Science

Ancient human DNA recovered from a Palaeolithic pendant

Published

2 years ago

May 3, 2023

Abstract

Artefacts made from stones, bones and teeth are fundamental to our understanding of human subsistence strategies, behaviour and culture in the Pleistocene. Although these resources are plentiful, it is impossible to associate artefacts to specific human individuals¹ who can be morphologically or genetically characterized, unless they are found within burials, which are rare in this time period. Thus, our ability to discern the societal roles of Pleistocene individuals based on their biological sex or genetic ancestry is limited^2,3,4,5. Here we report the development of a non-destructive method for the gradual release of DNA trapped in ancient bone and tooth artefacts. Application of the method to an Upper Palaeolithic 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.

Main

Palaeolithic assemblages typically contain a multitude of objects that may differ in age by hundreds or thousands of years, even when found in close proximity¹. Thus, it can be challenging to associate human remains with specific objects. Recent advances in the retrieval of human DNA from sediments^6,7,8 can be used to connect artefacts with genetic populations. However, precise identification of the specific makers or users of these objects would require the recovery of human DNA directly from the objects themselves, analogous to modern-day forensic investigations. In theory, such analyses are most promising for artefacts made from animal bones or teeth, not only because they are porous and thereby conducive to the penetration of body fluids (for example, sweat, blood or saliva) but also because they contain hydroxyapatite, which is known to adsorb DNA and reduce its degradation by hydrolysis and nuclease activity^9,10. Ancient bones and teeth may therefore function as a trap not only for DNA that is released within an organism during its lifetime and subsequent decomposition but also for exogenous DNA that enters the matrix post-mortem through microbial colonization¹¹ or handling by humans. However, DNA extraction from ancient skeletal material either requires destructive sampling, or risks alteration of specimens if they are directly submerged in extraction buffer^12,13. Conservation is a primary concern because of the scarcity of bone and tooth artefacts at Pleistocene sites, especially of pendants and other ornaments that were extensively handled or worn in close body contact. We therefore set out to develop a method for DNA isolation from bones and teeth that preserves the integrity of the material, including surface microtopography, and to investigate the possibility of DNA retrieval from bone and tooth artefacts.

A non-destructive DNA isolation method

To identify reagents compatible with non-destructive DNA extraction, we selected ten unmodified faunal remains from the Palaeolithic sites of Quinçay and Les Cottés in France (Extended Data Table 1 and Supplementary Information 1), which were similar in size and shape to material typically used for osseous artefact production, and submerged them in several reagents previously used in ancient DNA extraction, as well as in water for comparison. These included (1) a guanidinium thiocyanate-containing reagent previously suggested for non-destructive DNA extraction¹², (2) an ethylenediaminetetraacetate (EDTA) solution, which is a decalcifier commonly used in ancient DNA extraction^14,15,16, (3) a sodium hypochlorite (bleach) solution, which is an oxidizing reagent used to remove surface-exposed contaminant DNA^11,17, and (4) a sodium phosphate buffer supplemented with detergent¹¹, which has been recently shown to enable temperature-controlled DNA release from powdered bone samples¹⁸.

Mapping of the microtopography using quantitative 3D surface texture analysis^19,20 before and after the treatments revealed substantial surface alterations on all objects exposed to either the guanidinium thiocyanate reagent or EDTA (Extended Data Fig. 1 and Supplementary Information 2). By contrast, only sporadic and smaller alterations were detected with the other reagents, including sodium phosphate buffer (Fig. 1b), possibly due to the removal of traces of sediment and other small particles, as indicated by visible changes in coloration of some of the objects (Extended Data Fig. 2). On the basis of these results, we developed a non-destructive DNA isolation method for the stepwise release of DNA from the bone or tooth matrix using serial incubations in sodium phosphate buffer at 21, 37, 60 and 90 °C, with three incubations per temperature (Fig. 1a).

Fig. 1: Overview of the non-destructive DNA extraction method.

a, Workflow of the gradual, non-destructive DNA extraction method using sodium phosphate buffer at elevated temperatures. b, Four 3D surface texture measurements (1–4) indicated on the outline of a tooth used for testing (SP6649) before and after non-destructive DNA extraction showing no substantial surface alterations. c, Photographs of DCP1 before and after cleaning and non-destructive DNA extraction.

Full size image

We then applied this method to 11 osseous objects, labelled Q10 to Q19 as well as Q27, that were excavated several decades ago in the Châtelperronian layers of Quinçay Cave in France and that had potentially been used as tools some 35–45 thousand years ago (ka)²¹ (Extended Data Table 1 and Extended Data Fig. 3). We prepared single-stranded DNA libraries^22,23 from the first DNA fraction recovered at each temperature, and enriched the libraries for mammalian mitochondrial (mt) DNA²⁴. A metagenomic pipeline for assigning sequenced mtDNA fragments to mammalian taxa on the biological family level⁶ identified 1,628 cervid mtDNA fragments in the 60 °C and 90 °C fractions of object Q10, a reindeer bone (109 and 1,519 fragments, respectively; Fig. 2 and Supplementary Data 1). These fragments showed elevated frequencies of cytosine (C)-to-thymine (T) substitutions at their ends, consistent with deamination of cytosine residues seen in ancient DNA^6,25 (Supplementary Data 1). Another object, Q15, which was made of ivory, yielded 2,004 elephantid mtDNA sequences with elevated frequencies of C-to-T substitutions in the 37 °C, 60 °C and 90 °C fractions (248, 325 and 1,431 fragments, respectively). In addition, we identified hominid and suid mtDNA fragments with no evidence for ancient DNA base damage in every DNA fraction from the 11 objects, thus resulting from contamination with human and pig DNA after excavation. Human DNA contamination was particularly severe, amounting to between 70.9% and 98.3% of the identified mtDNA fragments (between 293 and 92,949 fragments in total; 17,627 on average), thereby potentially masking traces of ancient human or other mammalian DNA.

**Fig. 2: Number and taxonomic composition of mammalian and human mtDNA fragments recovered from six artefacts during stepwise, non-destructive DNA extraction.**

Studying freshly excavated artefacts

As present-day human DNA contamination seemed to be ubiquitous on surfaces of objects that were handled with bare hands during and after excavation, we collected artefacts from ongoing excavations at two Palaeolithic sites, using gloves and facemasks as soon as they became partly exposed to prevent contamination. At Bacho Kiro Cave in Bulgaria, we recovered three Upper Palaeolithic tooth pendants (henceforth ‘BKP1–BKP3’) from layers I, H/I and I/J of niche 1 (Extended Data Fig. 3). At Denisova Cave, a tooth pendant (‘DCP1’) was recovered from layer 11 of the south chamber (Fig. 1c and Extended Data Fig. 4).

Larger clumps of sediment adhering to the artefacts were manually removed, and the artefacts were subsequently cleaned by three successive water washes. DNA was then extracted from the sediment clumps, the water was used for washing and the sediment particles were collected in this process (‘sediment pellets’), as well as directly from the artefacts using the non-destructive method described above. Ancient mammalian mtDNA was detected in all fractions that were analysed, except for some of the water washes and the associated sediment pellets (Fig. 2). The trajectories of DNA released from the four artefacts were similar in that the highest yield of mammalian mtDNA was obtained at 90 °C in the phosphate-based DNA extraction (up to 734, 6,614, 456 and 77,910 mtDNA fragments for BKP1, BKP2, BKP3 and DCP1, respectively; Supplementary Data 1). However, library preparation efficiencies were low for the Bacho Kiro Cave material (less than 10% for many fractions), presumably due to the co-extraction of inhibitory substances (Fig. 2), indicating that more DNA was released than could be recovered and sequenced.

The phosphate DNA fractions obtained at 37 °C, 60 °C and 90 °C are dominated by ancient ursid mtDNA fragments for BKP2 and BKP3, and cervid mtDNA fragments for DCP1, in agreement with their morphological identification (Extended Data Table 1). For BKP1, which is morphologically indeterminate, the phosphate fractions are dominated by bovid mtDNA fragments. In contrast to the phosphate fractions, DNA recovered from the sediment adhering to the artefacts is taxonomically more heterogeneous (Fig. 2). In addition, substantially lower numbers of human mtDNA fragments (between 0 and 2,969 per fraction, 246 on average; Supplementary Data 1) were recovered from the freshly excavated artefacts than from the Quinçay material. Similarly, very few suid mtDNA fragments (15 or less) were recovered, indicating that little post-excavation contamination had occurred. Of note, significant signals of cytosine deamination were observed among the human mtDNA fragments recovered in one of the 90 °C fractions from DCP1.

To increase human DNA recovery, we enriched all libraries again using a capture probe set specifically targeting human mtDNA. For the Bacho Kiro Cave material, this enabled the detection of small traces of ancient human DNA in a sediment pellet from BKP3 (29 deaminated mtDNA fragments), but none of the other fractions. For DCP1, human mtDNA fragments with significant evidence for ancient DNA base damage were identified in the first two sediment pellets recovered from the water washes, the first 37 °C and 60 °C fractions, and all three 90 °C fractions (Fig. 2). The largest numbers of deaminated human mtDNA fragments were obtained in the second and third 90 °C fractions (971 and 1,096, respectively), indicating that extended incubation at high temperature enabled ancient human DNA release from the pendant.

Preparation of additional libraries from the second 90 °C fraction of DCP1, the fraction with the lowest estimate of present-day human contamination (0.1%, 95% CI: 0.0–2.8%), yielded 62-fold average coverage of the human mtDNA genome and a near-complete consensus sequence (Supplementary Information 5). This sequence, which falls together with mtDNA sequences assigned to haplogroup U in a phylogenetic tree (Fig. 3a), contains seven ‘diagnostic’ positions that distinguish it from the mtDNA sequences of other human individuals (Extended Data Table 2). Among the mtDNA fragments overlapping these positions, 86.6% (95% CI: 82.2–90.5%) match the state of DCP1 (Extended Data Table 3), suggesting that the mtDNA fragments recovered in this fraction originate predominantly, but not exclusively, from a single ancient human individual, presumably the user or the maker of the pendant. Support for the DCP1 consensus sequence is slightly lower in the first (77.8%, 95% CI: 40.0–97.2%) and third (82.8%, 95% CI: 78.9–86.2%) 90 °C phosphate fractions, consistent with the slightly higher estimates of present-day human contamination in these fractions (12.8%, 95% CI: 1.0–24.6% and 6.6%, 95% CI: 4.3–8.9%, respectively). By contrast, support for the DCP1 consensus is low in the preceding 60 °C fraction (37.5%, 95% CI: 8.5–75.5%), indeterminate in the 37 °C fraction and low in the first (20.0%, 95% CI: 5.7–43.7%) and second (9.5%, 95% CI: 1.2–30.4%) sediment pellets. These results indicate that the initial water washes and incubations in phosphate buffer at below 90 °C primarily released ancient human DNA from one or more other individuals, which were present in smaller quantities in the surrounding sediment or had adsorbed directly to the surface of DCP1.

**Fig. 3: Ancient human mtDNA and nuclear DNA isolated from DCP1.**

Phylogenetic analyses and dating

On the basis of the branch length of the DCP1 consensus sequence in a tree with other present-day and ancient human mtDNA genomes (Fig. 3a), we estimated its age to 18.5 thousand years (kyr), with a 95% highest posterior density interval ranging from 4.6 to 31.6 kyr (Supplementary Information 5). Furthermore, we used cervid mtDNA probes to reconstruct the complete mtDNA genome of the tooth, identified as wapiti (Cervus canadensis), at 635-fold coverage. A tree with eight additional ancient wapiti mtDNA genomes of known age generated in this study (Supplementary Information 6), estimates the age of DCP1 at 24.7 kyr (highest posterior density interval of 12.8–39.0 kyr). Both genetic ages are consistent with each other and with the younger of two radiocarbon dates that we obtained from charcoal discovered in the proximity of DCP1 in layer 11 (OxA-X-3089-11: 24,200–23,830 calibrated years before present (cal bp) and OxA-X-3089-12: 39,180–37,560 cal bp) at 95.4% probability (Supplementary Information 1). We therefore suggest that genetic dating obviates the need for direct radiocarbon dating of the pendant, although this remains technically possible after non-destructive DNA extraction (Supplementary Information 3).

For nuclear DNA analysis, hybridization capture was performed using libraries from the second and third 90 °C phosphate fractions, targeting sites in the human genome that are known to be polymorphic in modern or archaic humans and that are located in regions of high sequence divergence between humans and other mammals⁸. Sequence information was obtained for 336,429 of these sites (71.5% of the sites targeted), with estimates of present-day human and faunal contamination both below 1%. Comparisons with present-day human populations²⁶ using ƒ3-statistics and D-statistics^27,28 show high affinities to Native Americans (Extended Data Fig. 5). When projected into a principal component analysis with other ancient human individuals (Fig. 3c), DCP1 falls within a group of Ancient North Eurasian individuals from further east in Siberia, which includes the approximately 24 ka Mal’ta 1 and the approximately 17 ka Afontova Gora 3 individuals^29,30. Both of these individuals are genetically closer to DCP1 than non-Ancient North Eurasian individuals when tested with D-statistics (Extended Data Fig. 6b), and all three show similar affinities to ancient Siberians and Native Americans with ƒ3-statistics and D-statistics (Extended Data Fig. 6a,c). In addition, shotgun data were produced from one of the libraries to allow a comparison of sequence coverage for the X chromosome and the autosomes, which is compatible with the human DNA in the 90 °C fraction originating predominantly from a female individual (Fig. 3b and Supplementary Information 7).

Conclusions

In summary, our work highlights that artefacts made from bones or teeth are a previously untapped source of ancient human DNA that can provide insights about the ancestry and biological sex of the individuals who handled, carried or wore these objects in the deep past. The non-destructive DNA extraction method reported here allows a stepwise release of this DNA, making it possible to distinguish DNA that penetrated deeply into an object during its manufacture or use from DNA that may originate from the surrounding sediment. Of note, the coverage depth of targeted sites in the human nuclear genome achieved from DCP1 is similar to what has been obtained with hybridization capture from well-preserved Pleistocene human remains³⁰. Furthermore, the recovery of both human and faunal DNA enabled two independent genetic estimates of its age.

Further work is needed to determine how often human DNA can be recovered from Palaeolithic osseous artefacts. As surface DNA contamination can hamper these analyses, we urge archaeologists to apply protocols for minimizing handling during and after excavation. If this is done, it might become possible to systematically combine genetic and cultural analyses to study Pleistocene artefact use and uncover possible task specialization by individuals of a particular biological sex or genetic ancestry.

Methods

Sample collection

For reagent testing, we collected ten unmodified faunal remains, similar in and size and shape to material used in artefact production, from the late Middle Palaeolithic and early Upper Palaeolithic deposits of the French sites Quinçay (seven) and Les Cottés (three) (Extended Data Table 1 and Extended Data Fig. 2). The estimated ages of these specimens range from 55 to 35 kyr^21,37,38. Non-destructive DNA extraction was then applied to 15 osseous specimens excavated at Quinçay Cave, all from layers attributed to the Châtelperronian technocomplex and probably dating to 45–35 ka^21,37, to three tooth pendants excavated in the Initial Upper Palaeolithic (45–43 ka)³⁹ layers in the niche 1 area of Bacho Kiro Cave^40,41, as well as on one tooth pendant excavated in 2019 in layer 11 (39–24 ka), square E-3, of the south chamber of Denisova Cave (Extended Data Table 1 and Extended Data Fig. 3). Samples from Bacho Kiro and Denisova Cave were excavated and handled using sterile gloves and additional precautions were taken to minimize the introduction of modern DNA contamination. More information about the samples and the archaeological context in which they were recovered is provided in Supplementary Information 1.

Testing reagents for non-destructive DNA extraction using 3D surface texture measurements

We evaluated four reagents for their potential use in non-destructive DNA extraction by applying each to two Pleistocene samples comparable with the ones usually transformed into bone or tooth tools and ornaments, preferably choosing one bone and one tooth fragment for each reagent (Extended Data Table 1). As none of the objects was perfectly clean and sediment microparticles may be released upon exposure to liquids, we also performed treatments with water to obtain baseline measurements of changes in the microtopography that are independent of the chemical compositions of the reagents. Reagents and incubation conditions were as follows:

(1)Following a protocol⁴² further detailing the non-destructive DNA extraction method for museum specimens by Rohland et al. (2004)¹², samples were completely submerged in 10–15 ml guanidinium thiocyanate buffer (5 M guanidinium isothiocyanate, 50 mM Tris-HCl, pH 8.0, 25 mM NaCl, 1.3% Triton X-100, 20 mM EDTA, pH 8.0, and 50 mM DTT) and incubated for 5 days in the dark.
(2)Incubation in EDTA, supplemented with a detergent (0.45 M EDTA, pH 8.0, and 0.05% Tween-20), was performed for 15 min at room temperature. EDTA is widely used in bone extraction protocols as a decalcifying agent to dissolve hydroxyapatite, the main mineral component of bones^14,15,16. It is therefore expected to cause severe alterations to the sample material and was included to demonstrate the effect of such alterations on the quantitative 3D-surface texture analysis (3DST) measurements.
(3)Two samples were submerged for 15 min in 0.5% sodium hypochlorite (bleach) solution, a reagent previously used for decontamination of ancient skeletal remains^11,17. Bleach treatment destroys surface-bound DNA and is not suitable for DNA extraction. However, it may be used in later implementations of the method to remove contaminant DNA before non-destructive DNA extraction.
(4)Temperature-controlled release of DNA was performed following a modified version of the method by Essel et al. (2021)¹⁸ by submerging the samples in sodium phosphate buffer (0.5 M sodium phosphate, pH 7.0, and 0.1% Tween-20). Serial incubations were performed at 21 (room temperature), 37, 60 and 90 °C, each with incubation times of 30 min for a total of three incubations per temperature.

All treatments were performed in 50-ml Falcon tubes without agitation to avoid any mechanical damage to the sample. Reagent volumes were chosen individually for each sample (ranging from 5 ml to 17.5 ml) to ensure complete submergence. Incubations above room temperature were performed in a Heating-ThermoMixer MHR 11 (Hettich Benelux) equipped with inserts for 50-ml Falcon tubes. For the 90 °C incubation in phosphate buffer, the temperature of the device was set to 99 °C to ensure that 90 °C was reached inside the tube by the end of the incubation time. After the treatments, all samples were placed in fresh 50-ml Falcon tubes and incubated in water for 1 h at room temperature to remove residual reagents. Samples were dried at room temperature for 5 days before they were returned to their storage containers.

Changes in the microtopography of the bone or tooth objects were tracked using quantitative 3DST¹⁹ before and after extraction following established protocols^19,43. Meshed axiomatic 3D models were generated and the following ISO 25178 parameters were used for statistical testing (paired Student’s t-test, before and after the treatment, α ≤ 0.01): mean roughness (Sa), void volume (Vvv), peak curvature (Spc) and peak density (Spd). Further details regarding the 3DST measurements are provided in Supplementary Information 2.

In addition, we explored the compatibility of phosphate-based non-destructive DNA extraction with subsequent ¹⁴C dating (Supplementary Information 3).

Non-destructive DNA isolation from artefacts

As sodium phosphate buffer did not cause substantial alterations of ancient bones and teeth in 3DST measurements, we modified a previously described method for temperature-controlled gradual DNA release from bone and tooth powder using this reagent¹⁸ for non-destructive DNA extraction from complete bones and teeth. Stepwise extraction of DNA makes it possible to closely monitor the release of different DNA components during the extraction process (endogenous DNA, environmental DNA from the surrounding sediment, ancient human DNA and present-day contamination), potentially allowing inferences to be drawn about whether these components originate from traces of sediment that may still be adherent to the object, from its surface or its interior. We then applied this protocol to a total of 15 specimens from Quinçay, Bacho Kiro Cave and Denisova Cave. Temperature-controlled non-destructive DNA extraction was performed in an ancient DNA clean-room at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, according to the four steps below (see Fig. 1a for a schematic overview):

(1) Removal of sediment

This step was only performed for the freshly excavated material. First, clumps of sediment attached to the specimen were carefully removed by hand using a flexible disposable plastic microspatula. The specimen was then put into a 50-ml Falcon tube, rinsed by pouring between 20 ml and 50 ml of water into the tube, and transferred to a fresh Falcon tube. This procedure was repeated two to three times until no more sediment was released into the water. The tubes with water containing the sediment that had been washed off were then centrifuged at 16,400g for 5 min to pellet the sediment, and the clear supernatants were transferred to fresh tubes. All three types of material collected in this procedure (the manually removed sediment, the sediment pellet collected by centrifugation and the clear water) were subsequently subjected to DNA purification (see below).

(2) Temperature-controlled DNA release using sodium phosphate buffer

Each cleaned specimen was put into a 50-ml Falcon tube to which sodium phosphate buffer (0.5 M sodium phosphate, pH 7.0, and 0.1% Tween-20) was added until the specimen was completely submerged in the reagent (between 5 ml and 50 ml). After 30 min of incubation at room temperature (without agitation), the buffer was transferred to a fresh 50-ml Falcon tube. This step was repeated twice at room temperature (21 °C, for a total of three incubations) and then three times each at 37 °C, 60 °C and 90 °C (see above for details on the device and temperature settings used). A final incubation in water was performed at room temperature to remove residual reagent and the specimens were dried at room temperature for 5 days.

(3) DNA concentration

To facilitate subsequent DNA purification, half of the volumes of the water and phosphate buffer DNA fractions generated in steps (1) and (2) were reduced to between 50 µl and 75 µl by concentrating the DNA using Amicon Ultra-4 Centrifugal Filter Units with Ultracel-3 membranes (Millipore). For this, up to 4 ml or 15 ml of the respective sample was added to a filter unit, which was spun for 90 min at 4,000g in a centrifuge with an active cooling unit set to 21 °C. In cases in which the sample volume exceeded 4 ml or 15 ml, the flow-through was discarded and the filter unit was reloaded with remaining sample. Finally, a buffer exchange was performed by adding 4 ml or 15 ml TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) to the concentrated sample on top of the filter unit and spinning for 30 min at 4,000g. The supernatant was filled up to 300 µl with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and then transferred into a fresh 1.5-ml Eppendorf low-bind tube and stored at −20 °C until further processing.

(4) DNA purification

DNA was isolated from the concentrated DNA fractions prepared in step (3) using a column-based method for silica-based ancient DNA extraction detailed elsewhere⁴⁴. For this, 300 µl concentrated DNA was used as input for DNA purification using binding buffer ‘D’ and the purified DNA was recovered in 50 µl elution buffer. DNA extraction from the sediment samples removed from the artefacts manually or through water washes (sediment pellets) was performed using the same method but different input volumes. In brief, lysates were prepared by transferring up to 130 mg sediment to a 2-ml low-bind Eppendorf tube, adding up to 2 ml lysis buffer (0.45 M EDTA, pH 8.0, 0.05% Tween-20 and 0.25 mg ml⁻¹ proteinase K) (1 ml for samples of less than 100 mg or 0.5 ml for samples of less than 25 mg) and incubating overnight at 37 °C under rotation. DNA purification was performed using 500 µl or 1,000 µl lysate.

Negative controls containing sodium phosphate buffer or lysis buffer without sample material were carried alongside the samples through all steps of subsequent sample preparation and sequencing. For a subset of samples, DNA was extracted from only the first phosphate buffer fraction produced at each of the four incubation temperatures. Supplementary Data 1 provides an overview of the DNA extracts generated in this study.

Library preparation

Of the extract, 10 µl was then converted into single-stranded DNA libraries using the automated protocol described in Gansauge et al. (2020)²³. The number of unique library molecules obtained and the efficiency of library preparation were determined using two quantitative PCR assays⁴⁵. Libraries were then amplified and double-indexed by PCR⁴⁶. One library was prepared from each DNA fraction, except for the second and third 90 °C phosphate fractions from the DCP1 from which five libraries were prepared, each to maximize the yield of sequence data. Negative controls containing no sample material were carried along each set of extraction and library preparation. Note that there was substantial variation in library preparation efficiency across samples (Fig. 2 and Supplementary Data 1), ranging from 21.8% on average for the phosphate DNA fractions obtained from Bacho Kiro Cave specimens to 60.8% for DCP1 and 62.0% for the Quinçay specimens.

Enrichment of mitochondrial and nuclear DNA by hybridization capture

To determine the taxonomic composition of the DNA recovered from the artefacts, libraries prepared from nearly all extracts generated in this study (all samples and controls, with the exception of the second and third phosphate fractions from BKP3) were enriched for mammalian mtDNA by hybridization capture with a probe set (‘AA75’) encompassing the mtDNA genomes of 242 mammalian species²⁴. In cases in which several libraries were prepared from the same DNA extracts (that is, the 90 °C phosphate fractions from DCP1), only the first library was enriched for mammalian mtDNA. In addition, all libraries were enriched specifically for hominin mtDNA using a probe set (‘AA163’) designed in 1-bp tiling based on the revised Cambridge reference sequence of the human mitochondrial genome³². Both types of mtDNA captures were performed using two consecutive rounds of automated capture as detailed in Slon et al. (2017)⁶ or Zavala et al. (2022)⁴⁷. An overview of the capture reactions performed is provided in Supplementary Data 1.

In addition to mtDNA captures, 11 libraries prepared from the first, second and third 90 °C phosphate fractions of DCP1 were enriched for human nuclear DNA by two consecutive rounds of in-solution capture⁴⁸. This enrichment was performed using a subset of a previously designed capture probe panel⁸, named AA204, targeting two groups of SNPs: (1) 59,232 SNPs, randomly selected from set 6 ‘hominin diagnostic sites’⁸, which represent positions in the genome where primates differ from other mammals and are used to quantify faunal mis-alignments; and (2) 411,492 SNPs selected from the ‘1240k’ panel^30,49 (set 2 in Vernot et al. (2021)⁸). This set of SNPs is informative for investigating modern human population histories. Both SNP groups are located in regions of large evolutionary sequence divergence between humans and other mammals⁸.

Sequencing and raw sequence processing

Libraries enriched for mtDNA were combined into pools and sequenced on multiple lanes of a MiSeq sequencer (Illumina Technologies) in paired-end configuration with two index reads (2× 76 + 2× 8 cycles). Libraries enriched for human nuclear DNA were sequenced either on a HiSeq 2500 using the same configuration or on a HiSeq4000 (both Illumina Technologies) in single-read configuration with two index reads (1× 76 + 2× 8 cycles). In addition, one library prepared from the second 90 °C phosphate fraction of the DCP1 was sequenced directly (shotgun sequencing, without hybridization capture) on one lane of a HiSeq 4000 sequencer (Illumina Technologies) in single-read configuration. Base calling was performed using Illumina’s Bustard tool and sequences were assigned to the library that they originated from, requiring perfect matches to the expected index combinations. LeeHom (https://github.com/mpieva/leeHom/tree/v.1.1.5)⁵⁰ was used to trim adapters and, for paired-end data, to merge overlapping paired-end reads.

Taxonomic assignment of mtDNA sequences

Sequences resulting from mammalian or human mtDNA capture were assigned to mammalian taxa on the biology family level using a previously published computational pipeline⁶ based on BLAST and MEGAN (version 0.0.12)⁵¹, with modifications in data filtering detailed in Vernot et al. (2021)⁸ (Supplementary Data 1 and Supplementary Information 4). In brief, as described in the latter study, false identifications of taxa were minimized by requiring at least three unique sequences (covering at least 105 positions in the reference genome) to be assigned to a family, and by requiring these sequences to represent at least 1% of all taxonomically identified sequences. The presence of ancient DNA was determined individually for the sequences from each family (and each library) by computing the frequency of terminal C-to-T substitutions as a proxy for the deamination rate at the molecule ends. Substitution frequencies significantly higher than 10% on both molecule ends (based on 95% binomial CIs) were then taken as evidence for the presence of ancient DNA from the respective family.

Human and cervid mtDNA analysis

Present-day human contamination in the fractions from DCP1 that yielded ancient human mtDNA was estimated using the software tool AuthentiCT (version 1.0.0)⁵² and a near-complete consensus sequence called for the second 90 °C fraction from DCP1 using positions with at least tenfold coverage of the mtDNA genome. This consensus sequence was then used for haplogroup assignment with Haplogrep 2 (version2.4.0)⁵³ and to identify ‘diagnostic’ positions in the mtDNA genome, which were used to determine the support for the consensus sequence in each of the DNA fractions recovered from DCP1 (Extended Data Table 3). Tree building and genetic dating were performed using BEAST2 (version 2.6.6)⁵⁴. Further information on the mtDNA analyses are provided in Supplementary Information 5. For genetic dating of the deer DNA component, near-complete wapiti mtDNA genomes were reconstructed from the first 60 °C phosphate fraction obtained from DCP1, as well as eight ancient wapiti samples as described in detail in Supplementary Information 6.

Human nuclear DNA analysis

Human DNA capture data were processed as previously described⁸ and data from the libraries with the lowest estimates of modern human contamination (based on AuthentiCT) were merged for further analyses. Principal component analysis including sequence data from present-day and other ancient human individuals was performed using smartpca (from EIGENSOFT package version 8.0.0)⁵⁵. Using the R package admixr (version 0.7.1)²⁸, ƒ3-statistics were calculated to determine shared genetic drift between DCP1 and a selection of modern and ancient human populations and their relationships were further evaluated using D-statistics. Sex determination for the human DNA component recovered from DCP1 was performed by comparing the coverage of the X chromosome and the autosomes in shotgun data obtained from the second 90 °C phosphate fraction after filtering against faunal mis-mappings⁸. Further details are provided in Supplementary Information 7.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data that support the findings of this study are included in the Article and its Supplementary Information.

References

Bailey, G. Time perspectives, palimpsests and the archaeology of time. J. Anthropol. Archaeol. 26, 198–223 (2007).

Article

Google Scholar
Kuhn, S. & Stiner, M. What’s a mother to do? The division of labor among Neandertals and modern humans in Eurasia. Curr. Anthropol. 47, 953–981 (2006).

Article

Google Scholar
Nowell, A. Growing up in the Ice Age: Fossil and Archaeological Evidence of the Lived Lives of Plio-Pleistocene Children (Oxbow Books, 2021).
Owen, L. R. Distorting the Past: Gender and the Division of Labor in the European Upper Paleolithic (Kerns Verlag, 2005).
Villotte, S., Churchill, S. E., Dutour, O. J. & Henry-Gambier, D. Subsistence activities and the sexual division of labor in the European Upper Paleolithic and Mesolithic: evidence from upper limb enthesopathies. J. Hum. Evol. 59, 35–43 (2010).

Article
PubMed

Google Scholar
Slon, V. et al. Neandertal and Denisovan DNA from Pleistocene sediments. Science 356, 605–608 (2017).

Article
ADS
CAS
PubMed

Google Scholar
Zavala, E. I. et al. Pleistocene sediment DNA reveals hominin and faunal turnovers at Denisova Cave. Nature 595, 399–403 (2021).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Vernot, B. et al. Unearthing Neanderthal population history using nuclear and mitochondrial DNA from cave sediments. Science 372, eabf1667 (2021).

Article
CAS
PubMed

Google Scholar
Lindahl, T. Instability and decay of the primary structure of DNA. Nature 362, 709–715 (1993).

Article
ADS
CAS
PubMed

Google Scholar
Brundin, M., Figdor, D., Sundqvist, G. & Sjogren, U. DNA binding to hydroxyapatite: a potential mechanism for preservation of microbial DNA. J. Endod. 39, 211–216 (2013).

Article
PubMed

Google Scholar
Korlevic, P. et al. Reducing microbial and human contamination in DNA extractions from ancient bones and teeth. Biotechniques 59, 87–93 (2015).

Article
ADS
CAS
PubMed

Google Scholar
Rohland, N., Siedel, H. & Hofreiter, M. Nondestructive DNA extraction method for mitochondrial DNA analyses of museum specimens. Biotechniques 36, 814–821 (2004).

Article
CAS
PubMed

Google Scholar
Gomes, C. et al. Nondestructive extraction DNA method from bones or teeth, true or false? Forensic Sci. Int. Genet. 5, e279–e282 (2015).

Article

Google Scholar
Rohland, N. & Hofreiter, M. Ancient DNA extraction from bones and teeth. Nat. Protoc. 2, 1756–1762 (2007).

Article
CAS
PubMed

Google Scholar
Dabney, J. et al. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proc. Natl Acad. Sci. USA 110, 15758–15763 (2013).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Gamba, C. et al. Comparing the performance of three ancient DNA extraction methods for high-throughput sequencing. Mol. Ecol. Resour. 16, 459–469 (2016).

Article
CAS
PubMed

Google Scholar
Salamon, M., Tuross, N., Arensburg, B. & Weiner, S. Relatively well preserved DNA is present in the crystal aggregates of fossil bones. Proc. Natl Acad. Sci. USA 102, 13783–13788 (2005).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Essel, E., Korlevic, P. & Meyer, M. A method for the temperature-controlled extraction of DNA from ancient bones. Biotechniques 71, 382–386 (2021).

Article
CAS
PubMed

Google Scholar
Schulz, E., Calandra, I. & Kaiser, T. M. Applying tribology to teeth of hoofed mammals. Scanning 32, 162–182 (2010).

Article
PubMed

Google Scholar
Schulz-Kornas, E. et al. Everything matters: molar microwear texture in goats (Capra aegagrus hircus) fed diets of different abrasiveness. Palaeogeogr. Palaeoclimatol. Palaeoecol. 552, 109783 (2020).

Article

Google Scholar
Roussel, M., Soressi, M. & Hublin, J. J. The Chatelperronian conundrum: blade and bladelet lithic technologies from Quincay, France. J. Hum. Evol. 95, 13–32 (2016).

Article
CAS
PubMed

Google Scholar
Meyer, M. et al. A high-coverage genome sequence from an archaic Denisovan individual. Science 338, 222–226 (2012).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Gansauge, M. T., Aximu-Petri, A., Nagel, S. & Meyer, M. Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nat. Protoc. 15, 2279–2300 (2020).

Article
CAS
PubMed

Google Scholar
Slon, V. et al. Mammalian mitochondrial capture, a tool for rapid screening of DNA preservation in faunal and undiagnostic remains, and its application to Middle Pleistocene specimens from Qesem Cave (Israel). Quatern. Int. 398, 210–218 (2015).

Article

Google Scholar
Briggs, A. W. et al. Patterns of damage in genomic DNA sequences from a Neandertal. Proc. Natl Acad. Sci. USA 104, 14616–14621 (2007).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Mallick, S. et al. The Simons Genome Diversity Project: 300 genomes from 142 diverse populations. Nature 538, 201–206 (2016).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Patterson, N. et al. Ancient admixture in human history. Genetics 192, 1065–1093 (2012).

Article
PubMed
PubMed Central

Google Scholar
Petr, M., Vernot, B. & Kelso, J. admixr—R package for reproducible analyses using ADMIXTOOLS. Bioinformatics 35, 3194–3195 (2019).

Article
CAS
PubMed
PubMed Central

Google Scholar
Raghavan, M. et al. Upper Palaeolithic Siberian genome reveals dual ancestry of Native Americans. Nature 505, 87–91 (2014).

Article
ADS
PubMed

Google Scholar
Fu, Q. et al. The genetic history of Ice Age Europe. Nature 534, 200–205 (2016).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Green, R. E. et al. A draft sequence of the Neandertal genome. Science 328, 710–722 (2010).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Andrews, R. M. et al. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat. Genet. 23, 147 (1999).

Article
CAS
PubMed

Google Scholar
Green, R. E. et al. A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing. Cell 134, 416–426 (2008).

Article
CAS
PubMed
PubMed Central

Google Scholar
Hajdinjak, M. et al. Reconstructing the genetic history of late Neanderthals. Nature 555, 652–656 (2018).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Kay, P. C.A. A high-coverage Neandertal genome from Vindija Cave in Croatia. Science 358, 655–658 (2017).
Patterson, N., Price, A. L. & Reich, D. Population structure and eigenanalysis. PLoS Genet. 2, e190 (2006).
Welker, F. et al. Variations in glutamine deamidation for a Chatelperronian bone assemblage as measured by peptide mass fingerprinting of collagen. Sci. Technol. Archaeol. 3, 15–27 (2017).

Google Scholar
Rendu, W. et al. Subsistence strategy changes during the Middle to Upper Paleolithic transition reveals specific adaptations of human populations to their environment. Sci. Rep. 9, 15817 (2019).

Article
ADS
PubMed
PubMed Central

Google Scholar
Fewlass, H. et al. A ¹⁴C chronology for the Middle to Upper Palaeolithic transition at Bacho Kiro Cave, Bulgaria. Nat. Ecol. Evol. 4, 794–801 (2020).

Article
PubMed

Google Scholar
Hublin, J.-J. et al. Initial Upper Palaeolithic Homo sapiens from Bacho Kiro Cave, Bulgaria. Nature 581, 299–302 (2020).

Article
ADS
CAS
PubMed

Google Scholar
Tsanova, T. et al. The Initial Upper Palaeolithic lithic assemblage from Bacho Kiro Cave (Bulgaria). In 9th Annual Meeting of the European Society for the Study of Human Evolution. 120 (2020).
Hofreiter, M. Nondestructive DNA extraction from museum specimens. Methods Mol. Biol. 840, 93–100 (2012).

Article
CAS
PubMed

Google Scholar
Martisius, N. L. et al. Time wears on: assessing how bone wears using 3D surface texture analysis. PLoS ONE 13, e0206078 (2018).

Article
PubMed
PubMed Central

Google Scholar
Rohland, N., Glocke, I., Aximu-Petri, A. & Meyer, M. Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing. Nat. Protoc. 13, 2447–2461 (2018).

Article
CAS
PubMed

Google Scholar
Glocke, I. & Meyer, M. Extending the spectrum of DNA sequences retrieved from ancient bones and teeth. Genome Res. 27, 1230–1237 (2017).

Article
CAS
PubMed
PubMed Central

Google Scholar
Kircher, M., Sawyer, S. & Meyer, M. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic Acids Res. 40, e3 (2012).

Article
CAS
PubMed

Google Scholar
Zavala, E. I. et al. Quantifying and reducing cross-contamination in single- and multiplex hybridization capture of ancient DNA. Mol. Ecol. Resour. 22, 2196–2207 (2022).

Article
CAS
PubMed

Google Scholar
Fu, Q. et al. DNA analysis of an early modern human from Tianyuan Cave, China. Proc. Natl Acad. Sci. USA 110, 2223–2227 (2013).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Haak, W. et al. Massive migration from the steppe was a source for Indo-European languages in Europe. Nature 522, 207–211 (2015).

Article
ADS
CAS
PubMed
PubMed Central

Google Scholar
Renaud, G., Stenzel, U. & Kelso, J. leeHom: adaptor trimming and merging for Illumina sequencing reads. Nucleic Acids Res. 42, e141 (2014).

Article
PubMed
PubMed Central

Google Scholar
Huson, D. H., Auch, A. F., Qi, J. & Schuster, S. C. MEGAN analysis of metagenomic data. Genome Res. 17, 377–386 (2007).

Article
CAS
PubMed
PubMed Central

Google Scholar
Peyregne, S. & Peter, B. M. AuthentiCT: a model of ancient DNA damage to estimate the proportion of present-day DNA contamination. Genome Biol. 21, 246 (2020).

Article
CAS
PubMed
PubMed Central

Google Scholar
Weissensteiner, H. et al. HaploGrep 2: mitochondrial haplogroup classification in the era of high-throughput sequencing. Nucleic Acids Res. 44, W58–W63 (2016).

Article
CAS
PubMed
PubMed Central

Google Scholar
Bouckaert, R. et al. BEAST 2.5: an advanced software platform for Bayesian evolutionary analysis. PLoS Comput. Biol. 15, e1006650 (2019).

Article
CAS
PubMed
PubMed Central

Google Scholar
Price, A. L. et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat. Genet. 38, 904–909 (2006).

Article
CAS
PubMed

Google Scholar
R Core Team. R: A Language and Environment for Statistical Computing. http://www.R-project.org/ (R Foundation for Statistical Computing, 2013).

Download references

Acknowledgements

We thank V. Slon, C. Hopfe, M. Peresani and W. Roebroeks for their contributions in an early exploratory phase of the project; P. Kosintsev for providing samples; A. Hübner for help with data analysis; B. Schellbach and A. Weihmann for help with sequencing; S. Nagel, J. Richter, B. Nickel and A. Aximu-Petri for help in the laboratory; J. Visagie for data processing; L. Jauregui, A. Bossoms Mesa, A. Pelin Sümer, H. Temming, A. Lister, M. Meiri and Z. Rezek for comments on the manuscript and/or logistical support; the National Museum of Natural History in Sofia for cooperating; R. Spasov for help with excavation at Bacho Kiro Cave; F. Lévêque, A. Lévêque, C.-H. Bachelier and J. Bachelier for facilitating and supporting research at Quinçay and Les Cottés; S. Rigaud for help with morphological identification of Quinçay samples; the Max Planck Society for funding; and the French Ministry of Culture for allowing and funding research at Les Cottés and Quinçay. The archaeological studies at Denisova Cave were funded by the Russian Science Foundation (no. 22-28-00049). M.Soressi is funded by the NWO VICI award (Neandertal Legacy VI.C.191.07). The radiocarbon dating work received funding from the European Research Council under the European Union’s Horizon 2020 Research and Innovation Programme grant agreement no. 715069 (FINDER) to K.D. Work by E.I.Z. was partially funded by the Miller Institute for Basic Research in Science, University of California Berkeley.

Funding

Open access funding provided by Max Planck Society.

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Author notes

Elena I. Zavala

Present address: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
Tsenka Tsanova

Present address: Department of Chemistry “Giacomo Ciamician”, Alma Mater Studiorum, University of Bologna, Bologna, Italy
Jean-Jacques Hublin

Present address: Chaire de Paléoanthropologie, Collège de France, Paris, France
Mateja Hajdinjak

Present address: Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
These authors contributed equally: Elena Essel, Elena I. Zavala, Ellen Schulz-Kornas, Marie Soressi, Matthias Meyer

Authors and Affiliations

Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany

Elena Essel, Elena I. Zavala, Helen Fewlass, Benjamin Vernot, Anna Schmidt, Merlin Szymanski, Tsenka Tsanova, Shannon P. McPherron, Jean-Jacques Hublin, Janet Kelso, Svante Pääbo & Matthias Meyer
Department of Biology, San Francisco State University, San Francisco, CA, USA

Elena I. Zavala
Department of Cariology, Endodontology and Periodontology, University of Leipzig, Leipzig, Germany

Ellen Schulz-Kornas
Institute of Archaeology and Ethnography, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia

Maxim B. Kozlikin, Michael V. Shunkov & Anatoly P. Derevianko
Department of Evolutionary Anthropology, Faculty of Life Sciences, University of Vienna, Vienna, Austria

Katerina Douka
Human Evolution and Archaeological Sciences (HEAS) Research Network, University of Vienna, Vienna, Austria

Katerina Douka
Earth Sciences Department, Natural History Museum, London, UK

Ian Barnes
Maison de la Recherche, Université de Toulouse-Jean Jaurès, CNRS UMR 5608 TRACES, Toulouse, France

Marie-Cécile Soulier
National Institute of Archaeology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria

Nikolay Sirakov
National Museum of History, Sofia, Bulgaria

Elena Endarova
Ancient Genomics Laboratory, The Francis Crick Institute, London, UK

Mateja Hajdinjak
Faculty of Archaeology, Leiden University, Leiden, The Netherlands

Marie Soressi

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Contributions

E.Essel, M.Soressi and M.M. designed the study. E.Essel, E.S.-K., H.F., M.Soressi and M.M. developed and tested methods. M.B.K., M.V.S., A.P.D., I.B., M.-C.S., T.T., N.S., E.Endarova, S.P.M., J.-J.H. and M.Soressi excavated, identified or characterized archaeological material and/or provided archaeological context and interpretation. E.Essel, E.S.-K., H.F., K.D. and A.S. performed laboratory experiments or collected data. E.Essel, E.I.Z., E.S.-K., H.F., B.V., K.D., M.Szymanski, J.K., S.P., M.H., M.Soressi and M.M. performed, aided in or supervised data analysis. E.Essel, E.I.Z., M.Soressi and M.M. wrote the manuscript with input from all other authors. E.Essel, E.I.Z., E.S.-K., M.M. and M.Soressi contributed equally to this work.

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Elena Essel, Marie Soressi or Matthias Meyer.

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Extended data figures and tables

Extended Data Fig. 1 Microtopographic surface measurements.

Boxplots of the four selected surface texture parameters Sa (arithmetical mean height), Vvv (void volume of the valleys), Spc (arithmetic mean peak curvature), and Spd (density of peaks) by treatment (EDTA = ethylenediaminetetraacetic acid, GuSCN = guanidine thiocyanate reagent, Phos. = sodium phosphate buffer supplemented with detergent, Bleach = sodium hypochlorite, water = distilled water; e = enamel, d = dentine).

Extended Data Fig. 2 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 colors are not directly comparable, as the photographs were taken at slightly different angles, with different light settings and camera adjustments.

Extended Data Fig. 3 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.

Extended Data Fig. 4 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.

Extended Data Fig. 5 Shared genetic drift between DCP1 and different present-day and ancient human individuals.

Each point represents a distinct statistic using the calculation f3(X,Y; Mbuti) where Mbuti serves as an outgroup, population X is an ancient or present-day human population and Y is DCP1. Warmer colors on the map⁵⁶ represent more shared genetic drift.

Extended Data Fig. 6 Genetic affinity of DCP1 to the genomes of other modern and ancient humans.

(A) Genetic affinities of DCP1 and Ancient North Eurasian (ANE) individuals (Y) to different ancient Siberians (W and X) based on 9,297-787,534 SNPs from the “1240k” SNP panel. (B) D-statistics comparing the genetic affinity of DCP1 and Mal’ta1 to a selection of ancient modern humans based on 13,618-237,630 SNPs from the “1240k” SNP panel. Red color indicates all sequences, grey color deaminated sequences only. (C) Genetic affinities of DCP1 and ANE individuals (Y) to different ancient Americans (W and X) based on 42,548-574,391 SNPs from the “1240k” SNP panel. The calculations were performed using ADMIXTOOLS (version 5.1) via admixr (version 0.7.1) using all fragments or deaminated fragments only for DCP1. The error bars represent one standard error as calculated by a Weighted Block Jackknife. AG3 = Afontova Gora 3.

Extended Data Table 1 Description of the samples used in reagent testing and bone/tooth artefacts used for non-destructive DNA extraction

Full size table

Extended Data Table 2 Differences between the mtDNA consensus sequence of DCP1 and other human mtDNA genomes

Full size table

Extended Data Table 3 Support for the mtDNA consensus sequence reconstructed from DCP1

Full size table

Supplementary information

Supplementary Information

Reporting Summary

Peer Review File

Supplementary Data file 1

Overview of DNA lysates, extracts and libraries prepared in this study and results of the mammalian and human mtDNA captures.

Supplementary Data file 2

Overview and summary statistics of the human nuclear DNA captures targeting 470,724 positions in the genome.

Supplementary Data file 3

Calculated D-statistics by ADMIXTOOLS via admixr using D(W, X; ANE, Chimp) where W is a present-day African and X is a present-day non-African. The values in this table are visualized in Supplementary Figure 7.5.

Supplementary Data file 4

Calculated D-statistics by ADMIXTOOLS via admixr using D(W, X; ANE, Mbuti) where W is a present-day Americans and X is a present-day non-African/American. The values in this table are visualized in Supplementary Figure 7.6.

Supplementary Data file 5

Calculated D-statistics by ADMIXTOOLS via admixr using D(W, X; ANE, Mbuti) where W is an ancient Siberian and X is an ancient American. The values in this table are visualized in Supplementary Figure 7.7.

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Essel, E., Zavala, E.I., Schulz-Kornas, E. et al. Ancient human DNA recovered from a Palaeolithic pendant.
Nature (2023). https://doi.org/10.1038/s41586-023-06035-2

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DOI: https://doi.org/10.1038/s41586-023-06035-2

News

Here’s how Helene and other storms dumped a whopping 40 trillion gallons of rain on the South

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1 month ago

September 30, 2024

Anne Joseph

More than 40 trillion gallons of rain drenched the Southeast United States in the last week from Hurricane Helene and a run-of-the-mill rainstorm that sloshed in ahead of it — an unheard of amount of water that has stunned experts.

That’s enough to fill the Dallas Cowboys’ stadium 51,000 times, or Lake Tahoe just once. If it was concentrated just on the state of North Carolina that much water would be 3.5 feet deep (more than 1 meter). It’s enough to fill more than 60 million Olympic-size swimming pools.

“That’s an astronomical amount of precipitation,” said Ed Clark, head of the National Oceanic and Atmospheric Administration’s National Water Center in Tuscaloosa, Alabama. “I have not seen something in my 25 years of working at the weather service that is this geographically large of an extent and the sheer volume of water that fell from the sky.”

The flood damage from the rain is apocalyptic, meteorologists said. More than 100 people are dead, according to officials.

Private meteorologist Ryan Maue, a former NOAA chief scientist, calculated the amount of rain, using precipitation measurements made in 2.5-mile-by-2.5 mile grids as measured by satellites and ground observations. He came up with 40 trillion gallons through Sunday for the eastern United States, with 20 trillion gallons of that hitting just Georgia, Tennessee, the Carolinas and Florida from Hurricane Helene.

Clark did the calculations independently and said the 40 trillion gallon figure (151 trillion liters) is about right and, if anything, conservative. Maue said maybe 1 to 2 trillion more gallons of rain had fallen, much if it in Virginia, since his calculations.

Clark, who spends much of his work on issues of shrinking western water supplies, said to put the amount of rain in perspective, it’s more than twice the combined amount of water stored by two key Colorado River basin reservoirs: Lake Powell and Lake Mead.

Several meteorologists said this was a combination of two, maybe three storm systems. Before Helene struck, rain had fallen heavily for days because a low pressure system had “cut off” from the jet stream — which moves weather systems along west to east — and stalled over the Southeast. That funneled plenty of warm water from the Gulf of Mexico. And a storm that fell just short of named status parked along North Carolina’s Atlantic coast, dumping as much as 20 inches of rain, said North Carolina state climatologist Kathie Dello.

Then add Helene, one of the largest storms in the last couple decades and one that held plenty of rain because it was young and moved fast before it hit the Appalachians, said University of Albany hurricane expert Kristen Corbosiero.

“It was not just a perfect storm, but it was a combination of multiple storms that that led to the enormous amount of rain,” Maue said. “That collected at high elevation, we’re talking 3,000 to 6000 feet. And when you drop trillions of gallons on a mountain, that has to go down.”

The fact that these storms hit the mountains made everything worse, and not just because of runoff. The interaction between the mountains and the storm systems wrings more moisture out of the air, Clark, Maue and Corbosiero said.

North Carolina weather officials said their top measurement total was 31.33 inches in the tiny town of Busick. Mount Mitchell also got more than 2 feet of rainfall.

Before 2017’s Hurricane Harvey, “I said to our colleagues, you know, I never thought in my career that we would measure rainfall in feet,” Clark said. “And after Harvey, Florence, the more isolated events in eastern Kentucky, portions of South Dakota. We’re seeing events year in and year out where we are measuring rainfall in feet.”

Storms are getting wetter as the climate change s, said Corbosiero and Dello. A basic law of physics says the air holds nearly 4% more moisture for every degree Fahrenheit warmer (7% for every degree Celsius) and the world has warmed more than 2 degrees (1.2 degrees Celsius) since pre-industrial times.

Corbosiero said meteorologists are vigorously debating how much of Helene is due to worsening climate change and how much is random.

For Dello, the “fingerprints of climate change” were clear.

“We’ve seen tropical storm impacts in western North Carolina. But these storms are wetter and these storms are warmer. And there would have been a time when a tropical storm would have been heading toward North Carolina and would have caused some rain and some damage, but not apocalyptic destruction. ”

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Follow AP’s climate coverage at https://apnews.com/hub/climate

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Science

‘Big Sam’: Paleontologists unearth giant skull of Pachyrhinosaurus in Alberta

Published

1 month ago

September 25, 2024

Anne Joseph

It’s a dinosaur that roamed Alberta’s badlands more than 70 million years ago, sporting a big, bumpy, bony head the size of a baby elephant.

On Wednesday, paleontologists near Grande Prairie pulled its 272-kilogram skull from the ground.

They call it “Big Sam.”

The adult Pachyrhinosaurus is the second plant-eating dinosaur to be unearthed from a dense bonebed belonging to a herd that died together on the edge of a valley that now sits 450 kilometres northwest of Edmonton.

It didn’t die alone.

“We have hundreds of juvenile bones in the bonebed, so we know that there are many babies and some adults among all of the big adults,” Emily Bamforth, a paleontologist with the nearby Philip J. Currie Dinosaur Museum, said in an interview on the way to the dig site.

She described the horned Pachyrhinosaurus as “the smaller, older cousin of the triceratops.”

“This species of dinosaur is endemic to the Grand Prairie area, so it’s found here and nowhere else in the world. They are … kind of about the size of an Indian elephant and a rhino,” she added.

The head alone, she said, is about the size of a baby elephant.

The discovery was a long time coming.

The bonebed was first discovered by a high school teacher out for a walk about 50 years ago. It took the teacher a decade to get anyone from southern Alberta to come to take a look.

“At the time, sort of in the ’70s and ’80s, paleontology in northern Alberta was virtually unknown,” said Bamforth.

When paleontogists eventually got to the site, Bamforth said, they learned “it’s actually one of the densest dinosaur bonebeds in North America.”

“It contains about 100 to 300 bones per square metre,” she said.

Paleontologists have been at the site sporadically ever since, combing through bones belonging to turtles, dinosaurs and lizards. Sixteen years ago, they discovered a large skull of an approximately 30-year-old Pachyrhinosaurus, which is now at the museum.

About a year ago, they found the second adult: Big Sam.

Bamforth said both dinosaurs are believed to have been the elders in the herd.

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“It makes them look a little strange. It’s the one dinosaur that if you find it, it’s the only possible thing it can be.”

The genders of the two adults are unknown.

Bamforth said the extraction was difficult because Big Sam was intertwined in a cluster of about 300 other bones.

The skull was found upside down, “as if the animal was lying on its back,” but was well preserved, she said.

She said the excavation process involved putting plaster on the skull and wooden planks around if for stability. From there, it was lifted out — very carefully — with a crane, and was to be shipped on a trolley to the museum for study.

“I have extracted skulls in the past. This is probably the biggest one I’ve ever done though,” said Bamforth.

“It’s pretty exciting.”

This report by The Canadian Press was first published Sept. 25, 2024.

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The ancient jar smashed by a 4-year-old is back on display at an Israeli museum after repair

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2 months ago

September 11, 2024

Anne Joseph

TEL AVIV, Israel (AP) — A rare Bronze-Era jar accidentally smashed by a 4-year-old visiting a museum was back on display Wednesday after restoration experts were able to carefully piece the artifact back together.

Last month, a family from northern Israel was visiting the museum when their youngest son tipped over the jar, which smashed into pieces.

Alex Geller, the boy’s father, said his son — the youngest of three — is exceptionally curious, and that the moment he heard the crash, “please let that not be my child” was the first thought that raced through his head.

The jar has been on display at the Hecht Museum in Haifa for 35 years. It was one of the only containers of its size and from that period still complete when it was discovered.

The Bronze Age jar is one of many artifacts exhibited out in the open, part of the Hecht Museum’s vision of letting visitors explore history without glass barriers, said Inbal Rivlin, the director of the museum, which is associated with Haifa University in northern Israel.

It was likely used to hold wine or oil, and dates back to between 2200 and 1500 B.C.

Rivlin and the museum decided to turn the moment, which captured international attention, into a teaching moment, inviting the Geller family back for a special visit and hands-on activity to illustrate the restoration process.

Rivlin added that the incident provided a welcome distraction from the ongoing war in Gaza. “Well, he’s just a kid. So I think that somehow it touches the heart of the people in Israel and around the world,“ said Rivlin.

Roee Shafir, a restoration expert at the museum, said the repairs would be fairly simple, as the pieces were from a single, complete jar. Archaeologists often face the more daunting task of sifting through piles of shards from multiple objects and trying to piece them together.

Experts used 3D technology, hi-resolution videos, and special glue to painstakingly reconstruct the large jar.

Less than two weeks after it broke, the jar went back on display at the museum. The gluing process left small hairline cracks, and a few pieces are missing, but the jar’s impressive size remains.

The only noticeable difference in the exhibit was a new sign reading “please don’t touch.”

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