Genetic insights into the social organization of Neanderthals.

Genomic analyses of Neanderthals have previously provided insights into their population history and relationship to modern humans1-8, but the social organization of Neanderthal communities remains poorly understood. Here we present genetic data for 13 Neanderthals from two Middle Palaeolithic sites in the Altai Mountains of southern Siberia: 11 from Chagyrskaya Cave9,10 and 2 from Okladnikov Cave11-making this one of the largest genetic studies of a Neanderthal population to date. We used hybridization capture to obtain genome-wide nuclear data, as well as mitochondrial and Y-chromosome sequences. Some Chagyrskaya individuals were closely related, including a father-daughter pair and a pair of second-degree relatives, indicating that at least some of the individuals lived at the same time. Up to one-third of these individuals' genomes had long segments of homozygosity, suggesting that the Chagyrskaya Neanderthals were part of a small community. In addition, the Y-chromosome diversity is an order of magnitude lower than the mitochondrial diversity, a pattern that we found is best explained by female migration between communities. Thus, the genetic data presented here provide a detailed documentation of the social organization of an isolated Neanderthal community at the easternmost extent of their known range.

Age estimates for hominin fossils and the onset of the Upper Palaeolithic at Denisova Cave.

Denisova Cave in the Siberian Altai (Russia) is a key site for understanding the complex relationships between hominin groups that inhabited Eurasia in the Middle and Late Pleistocene epoch. DNA sequenced from human remains found at this site has revealed the presence of a hitherto unknown hominin group, the Denisovans1,2, and high-coverage genomes from both Neanderthal and Denisovan fossils provide evidence for admixture between these two populations3. Determining the age of these fossils is important if we are to understand the nature of hominin interaction, and aspects of their cultural and subsistence adaptations. Here we present 50 radiocarbon determinations from the late Middle and Upper Palaeolithic layers of the site. We also report three direct dates for hominin fragments and obtain a mitochondrial DNA sequence for one of them. We apply a Bayesian age modelling approach that combines chronometric (radiocarbon, uranium series and optical ages), stratigraphic and genetic data to calculate probabilistically the age of the human fossils at the site. Our modelled estimate for the age of the oldest Denisovan fossil suggests that this group was present at the site as early as 195,000 years ago (at 95.4% probability). All Neanderthal fossils-as well as Denisova 11, the daughter of a Neanderthal and a Denisovan4-date to between 80,000 and 140,000 years ago. The youngest Denisovan dates to 52,000-76,000 years ago. Direct radiocarbon dating of Upper Palaeolithic tooth pendants and bone points yielded the earliest evidence for the production of these artefacts in northern Eurasia, between 43,000 and 49,000 calibrated years before present (taken as AD 1950). On the basis of current archaeological evidence, it may be assumed that these artefacts are associated with the Denisovan population. It is not currently possible to determine whether anatomically modern humans were involved in their production, as modern-human fossil and genetic evidence of such antiquity has not yet been identified in the Altai region.

Reevaluating the timing of Neanderthal disappearance in Northwest Europe.

Elucidating when Neanderthal populations disappeared from Eurasia is a key question in paleoanthropology, and Belgium is one of the key regions for studying the Middle to Upper Paleolithic transition. Previous radiocarbon dating placed the Spy Neanderthals among the latest surviving Neanderthals in Northwest Europe with reported dates as young as 23,880 ± 240 B.P. (OxA-8912). Questions were raised, however, regarding the reliability of these dates. Soil contamination and carbon-based conservation products are known to cause problems during the radiocarbon dating of bulk collagen samples. Employing a compound-specific approach that is today the most efficient in removing contamination and ancient genomic analysis, we demonstrate here that previous dates produced on Neanderthal specimens from Spy were inaccurately young by up to 10,000 y due to the presence of unremoved contamination. Our compound-specific radiocarbon dates on the Neanderthals from Spy and those from Engis and Fonds-de-Forêt demonstrate that they disappeared from Northwest Europe at 44,200 to 40,600 cal B.P. (at 95.4% probability), much earlier than previously suggested. Our data contribute significantly to refining models for Neanderthal disappearance in Europe and, more broadly, show that chronometric models regarding the appearance or disappearance of animal or hominin groups should be based only on radiocarbon dates obtained using robust pretreatment methods.

Dating the last Middle Palaeolithic of the Crimean Peninsula: New hydroxyproline AMS dates from the site of Kabazi II.

Radiocarbon dating of bone and charcoal from sites dating to the Middle and Upper Paleolithic is challenging due to low residual levels of radiocarbon. This means that small amounts of contaminating carbon can wield a great influence over accuracy unless they are fully removed. The site of Kabazi II in the Crimea is important because radiocarbon dates previously obtained from bones in archaeological horizons that date to the Western Crimean Mousterian (WCM) are surprisingly young. We redated the same samples using a single compound dating method that focuses on extracting and dating the amino acid hydroxyproline. We show that single amino acid dates produce significantly older determinations than those that use bulk collagen pretreatment procedures. Our results suggest that instead of dating to 35,000-40,000 cal BP, the bones actually date to >50,000 cal BP. This implies that the WCM at this site is much older than previously thought. In light of these current findings, we considered the dates of other key Crimean sites and concluded that in the absence of reliable pretreatment methods, it would be wise to consider many of them minimum ages. We conclude that there is little robust evidence to suggest Neanderthals were present in the Crimea after 40,000 cal BP.

Reassessing the chronology of the archaeological site of Anzick.

Found in 1968, the archaeological site of Anzick, Montana, contains the only known Clovis burial. Here, the partial remains of a male infant, Anzick-1, were found in association with a Clovis assemblage of over 100 lithic and osseous artifacts-all red-stained with ochre. The incomplete, unstained cranium of an unassociated, geologically younger individual, Anzick-2, was also recovered. Previous chronometric work has shown an age difference between Anzick-1 and the Clovis assemblage (represented by dates from two antler rod samples). This discrepancy has led to much speculation, with some discounting Anzick-1 as Clovis. To resolve this issue, we present the results of a comprehensive radiocarbon dating program that utilized different pretreatment methods on osseous material from the site. Through this comparative approach, we obtained a robust chronometric dataset that suggests that Anzick-1 is temporally coeval with the dated antler rods. This implies that the individual is indeed temporally associated with the Clovis assemblage.

Direct dating of Neanderthal remains from the site of Vindija Cave and implications for the Middle to Upper Paleolithic transition.

Previous dating of the Vi-207 and Vi-208 Neanderthal remains from Vindija Cave (Croatia) led to the suggestion that Neanderthals survived there as recently as 28,000-29,000 B.P. Subsequent dating yielded older dates, interpreted as ages of at least ∼32,500 B.P. We have redated these same specimens using an approach based on the extraction of the amino acid hydroxyproline, using preparative high-performance liquid chromatography (Prep-HPLC). This method is more efficient in eliminating modern contamination in the bone collagen. The revised dates are older than 40,000 B.P., suggesting the Vindija Neanderthals did not live more recently than others across Europe, and probably predate the arrival of anatomically modern humans in Eastern Europe. We applied zooarchaeology by mass spectrometry (ZooMS) to find additional hominin remains. We identified one bone that is Neanderthal, based on its mitochondrial DNA, and dated it directly to 46,200 ± 1,500 B.P. We also attempted to date six early Upper Paleolithic bone points from stratigraphic units G1, Fd/d+G1 and Fd/d, Fd. One bone artifact gave a date of 29,500 ± 400 B.P., while the remainder yielded no collagen. We additionally dated animal bone samples from units G1 and G1-G3 These dates suggest a co-occurrence of early Upper Paleolithic osseous artifacts, particularly split-based points, alongside the remains of Neanderthals is a result of postdepositional mixing, rather than an association between the two groups, although more work is required to show this definitively.

New data for the Early Upper Paleolithic of Kostenki (Russia).

Several questions remain regarding the timing and nature of the Neanderthal-anatomically modern human (AMH) transition in Europe. The situation in Eastern Europe is generally less clear due to the relatively few sites and a dearth of reliable radiocarbon dates. Claims have been made for both notably early AMH and notably late Neanderthal presence, as well as for early AMH (Aurignacian) dispersal into the region from Central/Western Europe. The Kostenki-Borshchevo complex (European Russia) of Early Upper Paleolithic (EUP) sites offers high-quality data to address these questions. Here we revise the chronology and cultural status of the key sites of Kostenki 17 and Kostenki 14. The Kostenki 17/II lithic assemblage shares important features with Proto-Aurignacian material, strengthening an association with AMHs. New radiocarbon dates for Kostenki 17/II of ∼41-40 ka cal BP agree with new dates for the recently excavated Kostenki 14/IVw, which shows some similarities to Kostenki 17/II. Dates of ≥41 ka cal BP from other Kostenki sites cannot be linked to diagnostic archaeological material, and therefore cannot be argued to date AMH occupation. Kostenki 14's Layer in Volcanic Ash assemblage, on the other hand, compares to Early Aurignacian material. New radiocarbon dates targeting diagnostic lithics date to 39-37 ka cal BP. Overall, Kostenki's early EUP is in good agreement with the archaeological record further west. Our results are therefore consistent with models predicting interregional penecontemporaneity of diagnostic EUP assemblages. Most importantly, our work highlights ongoing challenges for reliably radiocarbon dating the period. Dates for Kostenki 14 agreed with the samples' chronostratigraphic positions, but standard pre-treatment methods consistently produced incorrect ages for Kostenki 17/II. Extraction of hydroxyproline from bone collagen using preparative high-performance liquid chromatography, however, yielded results consistent with the samples' chronostratigraphic position and with the layer's archaeological contents. This suggests that for some sites compound-specific techniques are required to build reliable radiocarbon chronologies.

Assessing the efficiency of supercritical fluid extraction for the decontamination of archaeological bones prior to radiocarbon dating.

Bone is one of the main sample types used for building chronologies in archaeology. It is also used in other research areas such as palaeodiet and palaeoenvironmental studies. However, for results to be accurate, samples must be free of exogenous carbon. Contamination can originate from a wide range of sources in the post-depositional environment but may also occur during excavation and post excavation activities (i.e. with the application of conservation materials) or during laboratory handling. Efficient procedures to remove contamination are therefore crucial prior to radiocarbon or stable isotope measurements. This work describes the development of an innovative sample pretreatment for bones, based on using supercritical CO2, which shows unique solvation properties. The effectiveness of supercritical fluid extraction (SFE) to remove conservation materials was compared with that obtained when applying a routine extraction based on the use of organic solvents (methanol, acetone and chloroform). The chemical composition of the bone samples before and after the two pre-treatments was then investigated using analytical pyrolysis-based techniques: EGA-MS (Evolved Gas Analysis-Mass Spectrometry) and Py-GC/MS (Pyrolysis-Gas Chromatography coupled with Mass Spectrometry). Collagen samples extracted from the same bones, prepared with the two cleaning protocols, were also radiocarbon dated by Accelerator Mass Spectrometry (AMS). The results of this study show that SFE is an efficient alternative method because it was as effective as the established treatment protocol. It removes contaminants such as conservation materials from bone samples with a minimum of handling and can be used routinely in radiocarbon dating laboratories. This work also demonstrates that analytical pyrolysis is not only a very efficient method to identify contaminants in bones but also to assess the effectiveness of the pretreatment prior to the radiocarbon measurement of the samples.

Supercritical Fluids for Higher Extraction Yields of Lipids from Archeological Ceramics.

The extraction and study of organic residues from ceramics has been a subject of interest for the last 50 years in archeology and archeological science. Lipids are among the best-preserved organic substances in archeological contexts and can provide information about the diets of ancient populations as well as past environments. Here, we present a method which demonstrates significantly improved extraction of lipids from archeological pots by replacing liquid organic solvents with supercritical fluids. Optimization of the procedure using response surface methodology (RSM) approach showed that, on our system, optimal conditions for supercritical extraction of lipids from synthetic fired clay ceramics could be achieved using carbon dioxide with 16 vol % of cosolvent EtOH-H2O (95:5 v/v) in 90 min at a flow rate of 2.3 mL/min, for a pressure of 30 MPa and a temperature of 50 °C. For all reference and archeological samples included in this study, lipid yields obtained by supercritical fluid extraction under these optimal conditions were systematically higher than by conventional solvent extraction. This study also highlighted a variability of the ratio of unsaturated versus saturated fatty acids depending on the extraction method. This can have important implications in the identification of the residue(s). The increased extraction efficiency provided by supercritical fluids, as well as their minimally destructive nature, enable new and refined approaches to residue analysis and dating of archeological ceramics.

New protocol for compound-specific radiocarbon analysis of archaeological bones.

RATIONALE: For radiocarbon results to be accurate, samples must be free of contaminating carbon. Sample pre-treatment using a high-performance liquid chromatography (HPLC) approach has been developed at the Oxford Radiocarbon Accelerator Unit (ORAU) as an alternative to conventional methods for dating heavily contaminated bones. This approach isolates hydroxyproline from bone collagen, enabling a purified bone-specific fraction to then be radiocarbon dated by accelerator mass spectrometry (AMS). METHODS: Using semi-preparative chromatography and non-carbon-based eluents, this technique enables the separation of underivatised amino acids liberated by hydrolysis of extracted bone collagen. A particular focus has been the isolation of hydroxyproline for single-compound AMS dating since this amino acid is one of the main contributors to the total amount of carbon in mammalian collagen. Our previous approach, involving a carbon-free aqueous mobile phase, required a two-step separation using two different chromatographic columns. RESULTS: This paper reports significant improvements that have been recently made to the method to enable faster semi-preparative separation of hydroxyproline from bone collagen, making the method more suitable for routine radiocarbon dating of contaminated and/or poorly preserved bone samples by AMS. All steps of the procedure, from the collagen extraction to the correction of the AMS data, are described. CONCLUSIONS: The modifications to the hardware and to the method itself have reduced significantly the time required for the preparation of each sample. This makes it easier for other radiocarbon facilities to implement and use this approach as a routine method for preparing contaminated bone samples.

Recipes of Ancient Egyptian kohls more diverse than previously thought.

Kohl, a dark eye cosmetic, is a well-known part of Ancient Egyptian culture. Modern chemical analyses of kohls have largely found lead-based inorganic constituents, whereas earlier studies argued for a much broader range of constituents. Furthermore, organic materials in kohls remain severely understudied. This raises questions regarding the true diversity of materials and recipes used to produce kohls. We analysed the contents of 11 kohl containers from the Petrie Museum collection in London. The objects selected cover a broad range of times and locations in Egypt. Our multi-analytical approach allowed us to characterise both inorganic and organic components. Our data show that inorganic ingredients in kohl recipes are not only lead-based but also manganese- and silicon-based. Our analyses also revealed that organic ingredients derived from both plant and animal sources were commonly used in kohl recipes and sometimes even represent the main constituent. All these findings point towards more varied recipes than initially thought and significantly shift our understanding of Ancient Egyptian kohls.

First chemical evidence of royal purple as a material used for funeral treatment discovered in a Gallo-Roman burial (Naintré, France, third century AD).

Violet-purple residues collected from a Gallo-Roman burial dated back to the second half of the third century A.D. and excavated at Naintré (France) were chemically investigated by multi-analytical methodology involving the use of Raman spectroscopy, direct exposure-mass spectrometry (DE-MS) and high-performance liquid chromatography (HPLC-UV-visible). Little is known about funeral treatment and rituals during Roman times. Retrieving valuable information on these by chemical analysis of organic residues was thus a key aspect of this work. Analyses demonstrated the presence of the very precious purple colorant obtained from shellfish glands commonly known as Tyrian or royal purple and its exceptional preservation. Chemical investigation and archaeological evidence have shown that purple was widely spread after the deposition of the body for burial. These results are the earliest chemical evidence of purple colorant used during funeral rituals (not as textile dye) and enabled us to highlight new aspects of funeral practices in Roman times.

Molecular profiling of Peru Balsam reveals active ingredients responsible for its pharmaceutical properties.

Peru Balsam, a resinous substance derived from Myroxylon balsamum var. pereirae, has historically been used as a topical ointment for various skin conditions such as scabies, poorly healing wounds, eczema, and haemorrhoids. The ingredients responsible for these properties are not fully elucidated. We investigated the chemical composition of two Peru Balsam samples, one historical and one modern, using gas chromatography/mass spectrometry to identify the active ingredients responsible for its pharmaceutical properties. Both Peru Balsam specimens investigated had similar compositions, showing the stability of the substance. Components identified are effective against scabies, exhibit antimicrobial activity and aid skin penetration. These properties are consistent with historical uses of Peru Balsam. Several ingredients are also known allergens. This study, combining chemical information with scientific literature related to pharmaceutical properties of natural substances, represents a breakthrough in the elucidation of active ingredients in Peru Balsam.

A genome sequence from a modern human skull over 45,000 years old from Zlatý kun in Czechia.

Modern humans expanded into Eurasia more than 40,000 years ago following their dispersal out of Africa. These Eurasians carried ~2-3% Neanderthal ancestry in their genomes, originating from admixture with Neanderthals that took place sometime between 50,000 and 60,000 years ago, probably in the Middle East. In Europe, the modern human expansion preceded the disappearance of Neanderthals from the fossil record by 3,000-5,000 years. The genetic makeup of the first Europeans who colonized the continent more than 40,000 years ago remains poorly understood since few specimens have been studied. Here, we analyse a genome generated from the skull of a female individual from Zlatý kun, Czechia. We found that she belonged to a population that appears to have contributed genetically neither to later Europeans nor to Asians. Her genome carries ~3% Neanderthal ancestry, similar to those of other Upper Palaeolithic hunter-gatherers. However, the lengths of the Neanderthal segments are longer than those observed in the currently oldest modern human genome of the ~45,000-year-old Ust'-Ishim individual from Siberia, suggesting that this individual from Zlatý kun is one of the earliest Eurasian inhabitants following the expansion out of Africa.

Birch bark tar in early Medieval England - Continuity of tradition or technological revival?

Birch bark tar is a manufactured product with a history of production and use that reaches back to the Palaeolithic. Its sticky, water resistant and biocidal properties mean that it has a wide range of applications, for example, as a multipurpose adhesive, sealant and in medicine. Archaeological evidence for birch bark tar in the old world covers a broad geographic range from the UK to the Baltic and from the Mediterranean to Scandinavia. In the east and north of this range there is continuity of use to modern times but in western Europe and the British Isles the use of birch bark tar has generally been viewed as limited to prehistory, with gradual displacement by pine tars during the Roman period. Here, we report new finds of birch bark tar from two early Medieval sites in the east of England. Analysis by HT-GC/MS to identify the tars also revealed fatty material, possibly added to modify the tar. The different contexts of the finds point to diverse applications of the material: in one case perhaps a medicine, the other associated with a ceramic container, possibly used for processing the tar. The results present the first identification of birch bark tar from early Medieval archaeological contexts in the UK. Together they indicate a later period of use for birch bark tar in the UK than has been previously observed and raise the question of whether this indicates evidence of a longer continuity of use than hitherto recognised or a later reintroduction of the technology in the Medieval period, in which case the similarities between the find sites, both early Anglo-Saxon cemeteries with comparable assemblages of grave goods, may be significant.

Denisovan ancestry and population history of early East Asians.

We present analyses of the genome of a ~34,000-year-old hominin skull cap discovered in the Salkhit Valley in northeastern Mongolia. We show that this individual was a female member of a modern human population that, following the split between East and West Eurasians, experienced substantial gene flow from West Eurasians. Both she and a 40,000-year-old individual from Tianyuan outside Beijing carried genomic segments of Denisovan ancestry. These segments derive from the same Denisovan admixture event(s) that contributed to present-day mainland Asians but are distinct from the Denisovan DNA segments in present-day Papuans and Aboriginal Australians.

Preparative HPLC Separation of Underivatized Amino Acids for Isotopic Analysis.

Single-compound analysis of stable or radioactive isotopes has found application in a number of fields ranging from archaeology to forensics. Often, the most difficult part of these analyses is the development of a method for isolating the compound(s) of interest, which can derive from a wide range of sample types including the hair, nails, and bone.Here we describe three complementary preparative HPLC techniques suitable for separating and isolating amino acids from bone collagen and hair keratin. Using preparative reversed-phase, ion-pair, or mixed-mode chromatography in aqueous carbon-free mobile phases, or those from which carbon can easily be removed, underivatized single amino acids can be isolated and further analyzed using mass spectrometric techniques.

Compound-specific radiocarbon dating and mitochondrial DNA analysis of the Pleistocene hominin from Salkhit Mongolia.

A skullcap found in the Salkhit Valley in northeast Mongolia is, to our knowledge, the only Pleistocene hominin fossil found in the country. It was initially described as an individual with possible archaic affinities, but its ancestry has been debated since the discovery. Here, we determine the age of the Salkhit skull by compound-specific radiocarbon dating of hydroxyproline to 34,950-33,900 Cal. BP (at 95% probability), placing the Salkhit individual in the Early Upper Paleolithic period. We reconstruct the complete mitochondrial genome (mtDNA) of the specimen. It falls within a group of modern human mtDNAs (haplogroup N) that is widespread in Eurasia today. The results now place the specimen into its proper chronometric and biological context and allow us to begin integrating it with other evidence for the human occupation of this region during the Paleolithic, as well as wider Pleistocene sequences across Eurasia.