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.

Million-year-old DNA sheds light on the genomic history of mammoths.

Temporal genomic data hold great potential for studying evolutionary processes such as speciation. However, sampling across speciation events would, in many cases, require genomic time series that stretch well back into the Early Pleistocene subepoch. Although theoretical models suggest that DNA should survive on this timescale1, the oldest genomic data recovered so far are from a horse specimen dated to 780-560 thousand years ago2. Here we report the recovery of genome-wide data from three mammoth specimens dating to the Early and Middle Pleistocene subepochs, two of which are more than one million years old. We find that two distinct mammoth lineages were present in eastern Siberia during the Early Pleistocene. One of these lineages gave rise to the woolly mammoth and the other represents a previously unrecognized lineage that was ancestral to the first mammoths to colonize North America. Our analyses reveal that the Columbian mammoth of North America traces its ancestry to a Middle Pleistocene hybridization between these two lineages, with roughly equal admixture proportions. Finally, we show that the majority of protein-coding changes associated with cold adaptation in woolly mammoths were already present one million years ago. These findings highlight the potential of deep-time palaeogenomics to expand our understanding of speciation and long-term adaptive evolution.

Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

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 and multimethod dating confirm the late arrival of anatomically modern humans in southern China.

The expansion of anatomically modern humans (AMHs) from Africa around 65,000 to 45,000 y ago (ca. 65 to 45 ka) led to the establishment of present-day non-African populations. Some paleoanthropologists have argued that fossil discoveries from Huanglong, Zhiren, Luna, and Fuyan caves in southern China indicate one or more prior dispersals, perhaps as early as ca. 120 ka. We investigated the age of the human remains from three of these localities and two additional early AMH sites (Yangjiapo and Sanyou caves, Hubei) by combining ancient DNA (aDNA) analysis with a multimethod geological dating strategy. Although U-Th dating of capping flowstones suggested they lie within the range ca. 168 to 70 ka, analyses of aDNA and direct AMS 14C dating on human teeth from Fuyan and Yangjiapo caves showed they derive from the Holocene. OSL dating of sediments and AMS 14C analysis of mammal teeth and charcoal also demonstrated major discrepancies from the flowstone ages; the difference between them being an order of magnitude or more at most of these localities. Our work highlights the surprisingly complex depositional history recorded at these subtropical caves which involved one or more episodes of erosion and redeposition or intrusion as recently as the late Holocene. In light of our findings, the first appearance datum for AMHs in southern China should probably lie within the timeframe set by molecular data of ca. 50 to 45 ka.

Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus.

Recent genomic data have revealed multiple interactions between Neanderthals and modern humans, but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this individual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)-the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution.

Ancient DNA Reconstructs the Genetic Legacies of Precontact Puerto Rico Communities.

Indigenous peoples have occupied the island of Puerto Rico since at least 3000 BC. Due to the demographic shifts that occurred after European contact, the origin(s) of these ancient populations, and their genetic relationship to present-day islanders, are unclear. We use ancient DNA to characterize the population history and genetic legacies of precontact Indigenous communities from Puerto Rico. Bone, tooth, and dental calculus samples were collected from 124 individuals from three precontact archaeological sites: Tibes, Punta Candelero, and Paso del Indio. Despite poor DNA preservation, we used target enrichment and high-throughput sequencing to obtain complete mitochondrial genomes (mtDNA) from 45 individuals and autosomal genotypes from two individuals. We found a high proportion of Native American mtDNA haplogroups A2 and C1 in the precontact Puerto Rico sample (40% and 44%, respectively). This distribution, as well as the haplotypes represented, supports a primarily Amazonian South American origin for these populations and mirrors the Native American mtDNA diversity patterns found in present-day islanders. Three mtDNA haplotypes from precontact Puerto Rico persist among Puerto Ricans and other Caribbean islanders, indicating that present-day populations are reservoirs of precontact mtDNA diversity. Lastly, we find similarity in autosomal ancestry patterns between precontact individuals from Puerto Rico and the Bahamas, suggesting a shared component of Indigenous Caribbean ancestry with close affinity to South American populations. Our findings contribute to a more complete reconstruction of precontact Caribbean population history and explore the role of Indigenous peoples in shaping the biocultural diversity of present-day Puerto Ricans and other Caribbean islanders.

2000-year-old pathogen genomes reconstructed from metagenomic analysis of Egyptian mummified individuals.

BACKGROUND: Recent advances in sequencing have facilitated large-scale analyses of the metagenomic composition of different samples, including the environmental microbiome of air, water, and soil, as well as the microbiome of living humans and other animals. Analyses of the microbiome of ancient human samples may provide insights into human health and disease, as well as pathogen evolution, but the field is still in its very early stages and considered highly challenging. RESULTS: The metagenomic and pathogen content of Egyptian mummified individuals from different time periods was investigated via genetic analysis of the microbial composition of various tissues. The analysis of the dental calculus' microbiome identified Red Complex bacteria, which are correlated with periodontal diseases. From bone and soft tissue, genomes of two ancient pathogens, a 2200-year-old Mycobacterium leprae strain and a 2000-year-old human hepatitis B virus, were successfully reconstructed. CONCLUSIONS: The results show the reliability of metagenomic studies on Egyptian mummified individuals and the potential to use them as a source for the extraction of ancient pathogen DNA.

Rapid size change associated with intra-island evolutionary radiation in extinct Caribbean "island-shrews".

BACKGROUND: The Caribbean offers a unique opportunity to study evolutionary dynamics in insular mammals. However, the recent extinction of most Caribbean non-volant mammals has obstructed evolutionary studies, and poor DNA preservation associated with tropical environments means that very few ancient DNA sequences are available for extinct vertebrates known from the region's Holocene subfossil record. The endemic Caribbean eulipotyphlan family Nesophontidae ("island-shrews") became extinct ~ 500 years ago, and the taxonomic validity of many Nesophontes species and their wider evolutionary dynamics remain unclear. Here we use both morphometric and palaeogenomic methods to clarify the status and evolutionary history of Nesophontes species from Hispaniola, the second-largest Caribbean island. RESULTS: Principal component analysis of 65 Nesophontes mandibles from late Quaternary fossil sites across Hispaniola identified three non-overlapping morphometric clusters, providing statistical support for the existence of three size-differentiated Hispaniolan Nesophontes species. We were also able to extract and sequence ancient DNA from a ~ 750-year-old specimen of Nesophontes zamicrus, the smallest non-volant Caribbean mammal, including a whole-mitochondrial genome and partial nuclear genes. Nesophontes paramicrus (39-47 g) and N. zamicrus (~ 10 g) diverged recently during the Middle Pleistocene (mean estimated divergence = 0.699 Ma), comparable to the youngest species splits in Eulipotyphla and other mammal groups. Pairwise genetic distance values for N. paramicrus and N. zamicrus based on mitochondrial and nuclear genes are low, but fall within the range of comparative pairwise data for extant eulipotyphlan species-pairs. CONCLUSIONS: Our combined morphometric and palaeogenomic analyses provide evidence for multiple co-occurring species and rapid body size evolution in Hispaniolan Nesophontes, in contrast to patterns of genetic and morphometric differentiation seen in Hispaniola's extant non-volant land mammals. Different components of Hispaniola's mammal fauna have therefore exhibited drastically different rates of morphological evolution. Morphological evolution in Nesophontes is also rapid compared to patterns across the Eulipotyphla, and our study provides an important new example of rapid body size change in a small-bodied insular vertebrate lineage. The Caribbean was a hotspot for evolutionary diversification as well as preserving ancient biodiversity, and studying the surviving representatives of its mammal fauna is insufficient to reveal the evolutionary patterns and processes that generated regional diversity.

New insights on Neolithic food and mobility patterns in Mediterranean coastal populations.

OBJECTIVES: The aims of this research are to explore the diet, mobility, social organization, and environmental exploitation patterns of early Mediterranean farmers, particularly the role of marine and plant resources in these foodways. In addition, this work strives to document possible gendered patterns of behavior linked to the neolithization of this ecologically rich area. To achieve this, a set of multiproxy analyses (isotopic analyses, dental calculus, microremains analysis, ancient DNA) were performed on an exceptional deposit (n = 61) of human remains from the Les Bréguières site (France), dating to the transition of the sixth to the fifth millennium BCE. MATERIALS AND METHODS: The samples used in this study were excavated from the Les Bréguières site (Mougins, Alpes-Maritimes, France), located along the southeastern Mediterranean coastline of France. Stable isotope analyses (C, N) on bone collagen (17 coxal bones, 35 craniofacial elements) were performed as a means to infer protein intake during tissue development. Sulfur isotope ratios were used as indicators of geographical and environmental points of origin. The study of ancient dental calculus helped document the consumption of plants. Strontium isotope analysis on tooth enamel (n = 56) was conducted to infer human provenance and territorial mobility. Finally, ancient DNA analysis was performed to study maternal versus paternal diversity within this Neolithic group (n = 30). RESULTS: Stable isotope ratios for human bones range from -20.3 to -18.1‰ for C, from 8.9 to 11.1‰ for N and from 6.4 to 15‰ for S. Domestic animal data range from -22.0 to -20.2‰ for C, from 4.1 to 6.9‰ for N, and from 10.2 to 12.5‰ for S. Human enamel 87 Sr/86 Sr range from 0.7081 to 0.7102, slightly wider than the animal range (between 0.7087 and 0.7096). Starch and phytolith microremains were recovered as well as other types of remains (e.g., hairs, diatoms, fungal spores). Starch grains include Triticeae type and phytolith includes dicotyledons and monocot types as panicoid grasses. Mitochondrial DNA characterized eight different maternal lineages: H1, H3, HV (5.26%), J (10.53%), J1, K, T (5.2%), and U5 (10.53%) but no sample yielded reproducible Y chromosome SNPs, preventing paternal lineage characterization. DISCUSSION: Carbon and nitrogen stable isotope ratios indicate a consumption of protein by humans mainly focused on terrestrial animals and possible exploitation of marine resources for one male and one undetermined adult. Sulfur stable isotope ratios allowed distinguishing groups with different geographical origins, including two females possibly more exposed to the sea spray effect. While strontium isotope data do not indicate different origins for the individuals, mitochondrial lineage diversity from petrous bone DNA suggests the burial includes genetically differentiated groups or a group practicing patrilocality. Moreover, the diversity of plant microremains recorded in dental calculus provide the first evidence that the groups of Les Bréguières consumed a wide breadth of plant foods (as cereals and wild taxa) that required access to diverse environments. This transdisciplinary research paves the way for new perspectives and highlights the relevance for novel research of contexts (whether recently discovered or in museum collections) excavated near shorelines, due to the richness of the biodiversity and the wide range of edible resources available.

The Neolithic Pitted Ware culture foragers were culturally but not genetically influenced by the Battle Axe culture herders.

OBJECTIVES: In order to understand contacts between cultural spheres in the third millennium BC, we investigated the impact of a new herder culture, the Battle Axe culture, arriving to Scandinavia on the people of the sub-Neolithic hunter-gatherer Pitted Ware culture. By investigating the genetic make-up of Pitted Ware culture people from two types of burials (typical Pitted Ware culture burials and Battle Axe culture-influenced burials), we could determine the impact of migration and the impact of cultural influences. METHODS: We sequenced and analyzed the genomes of 25 individuals from typical Pitted Ware culture burials and from Pitted Ware culture burials with Battle Axe culture influences in order to determine if the different burial types were associated with different gene-pools. RESULTS: The genomic data show that all individuals belonged to one genetic population-a population associated with the Pitted Ware culture-irrespective of the burial style. CONCLUSION: We conclude that the Pitted Ware culture communities were not impacted by gene-flow, that is, via migration or exchange of mates. These different cultural expressions in the Pitted Ware culture burials are instead a consequence of cultural exchange.

Specialized sledge dogs accompanied Inuit dispersal across the North American Arctic.

Domestic dogs have been central to life in the North American Arctic for millennia. The ancestors of the Inuit were the first to introduce the widespread usage of dog sledge transportation technology to the Americas, but whether the Inuit adopted local Palaeo-Inuit dogs or introduced a new dog population to the region remains unknown. To test these hypotheses, we generated mitochondrial DNA and geometric morphometric data of skull and dental elements from a total of 922 North American Arctic dogs and wolves spanning over 4500 years. Our analyses revealed that dogs from Inuit sites dating from 2000 BP possess morphological and genetic signatures that distinguish them from earlier Palaeo-Inuit dogs, and identified a novel mitochondrial clade in eastern Siberia and Alaska. The genetic legacy of these Inuit dogs survives today in modern Arctic sledge dogs despite phenotypic differences between archaeological and modern Arctic dogs. Together, our data reveal that Inuit dogs derive from a secondary pre-contact migration of dogs distinct from Palaeo-Inuit dogs, and probably aided the Inuit expansion across the North American Arctic beginning around 1000 BP.

Genetic resiliency and the Black Death: No apparent loss of mitogenomic diversity due to the Black Death in medieval London and Denmark.

OBJECTIVES: In the 14th century AD, medieval Europe was severely affected by the Great European Famine as well as repeated bouts of disease, including the Black Death, causing major demographic shifts. This high volatility led to increased mobility and migration due to new labor and economic opportunities, as evidenced by documentary and stable isotope data. This study uses ancient DNA (aDNA) isolated from skeletal remains to examine whether evidence for large-scale population movement can be gleaned from the complete mitochondrial genomes of 264 medieval individuals from England (London) and Denmark. MATERIALS AND METHODS: Using a novel library-conserving approach to targeted capture, we recovered 264 full mitochondrial genomes from the petrous portion of the temporal bones and teeth and compared genetic diversity across the medieval period within and between English (London) and Danish populations and with contemporary populations through population pairwise ΦST analysis. RESULTS: We find no evidence of significant differences in genetic diversity spatially or temporally in our dataset, yet there is a high degree of haplotype diversity in our medieval samples with little exact sequence sharing. DISCUSSION: The mitochondrial genomes of both medieval Londoners and medieval Danes suggest high mitochondrial diversity before, during and after the Black Death. While our mitochondrial genomic data lack geographically correlated signals, these data could be the result of high, continual female migration before and after the Black Death or may simply indicate a large female effective population size unaffected by the upheaval of the medieval period. Either scenario suggests a genetic resiliency in areas of northwestern medieval Europe.

The efficacy of whole human genome capture on ancient dental calculus and dentin.

OBJECTIVES: Dental calculus is among the richest known sources of ancient DNA in the archaeological record. Although most DNA within calculus is microbial, it has been shown to contain sufficient human DNA for the targeted retrieval of whole mitochondrial genomes. Here, we explore whether calculus is also a viable substrate for whole human genome recovery using targeted enrichment techniques. MATERIALS AND METHODS: Total DNA extracted from 24 paired archaeological human dentin and calculus samples was subjected to whole human genome enrichment using in-solution hybridization capture and high-throughput sequencing. RESULTS: Total DNA from calculus exceeded that of dentin in all cases, and although the proportion of human DNA was generally lower in calculus, the absolute human DNA content of calculus and dentin was not significantly different. Whole genome enrichment resulted in up to four-fold enrichment of the human endogenous DNA content for both dentin and dental calculus libraries, albeit with some loss in complexity. Recovering more on-target reads for the same sequencing effort generally improved the quality of downstream analyses, such as sex and ancestry estimation. For nonhuman DNA, comparison of phylum-level microbial community structure revealed few differences between precapture and postcapture libraries, indicating that off-target sequences in human genome-enriched calculus libraries may still be useful for oral microbiome reconstruction. DISCUSSION: While ancient human dental calculus does contain endogenous human DNA sequences, their relative proportion is low when compared with other skeletal tissues. Whole genome enrichment can help increase the proportion of recovered human reads, but in this instance enrichment efficiency was relatively low when compared with other forms of capture. We conclude that further optimization is necessary before the method can be routinely applied to archaeological samples.

East Anglian early Neolithic monument burial linked to contemporary Megaliths.

In the fourth millennium BCE a cultural phenomenon of monumental burial structures spread along the Atlantic façade. Megalithic burials have been targeted for aDNA analyses, but a gap remains in East Anglia, where Neolithic structures were generally earthen or timber. An early Neolithic (3762-3648 cal. BCE) burial monument at the site of Trumpington Meadows, Cambridgeshire, UK, contained the partially articulated remains of at least three individuals. To determine whether this monument fits a pattern present in megalithic burials regarding sex bias, kinship, diet and relationship to modern populations, teeth and ribs were analysed for DNA and carbon and nitrogen isotopic values, respectively. Whole ancient genomes were sequenced from two individuals to a mean genomic coverage of 1.6 and 1.2X and genotypes imputed. Results show that they were brothers from a small population genetically and isotopically similar to previously published British Neolithic individuals, with a level of genome-wide homozygosity consistent with a small island population sourced from continental Europe, but bearing no signs of recent inbreeding. The first Neolithic whole genomes from a monumental burial in East Anglia confirm that this region was connected with the larger pattern of Neolithic megaliths in the British Isles and the Atlantic façade.

Ancient DNA reveals the chronology of walrus ivory trade from Norse Greenland.

The importance of the Atlantic walrus ivory trade for the colonization, peak, and collapse of the medieval Norse colonies on Greenland has been extensively debated. Nevertheless, no studies have directly traced medieval European ivory back to distinct Arctic populations of walrus. Analysing the entire mitogenomes of 37 archaeological specimens from Europe, Svalbard, and Greenland, we here discover that Atlantic walrus comprises two monophyletic mitochondrial (MT) clades, which diverged between 23 400 and 251 120 years ago. Our improved genomic resolution allows us to reinterpret the geographical distribution of partial MT data from 306 modern and nineteenth-century specimens, finding that one of these clades was exclusively accessible to Greenlanders. With this discovery, we ascertain the biological origin of 23 archaeological specimens from Europe (most dated between 900 and 1400 CE). These results reveal a significant shift in trade from an early, predominantly eastern source towards a near exclusive representation of Greenland ivory. Our study provides empirical evidence for how this remote Arctic resource was progressively integrated into a medieval pan-European trade network, contributing to both the resilience and vulnerability of Norse Greenland society.

Proteomic evidence of dietary sources in ancient dental calculus.

Archaeological dental calculus has emerged as a rich source of ancient biomolecules, including proteins. Previous analyses of proteins extracted from ancient dental calculus revealed the presence of the dietary milk protein ß-lactoglobulin, providing direct evidence of dairy consumption in the archaeological record. However, the potential for calculus to preserve other food-related proteins has not yet been systematically explored. Here we analyse shotgun metaproteomic data from 100 archaeological dental calculus samples ranging from the Iron Age to the post-medieval period (eighth century BC to nineteenth century AD) in England, as well as 14 dental calculus samples from contemporary dental patients and recently deceased individuals, to characterize the range and extent of dietary proteins preserved in dental calculus. In addition to milk proteins, we detect proteomic evidence of foodstuffs such as cereals and plant products, as well as the digestive enzyme salivary amylase. We discuss the importance of optimized protein extraction methods, data analysis approaches and authentication strategies in the identification of dietary proteins from archaeological dental calculus. This study demonstrates that proteomic approaches can robustly identify foodstuffs in the archaeological record that are typically under-represented due to their poor macroscopic preservation.

Ancient DNA reveals temporal population structure within the South-Central Andes area.

OBJECTIVES: The main aim of this work was to contribute to the knowledge of pre-Hispanic genetic variation and population structure among the South-central Andes Area by studying individuals from Quebrada de Humahuaca, North-western (NW) Argentina. MATERIALS AND METHODS: We analyzed 15 autosomal STRs in 19 individuals from several archaeological sites in Quebrada de Humahuaca, belonging to the Regional Developments Period (900-1430 AD). Compiling autosomal, mitochondrial, and Y-chromosome data, we evaluated population structure and differentiation among eight South-central Andean groups from the current territories of NW Argentina and Peru. RESULTS: Autosomal data revealed a structuring of the analyzed populations into two clusters which seemed to represent different temporalities in the Andean pre-Hispanic history: pre-Inca and Inca. All pre-Inca samples fell into the same cluster despite being from the two different territories of NW Argentina and Peru. Also, they were systematically differentiated from the Peruvian Inca group. These results were mostly confirmed by mitochondrial and Y-chromosome analyses. We mainly found a clearly different haplotype composition between clusters. DISCUSSION: Population structure in South America has been mostly studied on current native groups, mainly showing a west-to-east differentiation between the Andean and lowland regions. Here we demonstrated that genetic population differentiation preceded the European contact and might have been more complex than thought, being found within the South-central Andes Area. Moreover, divergence among temporally different populations might be reflecting socio-political changes occurred in the evermore complex pre-Hispanic Andean societies.