A method for the temperature-controlled extraction of DNA from ancient bones

We here provide a protocol for the decontamination of ancient bones and teeth that is based on a temperature-controlled, sequential release of DNA. DNA can be extracted from all fractions generated with this method and the fraction with the highest proportion of endogenous DNA identified for further analysis. The protocol proceeds through repeated incubation of the sample powder in phosphate buffer at 37, 60 and 90 °C, followed by the complete lysis of the residual sample powder. As DNA is denatured at high temperature, subsequent DNA extraction and library preparation has to be performed using methods optimized for single-stranded DNA

Reducing microbial and human contamination in DNA extractions from ancient bones and teeth

Although great progress has been made in improving methods for generating DNA sequences from ancient biological samples, many, if not most, samples are still not amenable for analyses due to overwhelming contamination with microbial or modern human DNA. Here we explore different DNA decontamination procedures for ancient bones and teeth for use prior to DNA library preparation and high-throughput sequencing. Two procedures showed promising results: (i) the release of surface-bound DNA by phosphate buffer and (ii) the removal of DNA contamination by sodium hypochlorite treatment. Exposure to phosphate removes on average 64% of the microbial DNA from bone powder but only 37% of the endogenous DNA (from the organism under study), increasing the percentage of informative sequences by a factor of two on average. An average 4.6-fold increase, in one case reaching 24-fold, is achieved by sodium hypochlorite treatment, albeit at the expense of destroying 63% of the endogenous DNA preserved in the bone. While both pretreatment methods described here greatly reduce the cost of genome sequencing from ancient material due to efficient depletion of microbial DNA, we find that the removal of human DNA contamination remains a challenging problem

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

Genomic insights into the origin of farming in the ancient Near East

We report genome-wide ancient DNA from 44 ancient Near Easterners ranging in time between ~12,000 and 1,400 BC, from Natufian hunter–gatherers to Bronze Age farmers. We show that the earliest populations of the Near East derived around half their ancestry from a ‘Basal Eurasian’ lineage that had little if any Neanderthal admixture and that separated from other non-African lineages before their separation from each other. The first farmers of the southern Levant (Israel and Jordan) and Zagros Mountains (Iran) were strongly genetically differentiated, and each descended from local hunter–gatherers. By the time of the Bronze Age, these two populations and Anatolian-related farmers had mixed with each other and with the hunter–gatherers of Europe to greatly reduce genetic differentiation. The impact of the Near Eastern farmers extended beyond the Near East: farmers related to those of Anatolia spread westward into Europe; farmers related to those of the Levant spread southward into East Africa; farmers related to those of Iran spread northward into the Eurasian steppe; and people related to both the early farmers of Iran and to the pastoralists of the Eurasian steppe spread eastward into South Asia

Reduction of the contaminant fraction of DNA obtained from an ancient giant panda bone

Objective: A key challenge in ancient DNA research is massive microbial DNA contamination from the deposition site which accumulates post mortem in the study organism’s remains. Two simple and cost-effective methods to enrich the relative endogenous fraction of DNA in ancient samples involve treatment of sample powder with either bleach or Proteinase K pre-digestion prior to DNA extraction. Both approaches have yielded promising but varying results in other studies. Here, we contribute data on the performance of these methods using a comprehensive and systematic series of experiments applied to a single ancient bone fragment from a giant panda (Ailuropoda melanoleuca). Results: Bleach and pre-digestion treatments increased the endogenous DNA content up to ninefold. However, the absolute amount of DNA retrieved was dramatically reduced by all treatments. We also observed reduced DNA damage patterns in pre-treated libraries compared to untreated ones, resulting in longer mean fragment lengths and reduced thymine over-representation at fragment ends. Guanine–cytosine (GC) contents of both mapped and total reads are consistent between treatments and conform to general expectations, indicating no obvious biasing effect of the applied methods. Our results therefore confirm the value of bleach and pre-digestion as tools in palaeogenomic studies, providing sufficient material is available

The Beaker phenomenon and the genomic transformation of northwest Europe

From around 2750 to 2500 bc, Bell Beaker pottery became widespread across western and central Europe, before it disappeared between 2200 and 1800 bc. The forces that propelled its expansion are a matter of long-standing debate, and there is support for both cultural diffusion and migration having a role in this process. Here we present genome-wide data from 400 Neolithic, Copper Age and Bronze Age Europeans, including 226 individuals associated with Beaker-complex artefacts. We detected limited genetic affinity between Beaker-complex-associated individuals from Iberia and central Europe, and thus exclude migration as an important mechanism of spread between these two regions. However, migration had a key role in the further dissemination of the Beaker complex. We document this phenomenon most clearly in Britain, where the spread of the Beaker complex introduced high levels of steppe-related ancestry and was associated with the replacement of approximately 90% of Britain’s gene pool within a few hundred years, continuing the east-to-west expansion that had brought steppe-related ancestry into central and northern Europe over the previous centuries

Initial Upper Palaeolithic Homo sapiens from Bacho Kiro Cave, Bulgaria

The Middle to Upper Palaeolithic transition in Europe witnessed the replacement and partial absorption of local Neanderthal populations by Homo sapiens populations of African origin. However, this process probably varied across regions and its details remain largely unknown. In particular, the duration of chronological overlap between the two groups is much debated, as are the implications of this overlap for the nature of the biological and cultural interactions between Neanderthals and H. sapiens. Here we report the discovery and direct dating of human remains found in association with Initial Upper Palaeolithic artefacts, from excavations at Bacho Kiro Cave (Bulgaria). Morphological analysis of a tooth and mitochondrial DNA from several hominin bone fragments, identified through proteomic screening, assign these finds to H. sapiens and link the expansion of Initial Upper Palaeolithic technologies with the spread of H. sapiens into the mid-latitudes of Eurasia before 45 thousand years ago. The excavations yielded a wealth of bone artefacts, including pendants manufactured from cave bear teeth that are reminiscent of those later produced by the last Neanderthals of western Europe. These finds are consistent with models based on the arrival of multiple waves of H. sapiens into Europe coming into contact with declining Neanderthal populations

A mitogenomic timetree for Darwin’s enigmatic South American mammal Macrauchenia patachonica

The unusual mix of morphological traits displayed by extinct South American native ungulates (SANUs) confounded both Charles Darwin, who first discovered them, and Richard Owen, who tried to resolve their relationships. Here we report an almost complete mitochondrial genome for the litoptern Macrauchenia. Our dated phylogenetic tree places Macrauchenia as sister to Perissodactyla, but close to the radiation of major lineages within Laurasiatheria. This position is consistent with a divergence estimate of B66 Ma (95% credibility interval, 56.64–77.83 Ma) obtained for the split between Macrauchenia and other Panperissodactyla. Combined with their morphological distinctiveness, this evidence supports the positioning of Litopterna (possibly in company with other SANU groups) as a separate order within Laurasiatheria. We also show that, when using strict criteria, extinct taxa marked by deep divergence times and a lack of close living relatives may still be amenable to palaeogenomic analysis through iterative mapping against more distant relatives

A guide to ancient protein studies

Palaeoproteomics is an emerging neologism used to describe the application of mass spectrometry-based approaches to the study of ancient proteomes. As with palaeogenomics (the study of ancient DNA), it intersects evolutionary biology, archaeology and anthropology, with applications ranging from the phylogenetic reconstruction of extinct species to the investigation of past human diets and ancient diseases. However, there is no explicit consensus at present regarding standards for data reporting, data validation measures or the use of suitable contamination controls in ancient protein studies. Additionally, in contrast to the ancient DNA community, no consolidated guidelines have been proposed by which researchers, reviewers and editors can evaluate palaeoproteomics data, in part due to the novelty of the field. Here we present a series of precautions and standards for ancient protein research that can be implemented at each stage of analysis, from sample selection to data interpretation. These guidelines are not intended to impose a narrow or rigid list of authentication criteria, but rather to support good practices in the field and to ensure the generation of robust, reproducible results. As the field grows and methodologies change, so too will best practices. It is therefore essential that researchers continue to provide necessary details on how data were generated and authenticated so that the results can be independently and effectively evaluated. We hope that these proposed standards of practice will help to provide a firm foundation for the establishment of palaeoproteomics as a viable and powerful tool for archaeologists, anthropologists and evolutionary biologists