Ancient human DNA recovered from a Palaeolithic pendant.

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 limited2-5. 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.

Reconstructing the genetic history of late Neanderthals.

Although it has previously been shown that Neanderthals contributed DNA to modern humans, not much is known about the genetic diversity of Neanderthals or the relationship between late Neanderthal populations at the time at which their last interactions with early modern humans occurred and before they eventually disappeared. Our ability to retrieve DNA from a larger number of Neanderthal individuals has been limited by poor preservation of endogenous DNA and contamination of Neanderthal skeletal remains by large amounts of microbial and present-day human DNA. Here we use hypochlorite treatment of as little as 9 mg of bone or tooth powder to generate between 1- and 2.7-fold genomic coverage of five Neanderthals who lived around 39,000 to 47,000 years ago (that is, late Neanderthals), thereby doubling the number of Neanderthals for which genome sequences are available. Genetic similarity among late Neanderthals is well predicted by their geographical location, and comparison to the genome of an older Neanderthal from the Caucasus indicates that a population turnover is likely to have occurred, either in the Caucasus or throughout Europe, towards the end of Neanderthal history. We find that the bulk of Neanderthal gene flow into early modern humans originated from one or more source populations that diverged from the Neanderthals that were studied here at least 70,000 years ago, but after they split from a previously sequenced Neanderthal from Siberia around 150,000 years ago. Although four of the Neanderthals studied here post-date the putative arrival of early modern humans into Europe, we do not detect any recent gene flow from early modern humans in their ancestry.

Ancient DNA of narrow-headed vole reveal common features of the Late Pleistocene population dynamics in cold-adapted small mammals.

The narrow-headed vole, collared lemming and common vole were the most abundant small mammal species across the Eurasian Late Pleistocene steppe-tundra environment. Previous ancient DNA studies of the collared lemming and common vole have revealed dynamic population histories shaped by climatic fluctuations. To investigate the extent to which species with similar adaptations share common evolutionary histories, we generated a dataset comprised the mitochondrial genomes of 139 ancient and 6 modern narrow-headed voles from several sites across Europe and northwestern Asia covering approximately the last 100 thousand years (kyr). We inferred Bayesian time-aware phylogenies using 11 radiocarbon-dated samples to calibrate the molecular clock. Divergence of the main mtDNA lineages across the three species occurred during marine isotope stages (MIS) 7 and MIS 5, suggesting a common response of species adapted to open habitat during interglacials. We identified several time-structured mtDNA lineages in European narrow-headed vole, suggesting lineage turnover. The timing of some of these turnovers was synchronous across the three species, allowing us to identify the main drivers of the Late Pleistocene dynamics of steppe- and cold-adapted species.

Spatial analysis of the ancient proteome of archeological teeth using mass spectrometry imaging.

RATIONALE: Proteins extracted from archaeological bone and teeth are utilised for investigating the phylogeny of extinct and extant species, the biological sex and age of past individuals, as well as ancient health and physiology. However, variable preservation of proteins in archaeological materials represents a major challenge. METHODS: To better understand the spatial distribution of ancient proteins preserved within teeth, we applied matrix assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) for the first time to bioarchaeological samples to visualise the intensity of proteins in archaeological teeth thin sections. We specifically explored the spatial distribution of four proteins (collagen type I, of which the chains alpha-1 and alpha-2, alpha-2-HS-glycoprotein, haemoglobin subunit alpha and myosin light polypeptide 6). RESULTS: We successfully identified ancient proteins in archaeological teeth thin sections using mass spectrometry imaging. The data are available via ProteomeXchange with identifier PXD038114. However, we observed that peptides did not always follow our hypotheses for their spatial distribution, with distinct differences observed in the spatial distribution of several proteins, and occasionally between peptides of the same protein. CONCLUSIONS: While it remains unclear what causes these differences in protein intensity distribution within teeth, as revealed by MALDI-MSI in this study, we have demonstrated that MALDI-MSI can be successfully applied to mineralised bioarchaeological tissues to detect ancient peptides. In future applications, this technique could be particularly fruitful not just for understanding the preservation of proteins in a range of archaeological materials, but making informed decisions on sampling strategies and the targeting of key proteins of archaeological and biological interest.

Exceptionally high δ15N values in collagen single amino acids confirm Neandertals as high-trophic level carnivores.

Isotope and archeological analyses of Paleolithic food webs have suggested that Neandertal subsistence relied mainly on the consumption of large herbivores. This conclusion was primarily based on elevated nitrogen isotope ratios in Neandertal bone collagen and has been significantly debated. This discussion relies on the observation that similar high nitrogen isotopes values could also be the result of the consumption of mammoths, young animals, putrid meat, cooked food, freshwater fish, carnivores, or mushrooms. Recently, compound-specific C and N isotope analyses of bone collagen amino acids have been demonstrated to add significantly more information about trophic levels and aquatic food consumption. We undertook single amino acid C and N isotope analysis on two Neandertals, which were characterized by exceptionally high N isotope ratios in their bulk bone or tooth collagen. We report here both C and N isotope ratios on single amino acids of collagen samples for these two Neandertals and associated fauna. The samples come from two sites dating to the Middle to Upper Paleolithic transition period (Les Cottés and Grotte du Renne, France). Our results reinforce the interpretation of Neandertal dietary adaptations as successful top-level carnivores, even after the arrival of modern humans in Europe. They also demonstrate that high δ15N values of bone collagen can solely be explained by mammal meat consumption, as supported by archeological and zooarcheological evidence, without necessarily invoking explanations including the processing of food (cooking, fermenting), the consumption of mammoths or young mammals, or additional (freshwater fish, mushrooms) dietary protein sources.

Quantifying differences in hominin flaking technologies with 3D shape analysis.

Genetic and climate-driven estimates of past population dynamics are increasingly influential in broader models of hominin migration and adaptation, yet the contribution of stone artifact variability remains more contentious. Scientists are increasingly recognizing the potential of unretouched stone flakes ('flakes') in exploring existing models of hominin behavioral evolution. This is because flakes (1) were produced by all stone tool manufacturing groups in the past, (2) are abundant from the inception of the archaeological record up into the ethnographic present, and (3) preserve under most conditions. The statistical tools of 3D geometric morphometrics capture detailed approximations of flake form that are challenging to document with conventional artifact analyses. We analyze a collection of 717 3D scans of experimentally produced flakes from 5 production strategies that were practiced by hominins through large parts of the Pleistocene and that scientists have drawn on also to make demographic arguments about past human behavior (n = 45 reduction sequences, n = 3 knappers naive toward the study objectives). First, as a proof of concept, we demonstrate that we can estimate the strategies used to produce these flakes at a high success rate even when flakes from early stages of core reduction are included. We frame the significance of this finding against archaeological classifications from several key Middle Paleolithic assemblages in France (n = 4 sites, n = 28 layers, n = 16,467 flakes). Second, we show that 3D geometric morphometrics captures subtle differences in these strategies that influence flake formation on a flake-by-flake basis and that reflect decisions made by knappers about platform selection, preparation, and core-surface management. We explore the broader potential of our model with a cross-validation approach, and we describe a means of assessing flake form on a continuum wherein variability among assemblages separated by large expanses of space and time can be meaningfully explored.

Neandertals revised.

The last decade has seen a significant growth of our knowledge of the Neandertals, a population of Pleistocene hunter-gatherers who lived in (western) Eurasia between ∼400,000 and 40,000 y ago. Starting from a source population deep in the Middle Pleistocene, the hundreds of thousands of years of relative separation between African and Eurasian groups led to the emergence of different phenotypes in Late Pleistocene Europe and Africa. Both recently obtained genetic evidence and archeological data show that the biological and cultural gaps between these populations were probably smaller than previously thought. These data, reviewed here, falsify inferences to the effect that, compared with their near-modern contemporaries in Africa, Neandertals were outliers in terms of behavioral complexity. It is only around 40,000 y ago, tens of thousands of years after anatomically modern humans first left Africa and thousands of years after documented interbreeding between modern humans, Neandertals and Denisovans, that we see major changes in the archeological record, from western Eurasia to Southeast Asia, e.g., the emergence of representational imagery and the colonization of arctic areas and of greater Australia (Sahul).

The age of three Middle Palaeolithic sites: Single-grain optically stimulated luminescence chronologies for Pech de l'Azé I, II and IV in France.

Optically stimulated luminescence (OSL) measurements were made on individual, sand-sized grains of quartz from Middle Palaeolithic deposits at three sites (Pech de l'Azé I, II and IV) located close to one another in the Dordogne region of southwest France. We were able to calculate OSL ages for 69 samples collected from these three sites. These ages reveal periods of occupation between about 180 and 50 thousand years ago. Our single-grain OSL chronologies largely support previous age estimates obtained by thermoluminescence dating of burnt flints at Pech IV, electron spin resonance dating of tooth enamel at Pech I, II and IV and radiocarbon dating of bone at Pech I and IV, but provide a more complete picture due to the ubiquitous presence of sand-sized quartz grains used in OSL dating. These complete chronologies for the three sites have allowed us to compare the single-grain ages for similar lithic assemblages among the three sites, to test the correlations among them previously proposed by Bordes in the 1970s, and to construct our own correlative chronological framework for the three sites. This shows that similar lithic assemblages occur at around the same time, and that where a lithic assemblage is unique to one or found at two of the Pech sites, there are no deposits of chronologically equivalent age at the other Pech site(s). We interpret this to mean that, at least for these Pech de l'Azé sites, the Mousterian variants show temporal ordering. Whether or not this conclusion applies to the wider region and beyond, the hypothesis that Mousterian industrial variation is temporally ordered cannot be refuted at this time.

Neandertals made the first specialized bone tools in Europe.

Modern humans replaced Neandertals ∼40,000 y ago. Close to the time of replacement, Neandertals show behaviors similar to those of the modern humans arriving into Europe, including the use of specialized bone tools, body ornaments, and small blades. It is highly debated whether these modern behaviors developed before or as a result of contact with modern humans. Here we report the identification of a type of specialized bone tool, lissoir, previously only associated with modern humans. The microwear preserved on one of these lissoir is consistent with the use of lissoir in modern times to obtain supple, lustrous, and more impermeable hides. These tools are from a Neandertal context proceeding the replacement period and are the oldest specialized bone tools in Europe. As such, they are either a demonstration of independent invention by Neandertals or an indication that modern humans started influencing European Neandertals much earlier than previously believed. Because these finds clearly predate the oldest known age for the use of similar objects in Europe by anatomically modern humans, they could also be evidence for cultural diffusion from Neandertals to modern humans.

Multi-isotope analysis of bone collagen of Late Pleistocene ungulates reveals niche partitioning and behavioural plasticity of reindeer during MIS 3.

Here we present stable carbon, nitrogen and sulfur isotope ratios of collagen extracted from Rangifer, Equus and Bison bone (n = 128) from different stratigraphic levels at the chronologically well-constrained Middle and Upper Palaeolithic site of Les Cottés, France. Samples were taken from five phases of site use (US08, US06, US04 [upper and lower], and US02; ~ 45.8-35.3 ka cal BP) to explore the dietary and spatial palaeoecology of these ungulate species during MIS 3, and the contemporary climate. Temporal trends in δ15N values of all species broadly align with other climatic indicators at the site and the lowest values in US04 correspond to the Heinrich 4 cooling event, reflecting changes in the composition of soil/plant nitrogen at this time. Rangifer collagen is 13C-enriched compared to the other species throughout, consistent with lichen consumption. However, this isotopic niche partitioning between Rangifer and Equus/Bison is most extensive during US04, indicating plasticity in reindeer feeding behaviour, and potentially overall increased lichen biomass during this cooler/more arid phase. Rangifer δ34S values are consistently lower than Equus and Bison, which could be indicative of their more extensive spatial ranges incorporating greater inland areas. Equus and Bison demonstrate a significant decrease in δ34S values through time, which may be linked to contemporary climatic decline.

Neandertal mobility and large-game hunting: the exploitation of reindeer during the Quina Mousterian at Chez-Pinaud Jonzac (Charente-Maritime, France).

Neandertals were effective hunters of large ungulates throughout their geographic and temporal ranges. Equipped with this knowledge, researchers in paleoanthropology continue to seek insight on the relationships between hunting and subsistence strategies with other components of the Neandertals' niche, such as mobility, site use, and lithic technology. The Quina Mousterian deposits from the rockshelter site of Chez Pinaud Jonzac (Charente-Maritime, France; hereafter Jonzac) offer an excellent opportunity to pursue these issues. This paper focuses on the extensive and well-preserved skeletal remains of reindeer (Rangifer tarandus) recovered from recent excavations of the site, representing at least 18 individuals that were hunted by Neandertals during the fall through winter. Our zooarchaeological results indicate that all ages of reindeer were hunted but adult individuals predominate. No bias is evident in the comparable frequencies of males and females. These prey were butchered on-site, with abundant evidence of meat filleting and marrow exploitation. In the excavated sample, the absence of hearths and the almost complete lack of burned bones or stones suggest that Neandertals were not using fire to assist with processing the reindeer carcasses. The zooarchaeological results presented here indicate that reindeer were hunted during a restricted window of time when they were seasonally abundant in the local area near Jonzac. Taken together with the lithic industry based on bifacial elements, the evidence is consistent with a pattern of site use by highly mobile hunter-gatherers making frequent, short-term visits. Ongoing research at Jonzac and other Quina Mousterian localities will contribute to a better understanding of Neandertal behavior during cold climate phases.

Site-specific deamidation of glutamine: a new marker of bone collagen deterioration.

RATIONALE: Non-enzymatic deamidation accumulates in aging tissues in vivo and has been proposed to be potentially useful as a molecular clock. The process continues post mortem, and here we explore the increase in levels of deamidation in archaeological collagen, as measured during Zooarchaeology by Mass Spectrometry (ZooMS) analysis. METHODS: With the high sensitivity of current generation mass spectrometers, ZooMS provides a non-destructive and highly cost-effective method to characterise collagen peptides. Deamidation can be detected by mass spectrometry as a +0.984 Da mass shift; therefore, aside from its original purpose, peptide mass-fingerprinting for bone identification, ZooMS concurrently yields a 'thermal indicator' of the samples. RESULTS: By analysis of conventional ZooMS spectra, we determined the deamidation rate for glutamine residues in 911 bone collagen samples from 50 sites, with ages varying from medieval to Palaeolithic. The degree of deamidation was compared to diagenetic parameters and nearby sequence properties. CONCLUSIONS: The extent of deamidation was found to be influenced more by burial conditions and thermal age than, for example, chronological age, the extent of bioerosion or crystallinity. The method lends itself mostly to screening heterogenic deposits of bone to identify outliers.

Optimal linear estimation models predict 1400-2900 years of overlap between Homo sapiens and Neandertals prior to their disappearance from France and northern Spain.

Recent fossil discoveries suggest that Neandertals and Homo sapiens may have co-existed in Europe for as long as 5 to 6000 years. Yet, evidence for their contemporaneity at any regional scale remains highly elusive. In France and northern Spain, a region which features some of the latest directly-dated Neandertals in Europe, Protoaurignacian assemblages attributed to Homo sapiens appear to 'replace' Neandertal-associated Châtelperronian assemblages. Using the earliest and latest known occurrences as starting points, Bayesian modelling has provided indication that these occupations may in fact have been partly contemporaneous. The reality, however, is that we are unlikely to ever identify the 'first' or 'last' appearance of a species or cultural tradition in the archaeological and fossil record. Here, we use optimal linear estimation modelling to estimate the first appearance date of Homo sapiens and the extinction date of Neandertals in France and northern Spain by statistically inferring these 'missing' portions of the Protoaurignacian and Châtelperronian archaeological records. Additionally, we estimate the extinction date of Neandertals in this region using a dataset of directly-dated Neandertal fossil remains. Our total dataset consists of sixty-six modernly produced radiocarbon determinations which we recalibrated using the newest calibration curve (IntCal20) to produce updated age ranges. The results suggest that the onset of the Homo sapiens occupation of this region likely preceded the extinction of Neandertals and the Châtelperronian by up to 1400-2900 years. This reaffirms the Bayesian-derived duration of co-existence between these groups during the initial Upper Palaeolithic of this region using a novel independent method, and indicates that our understanding of the timing of these occupations may not be suffering from substantial gaps in the record. Whether or not this co-existence featured some form of direct interaction, however, remains to be resolved.

Strontium isotope evidence for migration in late Pleistocene Rangifer: implications for Neanderthal hunting strategies at the Middle Palaeolithic site of Jonzac, France.

In order to understand the behaviours and subsistence choices of Palaeolithic hunter-gatherers, it is essential to understand the behavioural ecology of their prey. Here, we present strontium isotope data from sequentially-sampled enamel from three reindeer (Rangifer tarandus ssp.) and a single bison (Bison cf. priscus) from the late Middle Palaeolithic site of Jonzac (Chez-Pinaud), France. The results are used to investigate the ranging and migratory behaviours of these important prey species. We found that the bison had isotope values most consistent with a local range, while the three reindeer had values indicating a seasonal migration pattern. Due to the similarity of the patterning of two of the three reindeer and in conjunction with zooarchaeological results, we suggest that they may have been from the same herd, were likely killed around the same point during their seasonal round and may therefore be the product of a single hunting event or a small number of successive hunting events. The isotope analyses complement the zooarchaeological data and have allowed greater insight into the palaeoecology of these species, the palaeoenvironment, and Neanderthal site use and hunting strategies.

The Technological Condition of Human Evolution: Lithic Studies as Basic Science.

The recent elaboration and rapid expansion of aDNA, paleoproteomics, and related fields have propelled a profound "biomolecular turn" in archaeology and fundamentally changed the topology of archaeological knowledge production. Such a transformation of the archaeological research landscape is not without consequence for long-standing research practices in the field, such as lithic analysis. This special issue derives from the session Old Stones, New Eyes? organized by the authors at the UISPP World Congress in Paris in 2018, which aimed to explore the future of lithic studies. An underlying theme of our session was the felt need to respond to the increasing marginalization of lithic research in terms of its capacity to (1) contribute to the grand narratives of early human evolution and (2) better articulate the role and significance of lithic studies in interdisciplinary human origins research. In this editorial, we briefly outline some of the questions and challenges raised by the biomolecular turn and advocate for a more self-conscious and reflexive stance among lithic experts. We argue that lithic studies fulfill all necessary requirements to act as a basic science for human origins research and that its role and status depends less on technological advances, such as, e.g., improved computing facilities, novel analytical software, or automated shape capture technologies, than on continuous work on the conceptual and methodological foundations of inquiry. We finally draw attention to the unique capability of lithic studies to shed light on the human technological condition and illustrate this potential by introducing and briefly discussing the papers included in this issue.

Non-destructive ZooMS identification reveals strategic bone tool raw material selection by Neandertals.

Five nearly identical fragments of specialized bone tools, interpreted as lissoirs (French for "smoothers"), have been found at two Middle Paleolithic sites in southwest France. The finds span three separate archaeological deposits, suggesting continuity in the behavior of late Neandertals. Using standard morphological assessments, we determined that the lissoirs were produced on ribs of medium-sized ungulates. However, since these bones are highly fragmented and anthropogenically modified, species determinations were challenging. Also, conservative curation policy recommends minimizing destructive sampling of rare, fragile, or small artifacts for molecular identification methods. To better understand raw material selection for these five lissoirs, we reassess their taxonomy using a non-destructive ZooMS methodology based on triboelectric capture of collagen. We sampled four storage containers and obtained identifiable MALDI-TOF MS collagen fingerprints, all indicative of the same taxonomic clade, which includes aurochs and bison (Bos sp. and Bison sp.). The fifth specimen, which was stored in a plastic bag, provided no useful MALDI-TOF MS spectra. We show that the choice of large bovid ribs in an archaeological layer dominated by reindeer (Rangifer tarandus) demonstrates strategic selection by these Neandertals. Furthermore, our results highlight the value of a promising technique for the non-destructive analysis of bone artifacts.

Subsistence strategy changes during the Middle to Upper Paleolithic transition reveals specific adaptations of Human Populations to their environment.

The transition from Middle to Upper Paleolithic is a major biological and cultural threshold in the construction of our common humanity. Technological and behavioral changes happened simultaneously to a major climatic cooling, which reached its acme with the Heinrich 4 event, forcing the human populations to develop new strategies for the exploitation of their environment. The recent fieldwork at Les Cottés (France) transitional site offers a good opportunity to document subsistence strategies for this period and to provide for the first time high-resolution insights on its evolution. We present the results of the complete zooarchaeological and taphonomic analysis of the transitional sequence, associated with a large regional synthesis of the subsistence strategy evolution during the Middle to Upper Paleolithic. We conclude that, while there is no major change in the hunting strategies, the butchery activities evolved in strict correlation with the development of range weapons. In addition, the demise of carnivore seems to be a consequence of the human pressure on the environment. Our study demonstrates how the faunal component of the environment became a structuring element of the human social organization, being at the base of future cultural evolutions.

Neandertal and Denisovan DNA from Pleistocene sediments.

Although a rich record of Pleistocene human-associated archaeological assemblages exists, the scarcity of hominin fossils often impedes the understanding of which hominins occupied a site. Using targeted enrichment of mitochondrial DNA, we show that cave sediments represent a rich source of ancient mammalian DNA that often includes traces of hominin DNA, even at sites and in layers where no hominin remains have been discovered. By automation-assisted screening of numerous sediment samples, we detected Neandertal DNA in eight archaeological layers from four caves in Eurasia. In Denisova Cave, we retrieved Denisovan DNA in a Middle Pleistocene layer near the bottom of the stratigraphy. Our work opens the possibility of detecting the presence of hominin groups at sites and in areas where no skeletal remains are found.