phylogeographic studies in this region have uncovered substantial taxa diversification induced by the climate shifts that occurred during the Pliocene-Pleistocene interval (from about million years ago onwards) and the successive range shifts of the Sahara Desert. These climatic fluctuations caused significant movements of the Sahara-Sahel boundaries, leading to alterations in the ecological composition of landscapes. Such dynamics resulted in new selective pressures and/or geographic isolation within lineages, causing events of genetic diversification, adaptation, and eventually speciation.

Moutinho et al. sampled mitochondrial DNA from both putative species from eleven countries across the Saharan, Sahelian and Arabian regions; Mauritania, Morocco, Mali, Niger, Tunisia, Libya, Algeria, Senegal, Egypt, Kuwait, and Israel. 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 (it is also possible to trace direct ancestry through the male line, using DNA from the Y chromosome, which is passed directly from father to son without sexual recombination).

A Lesser Egyptian Jerboa, Jaculus jaculus, (top) and an African Hammada Jerboa, Jaculus hirtipes, (bottom). Moutinho et al. (2020).

Moutinho et al. found that all of the Jerboa's sampled belonged to two haplogroups (a haplogroup is a group of individuals shown to share a common ancestry through the male or female line, using either mitochondrial or Y chromosome DNA), which corresponded to the two species, strongly supporting the idea that these are true biological species (i.e. species which do not interbreed). 

Phylogenetic relationship of Jaculus individuals and their geographic distribution across North Africa. a Phylogenetic tree based on Bayesian inference showing the relationship among the haplotypes of two Jaculus species for the cytb gene. Values on branches indicate Bayesian posterior probabilities support and bootstrap values of Maximum-Likelihood analysis, respectively. White circles indicate posterior probabilities and bootstrap values above 0.91/91, respectively, for internal nodes. On each clade, the respective species is indicated. Jaculus orientalis (seven individuals, belonong to two haplotypes) was used as outgroup. Each tip of the tree branches is coloured according to the country of origin of each individual belonging to a haplotype. (b) Geographical locations of all Jaculus individuals used in this study. Red (circles) and green (triangles) samples denote, respectively, Jaculus jaculus and Jaculus hirtipes. Moutinho et al. (2020).

Both species were highly variable across their range, suggesting degree of environmental flexibility, with populations adapting to local environmental conditions. Interestingly, for both species individuals from Israel, where it has previously been claimed that the two species are more easily differentiated than in other areas, form distinct clusters, which may indicate some level of geographic isolation from other populations, and potentially niche partitioning between the two species (i.e. the two species may avoid competing with one another by occupying different ecological niches). 

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Andrias davidianus: How failure to recognise a species complex may be undermining efforts to preserve the Chinese Giant Salamander.

The Chinese Giant Salamander, Andrias davidianus, is the largest extant species of Amphibian, reaching about 1.8 m in length. It is one of only three species of Giant Salamander, Cryptobranchidae, along with the Japanese Giant Salamander, Andrias japonicus, and the Hellbender, Cryptobranchus alleganiensis, which is found in the eastern United States, which are thought to have diverged from their closest relatives during the Middle Jurassic, around 170 million years ago. The species was once widespread across China, but it's wild population is thought to have declined by about 80% since the 1950s, due to a combination of habitat loss, disease, and consumption by Humans. For this reason the species is considered to be Critically Endangered under the terms of the International Union for the Conservation of Nature’s Red List of Threatened Species. As such the species has become a focus for conservation efforts in China, which have focused on the release of large numbers of farmed Giant Salamanders back into the wild, with over 78 000 farmed Salamanders released into the wild since 2008.

In a paper published in the journal Current Biology on 21 May 2018, a group of scientists led by Fang Yan of the State Key Laboratory of Genetic Resources and Evolution at the Kunming Institute of Zoology of the Chinese Academy of Sciences present evidence that the Chinese Giant Salamander should in fact be considered to be a species cluster (group of morphologically identical but genetically distinct species, known as 'cryptic species') rather than a distinct species, and discuss the implication of this for conservation efforts.

A Chinese Giant Salamander, Andrias davidianus, in the Shanghai Aquarium. Patrick Fischer/Wikimedia Commons.

Yan et al. collected genetic samples from 70 wild caught and 1034 farmed Salamanders from across the species' range. They found that the wild Salamanders could be grouped into five distinct genetic populations, which appear to have diverged from one-another between 4.71 and 10.25 million years ago, sufficient to be considered to be separate species under most classification systems (Humans and Chimpanzees are thought to have diverged between 4 and 13 million years ago). This is not greatly surprising, as the Salamander favour upland environments, and therefore have a scattered distribution, with wide areas in between that the Salamanders cannot easily cross. 

The farmed Salamanders, in contrast, appeared to be largely hybrids, with the majority of their DNA derived from a population in the Yellow River in Shaanxi Province, the area where they were first farmed. The ability of the separate species to hybridise is not considered surprising, as the Chinese Giant Salamander is known to be able to hybridise with the (more distantly related) Japanese Giant Salamander. However the resultant hybrid Salamanders show much lower genetic diversity than wild Salamanders, and may be less well adapted to surviving in the wild. 

mtDNA haplotype clades and genetic structure of wild-caught and farm-bred Chinese Giant Salamanders. (A) A simplified Bayesian inference tree based on concatenated mtDNA haplotypes. Numbers near branches are posterior probabilities (BPP ≥ 0.90). A–E are wild caught populations and U1 and U2 are known from farms only.  (B) Sampling sites for wild-caught individuals. Lower-right insert shows the best genetic clustering (K = 5) based on genomic SNPs from localities circled in dashed line. (C) Sampling sites for farm-bred individuals where pie charts show the proportions of mitochondrial haplotypes as grouped in (A). Lower-right insert shows the second-best cluster (K = 3; optimal K = 1) based on microsatellite data for farm-bred individuals from Guizhou (farms circled in dashed line). Colours denote cryptic species lineages. Yan et al. (2018).

Yan et al. express concern that the practise of releasing farmed Salamanders (from populations originally intended form commercial exploitation as a food animal) into the wild may have a strongly adverse effect on the wild populations, potentially leading to some species becoming extinct; indeed they could find no pure indigenous Salamanders in some tributaries of the Pearl and Yangtze rivers, where all captured individuals had mtDNA (mitochondrial DNA, which is passed on only through the female line) from Yellow River Salamanders. They recommend that in future conservationists should avoid the release of captive-raised animals until a full study of the genetic structure of the subject population is carried out.

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Identifying the clothes of Ötzi the Iceman.

In 1991 two German hikers in the Ötztal Alps of the South Tyrol Region of Italy discovered a Human body partially exposed on the surface of a glacier. Careful examination of this body revealed it to be, not as originally thought a modern corpse, but the mummified remains of a 5300-year-old Chalcolithic (Copper Age) hunter, the oldest mummified remains ever discovered in Europe. The mummy, dubbed Ötzi the Iceman, became an overnight sensation, and has been the subject of numerous studies, due to the unique insight he offers into the lives of Chalcolithic Europeans.

Ötzi the Iceman as he was originally discovered. South Tyrol Museum of Archaeology.

As well as the body of Ötzi himself, the site yielded numerous items of personal effects, the clothes worn, and goods carried by, Ötzi at the time of his death. One area that has interested scientists since the time of the discovery has been the animals from which the Ötzi's cloths were made. A variety of approaches have been tried in trying to solve this puzzle, including DNA analysis, proteomic analyses (analyses of proteins) and study of the microstructure of the leather, but all have been thwarted by the advanced state of decomposition of the material.

In a paper published in the journal Scientific Reports on 18 August 2016, Niall O’Sullivan of the Institute for Mummies and the Iceman and the School of Archaeology and Earth Institute at University College Dublin, Matthew Teasdale and Valeria Mattiangeli of the Smurfit Institute of Genetics at the Trinity College, Dublin, Frank Maixner, also of the Institute for Mummies and the Iceman, Ron Pinhasi, also of the School of Archaeology and Earth Institute at University College Dublin, Daniel Bradley, also of the Smurfit Institute of Genetics at the University of Dublin, and Albert Zink, again of the Institute for Mummies and the Iceman, describe the results of a new study of the clothes of Ötzi the Iceman, which used mitochondrial DNA capture in oder to identify the animals from which the skins had come.

Mitochondrial DNA (mtDNA) 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. Importantly, for the purposes of this study, mtDNA comes in much shorter packages than chromosomal DNA (it contains the code for making and operating the mitochondria only, not that for the whole body), and there is much more of it, since each mitochondria has its own DNA and there are typically 1000-2000 mitochondria in each cell, making it much more likely that enough mtDNA will survive for useful analysis.

O'Sullivan et al. were able to extract mtDNA from nine fragments of leather; four fragments of coat, one piece of loincloth, one piece of leggings, a shoelace, a piece of hat and a piece of leather from a quiver (arrow carrying device). Of these pieces thre fragments of the coat and the loincloth were found to be Sheepskin, the remaining piece of the coat and the leggings were Goatskin, the shoelace was Cow leather, the hat Bearskin and the quiver made from the skin of a Roe Deer.

The remains of Ötzi's coat, made from pieces of different skins. South Tyrol Museum of Archaeology.

At first sight the number of different skins, and the fact that these skins came from different animals, in the coat appears to suggest that clothing was assembled in a haphazard way from whatever materials were at hand. However making such a garment from different cuts of leather or cloth would not be unusual today. The fact that Ötzi's leggings were made out of Goatskin is illuminating, since a similar pair of leggings from a 4500-year-old Chalcolithic site in Switzerland were also made of Goatskin, suggesting that people were consistently using the same material to make the same garments, implying that the skins were being actively chosen for properties that made them suitable for certain garments.

Ötzi's leggings were made from several different peieces of skin, although mtDNA was obtained from only one fragment. South Tyrol Museum of Archaeology.

The Sheep and Goat skins used to make Ötzi's clothing, as well as the Cow leather of his shoelace all appear to have come from domestic animals, but the Deerskin of the quiver and Bearskin of the hat were almost certainly from wild animals, indicating that the Chalcolithic Alpine people were utilizing material from both sources. Interestingly an earlier study had previously revealed that Ötzi's stomach contents included meat from a Red Deer, as well as identifying several hairs found on Ötzi's body as also having come from Red Deer, indicating that he was able to utilize at least two Deer species.

Ötzi's quiver, made from the skin of a Roe Deer. South Tyrol Museum of Archaeology.

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Determining the diets of Late Mesolithic Humans at Cnoc Coig, Scotland.                   In recent years isotopic and molecular analysis methods have transformed archaeology, enabling modern archaeologists to study the diets of and relationships between ancient peoples in a way that would have been unthinkable a few decades ago...

Animal remains from Middle Neolithic deposits at the Pena d’Água Rock-shelter of Portugal.                                                   Middle Neolithic remains are known from a number of archaeological sites across Portugal, and have been studied since the nineteenth century. However historically almost all studies of this material have concentrated on funerary behavior rather than...

The first dairy farmers in Finland.                  Dairy farming (keeping Mammals in order to consume their milk or products derived from it) spread through Europe as part of the ‘Neolithic Package’ of technologies, which originated in the Middle East about 11 000 years ago. Despite this development...

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