Abstract
We generated genome-wide data from 69 Europeans who lived between 8,000-3,000 years ago by enriching ancient DNA libraries for a target set of almost four hundred thousand polymorphisms. Enrichment of these positions decreases the sequencing required for genome-wide ancient DNA analysis by a median of around 250-fold, allowing us to study an order of magnitude more individuals than previous studies1–⇓⇓⇓⇓⇓⇓8 and to obtain new insights about the past. We show that the populations of western and far eastern Europe followed opposite trajectories between 8,000-5,000 years ago. At the beginning of the Neolithic period in Europe, ~8,000-7,000 years ago, closely related groups of early farmers appeared in Germany, Hungary, and Spain, different from indigenous hunter-gatherers, whereas Russia was inhabited by a distinctive population of hunter-gatherers with high affinity to a ~24,000 year old Siberian6. By ~6,000-5,000 years ago, a resurgence of hunter-gatherer ancestry had occurred throughout much of Europe, but in Russia, the Yamnaya steppe herders of this time were descended not only from the preceding eastern European hunter-gatherers, but from a population of Near Eastern ancestry. Western and Eastern Europe came into contact ~4,500 years ago, as the Late Neolithic Corded Ware people from Germany traced ~3/4 of their ancestry to the Yamnaya, documenting a massive migration into the heartland of Europe from its eastern periphery. This steppe ancestry persisted in all sampled central Europeans until at least ~3,000 years ago, and is ubiquitous in present-day Europeans. These results provide support for the theory of a steppe origin9 of at least some of the Indo-European languages of Europe.
Genome-wide analysis of ancient DNA has emerged as a transformative technology for studying prehistory, providing information that is comparable in power to archaeology and linguistics. Realizing its promise, however, requires collecting genome-wide data from an adequate number of individuals to characterize population changes over time, which means not only sampling a succession of archaeological cultures2, but also multiple individuals per culture. To make analysis of large numbers of ancient DNA samples practical, we used in-solution hybridization capture10,11 to enrich next generation sequencing libraries for a target set of 394,577 single nucleotide polymorphisms (SNPs) (“390k capture”), 354,212 of which are autosomal SNPs that have also been genotyped using the Affymetrix Human Origins array in 2,345 humans from 203 populations4,12. This reduces the amount of sequencing required to obtain genome-wide data by a minimum of 45-fold and a median of 262-fold (Online Table 1). This strategy allows us to report genomic scale data on more than twice the number of ancient Eurasians as the entire preceding literature1–⇓⇓⇓⇓⇓⇓8 (Extended Data Table 1).
Only studies that produced at least one sample at ≥0.05× coverage are listed.
We used this technology to study population transformations in Europe. We began by preparing 212 DNA libraries from 119 ancient samples in dedicated clean rooms, and testing these by light shotgun sequencing and mitochondrial genome capture (SI1, Online Table 1). We restricted to libraries with molecular signatures of authentic ancient DNA (elevated damage in the terminal nucleotide), negligible evidence of contamination based on mismatches to the mitochondrial consensus13, and, where available, a mitochondrial DNA haplogroup that matched previous results using PCR4,14,15 (SI2). For 123 libraries prepared in the presence of Uracil-DNA-glycosylase16 to reduce errors due to ancient DNA damage17, we performed 390k capture, carried out paired end sequencing, and mapped to the human genome. We restricted analysis to 95 libraries from 69 samples that had at least 0.06-fold average target coverage (average of 3.8-fold), and used majority rule to call an allele at each SNP covered at least once (Online Table 1). After combining our data (SI3) with 25 ancient samples from the literature — three Upper Paleolithic samples from Russia1,7,6, seven people of European hunter gatherer ancestry4,5,8,2, and fifteen European farmers2,8,4,3, — we had data from 94 ancient Europeans. Geographically, these came from Germany (n=41), Spain (n=10), Russia (n=14), Sweden (n=12), Hungary (n=15), Italy (n=1) and Luxembourg (n=1) (Extended Data Table 2). Following the central European chronology, these included 19 hunter-gatherers (>5,500 BCE), 28 Early Neolithic farmers (EN: ~6,000-4,000 BCE), 11 Middle Neolithic farmers (MN: ~4,000-3,000 BCE) including the Tyrolean Iceman3, 9 Late Copper/Early Bronze Age individuals (Yamnaya: ~3,300-2,700 BCE), 15 Late Neolithic individuals (LN: ~2,500-2,200 BCE), 9 Early Bronze Age individuals (~2,200-1,500 BCE), two Late Bronze Age individuals (~1,200-1,100 BCE) and one Iron Age individual (~900 BCE). Two individuals were excluded from analyses as they were related to others from the same population. The average number of SNPs covered at least once was 212,375 and the minimum was 22,869 (Fig. 1).
Samples with direct radiocarbon dates are indicated by a calibrated date “cal BCE” along with associated laboratory numbers. Dates that are estimated based on faunal elements associated with the samples are not indicated with “cal” (although they are still calibrated, absolute dates).
(a) Geographic location and time-scale (central European chronology) of the 69 newly typed ancient individuals from this study (black outline) and 25 from the literature for which shotgun sequencing data was available (no outline). (b) Number of SNPs covered at least once in the analysis dataset of 94 individuals.
We determined that 34 of the 69 newly analyzed individuals were male and used 2,258 Y chromosome SNPs targets included in the capture to obtain high resolution Y chromosome haplogroup calls (SI4). Outside Russia, and before the Late Neolithic period, only a single R1b individual was found (early Neolithic Spain) in the combined literature (n=70). By contrast, haplogroups R1a and R1b were found in 60% of Late Neolithic/Bronze Age Europeans outside Russia (n=10), and in 100% of the samples from European Russia from all periods (7,500-2,700 BCE; n=9). R1a and R1b are the most common haplogroups in many European populations today18,19, and our results suggest that they spread into Europe from the East after 3,000 BCE. Two hunter-gatherers from Russia included in our study belonged to R1a (Karelia) and R1b (Samara), the earliest documented ancient samples of either haplogroup discovered to date. These two hunter-gatherers did not belong to the derived lineages M417 within R1a and M269 within R1b that are predominant in Europeans today18,19, but all 7 Yamnaya males did belong to the M269 subclade18 of haplogroup R1b.
Principal components analysis (PCA) of all ancient individuals along with 777 present-day West Eurasians4 (Fig. 2a, SI5) replicates the positioning of present-day Europeans between the Near East and European hunter-gatherers4,20, and the clustering of early farmers from across Europe with present day Sardinians3,4,27, suggesting that farming expansions across the Mediterranean to Spain and via the Danubian route to Hungary and Germany descended from a common stock. By adding samples from later periods and additional locations, we also observe several new patterns. All samples from Russia have affinity to the ~24,000 year old MA16, the type specimen for the Ancient North Eurasians (ANE) who contributed to both Europeans4 and Native Americans4,6,8. The two hunter-gatherers from Russia (Karelia in the northwest of the country and Samara on the steppe near the Urals) form an “eastern European hunter-gatherer” (EHG) cluster at one end of a hunter-gatherer cline across Europe; people of hunter-gatherer ancestry from Luxembourg, Spain, and Hungary sit at the opposite “western European hunter-gatherer4” (WHG) end, while the hunter-gatherers from Sweden4,8 (SHG) are intermediate. Against this background of differentiated European hunter-gatherers and homogeneous early farmers, multiple events transpired in all parts of Europe included in our study. Middle Neolithic Europeans from Germany, Spain, Hungary, and Sweden from the period ~4,000-3,000 BCE are intermediate between the earlier farmers and the WHG, suggesting an increase of WHG ancestry throughout much of Europe. By contrast, in Russia, the later Yamnaya steppe herders of ~3,000 BCE plot between the EHG and the Near East / Caucasus, suggesting a decrease of EHG ancestry during the same time period. The Late Neolithic and Bronze Age samples from Germany and Hungary2 are distinct from the preceding Middle Neolithic and plot between them and the Yamnaya. This pattern is also seen in ADMIXTURE analysis (Fig. 2b, SI6), which implies that the Yamnaya have ancestry from populations related to the Caucasus and South Asia that is largely absent in 38 Early or Middle Neolithic farmers but present in all 25 Late Neolithic or Bronze Age individuals. This ancestry appears in Central Europe for the first time in our series with the Corded Ware around 2,500 BCE (SI6, Fig. 2b, Extended Data Fig. 1). The Corded Ware shared elements of material culture with steppe groups such as the Yamnaya although whether this reflects movements of people has been contentious21. Our genetic data provide direct evidence of migration and suggest that it was relatively sudden. The Corded Ware are genetically closest to the Yamnaya ~2,600 kilometers away, as inferred both from PCA and ADMIXTURE (Fig.2) and FST (0.011 ± 0.002) (Extended Data Table 3). If continuous gene flow from the east, rather than migration, had occurred, we would expect successive cultures in Europe to become increasingly differentiated from the Middle Neolithic, but instead, the Corded Ware are both the earliest and most strongly differentiated from the Middle Neolithic population.
FST (below the diagonal), standard deviation (above the diagonal).
“Outgroup” f3-statistics6 (SI7), which measure shared genetic drift between a pair of populations (Extended Data Fig. 2), support the clustering of hunter-gatherers, Early/Middle Neolithic, and Late Neolithic/Bronze Age populations into different groups as in the PCA (Fig. 2a). We also analyzed f4-statistics, which allow us to test whether pairs of populations are consistent with descent from common ancestral populations, and to assess significance using a normally distributed Z-score. Early European farmers from the Early and Middle Neolithic were closely related but not identical. This is reflected in the fact that Loschbour shared more alleles with post-4,000 BCE European farmers from Germany, Spain, Hungary, Sweden, and Italy than with early farmers of Germany, Spain, and Hungary, documenting an increase of hunter-gatherer ancestry in multiple regions of Europe during the course of the Neolithic. The two EHG form a clade with respect to all other present-day and ancient populations (|Z|<1.9), and MA1 shares more alleles with them (|Z|>4.7) than with other ancient or modern populations, suggesting that they may be a source for the ANE ancestry in present Europeans4,12,22 as they are geographically and temporally more proximate than Upper Paleolithic Siberians. The Yamnaya differ from the EHG by sharing fewer alleles with MA1 (|Z|=6.7) suggesting a dilution of ANE ancestry between 5,000-3,000 BCE on the European steppe. This was likely due to admixture of EHG with a population related to present-day Near Easterners, as the most negative f3-statistic in the Yamnaya (giving unambiguous evidence of admixture) is observed when we model them as a mixture of EHG and present-day Near Eastern populations like Armenians (Z = −6.3; SI7). The Late Neolithic / Bronze Age groups of central Europe share more alleles with Yamnaya than the Middle Neolithic populations do (|Z|=12.4) and more alleles with the Middle Neolithic than the Yamnaya do (|Z|=12.5), and have a negative f3-statistic with the Middle Neolithic and Yamnaya as references (Z=-20.7), proving that they were descended from a mixture of the local European populations and new migrants from the east. Moreover, the Yamnaya share more alleles with the Corded Ware (|Z|≥3.6) than with any other Late Neolithic/ Early Bronze age group with at least two individuals (SI7), proving that they had more eastern ancestry, consistent with the PCA and ADMIXTURE patterns (Fig. 2).
(a) PCA analysis, (b) ADMIXTURE analysis. The full ADMIXTURE analysis including present-day humans is shown in Extended Data Fig. 1.
Modeling of the ancient samples shows that while Karelia is genetically intermediate between Loschbour and MA1, the topology that considers Karelia as a mixture of these two elements is not the only one that can fit the data (SI8). To avoid biasing our inferences by fitting an incorrect model, we developed new statistical methods that are substantial extensions of a previously reported approach4, which allow us to obtain precise estimates of the proportion of mixture in later Europeans without requiring a formal model for the relationship among the ancestral populations. The method (SI9) is based on the idea that if a Test population has ancestry related to reference populations Ref1, Ref2, …, RefN in proportions α1, α2, …,αN, and the references are themselves differentially related to a triple of outgroup populations A, B, C, then 
. By using a large number of outgroup populations we can fit the admixture coefficients αi and estimate mixture proportions (SI9, Extended Data Fig. 3). Using 15 outgroups from Africa, South/East/North Asia, and the Americas, we obtain good fits as assessed by a formal test (SI10), and estimate that the Middle Neolithic populations of Germany and Spain have ~18-34% more WHG-related ancestry than Early Neolithic ones and that the Late Neolithic and Early Bronze Age populations of Germany have ~22-39% more EHG-related ancestry than the Middle Neolithic ones (SI9). If we model them as mixtures of Yamnaya-related and Middle Neolithic populations, the inferred degree of population turnover is doubled to 48-80% (SI9, SI10).
We estimate mixture proportions using a method that gives unbiased estimates even without an accurate model for the relationships between the test populations and the outgroup populations (SI9). Population samples are grouped according to chronology (ancient) and Yamnaya ancestry (present-day humans).
To distinguish whether a Yamnaya or an EHG source fits the data better, we added ancient samples as outgroups (SI9). Adding any Early or Middle Neolithic farmer results in EHG-related genetic input into Late Neolithic populations being a poor fit to the data (SI9); thus, Late Neolithic populations have ancestry that cannot be explained by a mixture of EHG and Middle Neolithic. When using Yamnaya instead of EHG, however, we obtain a good fit (SI9, SI10). These results can be explained if the new genetic material that arrived in Germany was a composite of two elements: EHG and a type of Near Eastern ancestry different from that which was introduced by early farmers (also suggested by PCA and ADMIXTURE; Fig. 2, SI5, SI6). We estimate that these two elements each contributed about half the ancestry each of the Yamnaya (SI6, SI9), explaining why the population turnover inferred using Yamnaya as a source is about twice as high compared to the undiluted EHG. The estimate of Yamnaya-related ancestry in the Corded Ware is consistent when using either present populations or ancient Europeans as outgroups (SI9, SI10), and is 73.1 ± 2.2% when both sets are combined (SI10). The best proxies for ANE ancestry in Europe4 were initially Native Americans12,22, and then the Siberian MA16, but both are geographically and temporally too remote for what appears to be a recent migration into Europe4. We can now add three new pieces to the puzzle of how ANE ancestry was transmitted to Europe: first the EHG, then the Yamnaya formed by mixture between EHG and a Near Eastern related population, and then the Corded Ware who were formed by a mixture of the Yamnaya with Middle Neolithic Europeans. We caution that the sampled Yamnaya individuals from Samara might not be directly ancestral to Corded Ware individuals from Germany. It is possible that a more western Yamnaya population, or an earlier (pre-Yamnaya) steppe population may have migrated into central Europe, and future work may uncover more missing links in the chain of transmission of steppe ancestry.
By extending our model to a three way mixture of WHG, Early Neolithic and Yamnaya, we estimate that the ancestry of the Corded Ware was 79% Yamnaya-like, 4% WHG, and 17% Early Neolithic (Fig. 3). A small contribution of the first farmers is also consistent with uniparentally inherited DNA: e.g. mitochondrial DNA haplogroup N1a and Y chromosome haplogroup G2a, common in early central European farmers14,23, almost disappear during the Late Neolithic and Bronze Age, when they are largely replaced by Y haplogroups R1a and R1b (SI4) and mtDNA haplogroups I, T1, U2, U4, U5a, W, and subtypes of H14,23,24 (SI2).
The uniparental data not only confirm a link to the steppe populations but also suggest that both sexes participated in the migrations (SI2, SI4, Extended Data Table 3). The magnitude of the population turnover that occurred becomes even more evident if one considers the fact that the steppe migrants may well have mixed with eastern European agriculturalists on their way to central Europe. Thus, we cannot exclude a scenario in which the Corded Ware arriving in today’s Germany had no ancestry at all from local populations.
Our results support a view of European pre-history punctuated by two major migrations: first, the arrival of first farmers during the Early Neolithic from the Near East, and second of Yamnaya pastoralists during the Late Neolithic from the steppe (Extended Data Fig. 5). Our data further show that both migrations were followed by resurgences of the previous inhabitants: first, during the Middle Neolithic, when hunter-gatherer ancestry rose again after its Early Neolithic decline, and then between the Late Neolithic and the present, when farmer and hunter-gatherer ancestry rose after its Late Neolithic decline. This second resurgence must have started during the Late Neolithic/Bronze Age period itself, as the Bell Beaker and Unetice groups had reduced Yamnaya ancestry compared to the earlier Corded Ware, and comparable levels to that in some present-day Europeans (Fig. 3). Today, Yamnaya related ancestry is lower in southern Europe and higher in northern Europe. Further data are needed to determine whether the steppe ancestry arrived in southern Europe at the time of the Late Neolithic / Bronze Age, or is due to migrations in historical times from northern Europe25,26.
Our results provide new data relevant to debates on the origin and expansion of Indo-European languages in Europe (SI11). Although ancient DNA is silent on the question of the languages spoken by preliterate populations, it does carry evidence about processes of migration which are invoked by theories on Indo-European language dispersals. Such theories make predictions about movements of people to account for the spread of languages and material culture. The technology of ancient DNA makes it possible to reject or confirm the proposed migratory movements, as well as to identify new movements that were not previously known. The best argument for the “Anatolian hypothesis27” that Indo-European languages arrived in Europe from Anatolia ~8,500 years ago is that major language replacements are thought to require major migrations, and that after the Early Neolithic when farmers established themselves in Europe, the population base was likely to have been so large as to be impervious to subsequent turnover27,28. However, our study shows that a later major turnover did occur, and that steppe migrants replaced ~3/4 of the ancestry of central Europeans. An alternative theory is the “Steppe hypothesis”, which proposes that early Indo-European speakers were pastoralists of the grasslands north of the Black and Caspian Seas, and that their languages spread into Europe after the invention of wheeled vehicles9. Our results make a compelling case for the steppe as a source of at least some of the Indo-European languages in Europe by documenting a massive migration ~4,500 years ago associated with the Yamnaya and Corded Ware cultures, which are identified by proponents of the Steppe hypothesis as vectors for the spread of Indo-European languages into Europe.
These results challenge the Anatolian hypothesis by showing that not all Indo-European languages in Europe can plausibly derive from the first farmer migrations thousands of years earlier (SI11). We caution that the location of the Proto-Indo-European9,27,29,30 homeland that also gave rise to the Indo-European languages of Asia, as well as the Indo-European languages of southeastern Europe, cannot be determined from the data reported here (SI11).
Studying the mixture in the Yamnaya themselves, and understanding the genetic relationships among a broader set of ancient and present-day Indo-European speakers, may lead to new insight about the shared homeland.
Online Methods
Screening of libraries (shotgun sequencing and mitochondrial capture)
The 212 libraries screened in this study (SI1) from a total of 119 samples (SI3) were produced at Adelaide (n=151), Tübingen (n=16), and Boston (n=45) (Online Table 1).
The libraries from Adelaide and Boston had internal barcodes directly attached to both sides of the molecules from the DNA extract so that each sequence begins with the barcode10. The Adelaide libraries had 5 base pair (bp) barcodes on both sides, while the Boston libraries had 7 bp barcodes. Libraries from Tübingen had no internal barcodes, but were differentiated by the sequence of the indexing primer31.
We adapted a reported protocol for enriching for mitochondrial DNA10, with the difference that we adjusted the blocking oligonucleotides and PCR primers to fit our libraries with shorter adapters. Over the course of this project, we also lowered the hybridization temperature from 65°C to 60°C and performed stringent washes at 55°C instead of 60°C32.
We used an aliquot of approximately 500ng of each library for target enrichment of the complete mitochondrial genome in two consecutive rounds32, using a bait set for human mtDNA32. We performed enrichment in 96-well plates with one library per well, and used a liquid handler (Evolution P3, Perkin Elmer) for the capture and washing steps33. We used blocking oligonucleotides in hybridization appropriate to the adapters of the truncated libraries. After either of the two enrichment rounds, we amplified the enriched library molecules with the primer pair that keeps the adapters short (PreHyb) using Herculase Fusion II PCR Polymerase. We performed an indexing PCR of the final capture product using one or two indexing primers31. We cleaned up all PCR’s using SPRI technology34 and the liquid handler. Libraries from Tübingen were amplified with the primer pair IS5/IS631.
For libraries from Boston and Adelaide, we used a second aliquot of each library for shotgun sequencing after performing an indexing PCR31. We used unique index combinations for each library and experiment, allowing us to distinguish shotgun sequencing and mitochondrial DNA capture data, even if both experiments were in the same sequencing run. We sequenced shotgun libraries and mtDNA captured libraries from Tübingen in independent sequencing runs since the index was already attached at the library preparation stage.
We quantified the sequencing pool with the BioAnalyzer (Agilent) and/or the KAPA Library Quantification kit (KAPA biosystems) and sequenced on Illumina MiSeq, HiSeq2500 or NextSeq500 instruments for 2×75, 2×100 or 2×150 cycles along with the indexing read(s).
Enrichment for 394,577 SNP targets (“390k capture”)
The protocol for enrichment for SNP targets was similar to the mitochondrial DNA capture, with the exception that we used another bait set (390k) and about twice as much library (up to 1000ng) compared to the mtDNA capture.
The specific capture reagent used in this study is described for the first time here. To target each SNP, we used a different oligonucleotide probe design compared to ref. 1. We used four 52 base pair probes for each SNP target. One probe ends just before the SNP, and one starts just after. Two probes are centered on the SNP, and are identical except for having the alternate alleles. This probe design avoids systematic bias toward one SNP allele or another. For the template sequence for designing the San and Yoruba panels baits, we used the sequence that was submitted for these same SNPs during the design of the Affymetrix Human Origins SNP array12. For SNPs that were both in the San and Yoruba panels, we used the Yoruba template sequence in preference. For all other SNPs, we used the human genome reference sequence as a template. Online Table 2 gives the list of SNPs that we targeted, along with details of the probes used. The breakdown of SNPs into different classes is:
124,106 “Yoruba SNPs”: All SNPs in “Panel 5” of the Affymetrix Human Origins array (discovered as heterozygous in a Yoruba male: HGDP00927)12 that passed the probe design criteria specified in ref. 11.
146,135 “San SNPs”: All SNPs in “Panel 4” of the Affymetrix Human Origins array (discovered as heterozygous in a San male: HGDP01029)12 that passed probe design criteria11. The full Affymetrix Human Origins array Panel 4 contains several tens of thousands of additional SNPs overlapping those from Panel 5, but we did not wish to redundantly capture Panel 5 SNPs.
98,166 “Compatibility SNPs”: SNPs that overlap between the Affymetrix Human Origins the Affymetrix 6.0, and the Illumina 610 Quad arrays, which are not already included in the “Yoruba SNPs” or “San SNPs” lists12 and that also passed the probe design design criteria11.
26,170 “Miscellaneous SNPs”: SNPs that did not overlap the Human Origins array. The subset analyzed in this study were 2,258 Y chromosome SNPs (http://isogg.org/tree/ISOGG_YDNA_SNP_Index.html) that we used for Y haplogroup determination.
Processing of sequencing data
We restricted analysis to read pairs that passed quality control according to the Illumina software (“PF reads”).
We assigned read pairs to libraries by searching for matches to the expected index and barcode sequences (if present, as for the Adelaide and Boston libraries). We allowed no more than 1 mismatch per index or barcode, and zero mismatches if there was ambiguity in sequence assignment or if barcodes of 5 bp length were used (Adelaide libraries).
We used Seqprep (https://github.com/jstjohn/SeqPrep) to search for overlapping sequence between the forward and reverse read, and restricted to molecules where we could identify a minimum of 15 bp of overlap. We collapsed the two reads into a single sequence, using the consensus nucleotide if both reads agreed, and the read with higher base quality in the case of disagreement. For each merged nucleotide, we assigned the base quality to be the higher of the two reads. We further used Seqprep to search for the expected adapter sequences at either ends of the merged sequence, and to produce a trimmed sequence for alignment.
We mapped all sequences using BWA-0.6.135. For mitochondrial analysis we mapped to the mitochondrial genome RSRS36. For whole genome analysis we mapped to the human reference genome hg19. We restricted all analyses to sequences that had a mapping quality of MAPQ≥37.
We sorted all mapped sequences by position, and used a custom script to search for mapped sequences that had the same orientation and start and stop positions. We stripped all but one of these sequences (keeping the best quality one) as duplicates.
Mitochondrial sequence analysis and assessment of ancient DNA authenticity
For each library for which we had average coverage of the mitochondrial genome of at least 10-fold after removal of duplicated molecules, we built a mitochondrial consensus sequence, assigning haplogroups for each library as described in SI2.
We used contamMix-1.0.9 to search for evidence of contamination in the mitochondrial DNA13. This software estimates the fraction of mitochondrial DNA sequences that match the consensus more closely than a comparison set of 311 worldwide mitochondrial genomes. This is done by taking the consensus sequence of reads aligning to the RSRS mitochondrial genome, and requiring a minimum coverage of 5 after filtering bases where the quality was <30. Raw reads are then realigned to this consensus. In addition, the consensus is multiply aligned with the other 311 mitochondrial genomes using kalign (2.0.4)37 to build the necessary inputs for contamMix, trimming the first and last 5 bases of every read to mitigate against the confounding factor of ancient damage. This software had difficulty running on datasets with higher coverage, and for these datasets, we down-sampled to 50,000 reads, which we found produced adequate contamination estimation.
For all sequences mapping to the mitochondrial DNA that had a cytosine at the terminal nucleotide, we measured the proportion of sequences with a thymine at that position. For population genetic analysis, we only used partially UDG-treated libraries with a minimum of 3% C→T substitutions as recommended by ref. 33. In cases where we used a fully UDG-treated library for 390k analysis, we examined mitochondrial capture data from a non-UDG-treated library made from the same extract, and verified that the non-UDG library had a minimum of 10% C→T at the first nucleotide as recommended by ref. 38. Metrics for the mitochondrial DNA analysis on each library are given in Online Table 1.
390k capture, sequence analysis and quality control
For 390k analysis, we restricted to reads that not only mapped to the human reference genome hg19 but that also overlapped the 354,212 autosomal SNPs genotyped on the Human Origins array4. We trimmed the last two nucleotides from each sequence because we found that these are highly enriched in ancient DNA damage even for UDG-treated libraries. We further restricted analyses to sites with base quality≥30.
We made no attempt to determine a diploid genotype at each SNP in each sample. Instead, we used a single allele – randomly drawn from the two alleles in the individual – to represent the individual at that site20,39. Specifically, we made an allele call at each target SNP using majority rule over all sequences overlapping the SNP. When each of the possible alleles was supported by an equal number of sequences, we picked an allele at random. We set the allele to “no call” for SNPs at which there was no read coverage.
We restricted population genetic analysis to libraries with a minimum of 0.06-fold average coverage on the 390k SNP targets, and for which there was an unambiguous sex determination based on the ratio of X to Y chromosome reads (SI4) (Online Table 1). For individuals for whom there were multiple libraries per sample, we performed a series of quality control analysis. First, we used the ADMIXTURE software40,41 in supervised mode, using Kharia, Onge, Karitiana, Han, French, Mbuti, Ulchi and Eskimo as reference populations. We visually inspected the inferred ancestry components in each individual, and removed individuals with evidence of heterogeneity in inferred ancestry components across libraries. For all possible pairs of libraries for each sample, we also computed statistics of the form D(Library1, Library2; Probe, Mbuti), where Probe is any of a panel of the same set of eight reference populations), to determine whether there was significant evidence of the Probe population being more closely related to one library from an ancient individual than another library from that same individual. None of the individuals that we used had strong evidence of ancestry heterogeneity across libraries. For samples passing quality control for which there were multiple libraries per sample, we merged the sequences into a single BAM.
We called alleles on each merged BAM using the same procedure as for the individual libraries. We used ADMIXTURE41 as well as PCA as implemented in EIGENSOFT42 (using the lsqproject: YES option to project the ancient samples) to visualize the genetic relationships of each set of samples with the same culture label with respect to 777 diverse present-day West Eurasians4. We visually identified outlier individuals, and renamed them for analysis either as outliers or by the name of the site at which they were sampled (Extended Data Table 1). We also identified two pairs of related individuals based on the proportion of sites covered in pairs of ancient samples from the same population that had identical allele calls using PLINK43. From each pair of related individuals, we kept the one with the most SNPs.
Population genetic analyses
We determined genetic sex using the ratio of X and Y chromosome alignments44 (SI4), and mitochondrial haplogroup for all samples (SI2), and Y chromosome haplogroup for the male samples (SI4). We studied population structure (SI5, SI6). We used f-statistics to carry out formal tests of population relationships (SI6) and built explicit models of population history consistent with the data (SI7). We estimated mixture proportions in a way that was robust to uncertainty about the exact population history that applied (SI8). We estimated the minimum number of streams of migration into Europe needed to explain the data (SI9, SI10). The estimated mixture proportions shown in Fig. 3 were obtained using the lsqlin function of Matlab and the optimization method described in SI9 with 15 world outgroups.
Supplementary Information 3 Description of ancient samples and archaeological context
Overview
The archaeology of prehistoric Europe has revealed numerous material cultures that differed from each other over space and time. Some cultures left behind artifacts similar to those that preceded them. Other cultures left behind artifacts that mark a clear break from preceding local traditions.
The largest single group of samples analyzed in this study was from the transect through time study from the Early Neolithic to the Early Bronze Age reported in Brandt et al. 20131. We selected samples from that study based on two criteria: We predominantly chose samples based on overall DNA quality, which we assessed based on performance in our previous study (largely PCR success rates, replication of results, and completeness of SNP typing). From the subset of samples that performed well, we chose ones that could be assigned most confidently to an archaeological culture via accompanying grave goods/context, orientation of the burials, available radiocarbon dates, and seemingly typical mtDNA haplotype. In addition, we added to this study some individuals for which the cultural attribution was less clear, in that there were no characteristic grave goods or context. These individuals were included to test the ability of the 390k SNP capture strategy to assign a potentially unknown or unprovenanced sample to a candidate population in combination with absolute dating. For the latter set of samples, we obtained 19 new radiocarbon dates at the Curt-Engelholm-Centre for Archaeometry, Mannheim, Germany.
In what follows we provide archaeological context information for the 69 samples for which genome-wide data is newly presented in this study (a total of 119 individuals were screened as detailed in Online Table 1, but here we discuss only the subset that produced genome-wide data). We present the samples in chronological order, largely following the central European terminology, i.e. Mesolithic (>6000-5000 BCE), Early Neolithic (6000-4000 BCE), Middle Neolithic (4000-2800 BCE), Late Neolithic (2800-2200 BCE), and Early Bronze Age (2200-1600 BCE; Figure 1a). We note that there are different traditional terminology for samples from the Russian steppe and regions of southern Europe.
We use “BCE” (Before Common Era) to refer to a calibrated date that is estimated based on archaeological context (e.g. radiocarbon dates from associated faunal remains), and “calBCE” to refer to a calibrated radiocarbon date range (1σ) directly from human skeletal remains, followed by the respective dating laboratory number.
Hunter-gatherer samples
We generated data on two hunter-gatherer samples from western Russia (the easternmost part of Europe) to bridge the geographical gap between the West European site Loschbour (WHG)2 and the Siberian site Mal’ta in Siberia, Russia3, from which hunter-gatherer genetic data are available from the literature.
The individual we refer to as ‘Karelia’ in this study is
UZ0074/I0061 (MAE RAS collection number 5773-74, grave number 142) from the ~5500 BCE Mesolithic site Yuzhnyy Oleni Ostrov (island in Lake Onega) in Karelia, Western Russia, for which a complete mitochondrial genome was published recently4. Mitochondrial HVS I data from eight other individuals from the same site have also been described5.
The individual we refer to as ‘Samara hunter-gatherer’
I0124/SVP44 (5640-5555 calBCE, Beta-392490) is an adult male from grave 1 in a Neolithic-Eneolithic settlement producing artifacts from the Elshanka, Samara, and Repin cultures. The specific site is Lebyazhinka IV, on the Sok River, Samara oblast, Russia. (‘Neolithic’ here refers to the presence of ceramics, not to domesticated animals or plants.) The radiocarbon date of this individual, based on a femur, is centuries before the appearance of domesticated animals in the middle Volga region. Lebyazhinka IV and the neighboring Lebyazhinka V site were occupied seasonally by multiple cultures between 7000-3500 BCE; a few graves were found in the settled areas6.
Early Neolithic
The Early Neolithic in Europe in this study is represented by new samples reported from sites in Hungary, Germany and Spain.
The central European Neolithic (6200–3950 BCE) is first manifested in the Starčevo culture, the Transdanubian Linienbandkeramik (LBKT), and the central European distribution of the Linienbandkeramik (LBK); these were the first people in the region to exploit agriculture and animal husbandry7. The Linienbandkeramik appears earliest in the archaeological record of western Hungary (Transdanubia), where it incorporated novel technologies and cultural elements from the preceding, southeastern Starčevo culture, which in turn also shows similarities in material culture to early farming groups further southeast, including Anatolia, the Levant and the Near East. During the Neolithic transition, the LBK expanded relatively rapidly along the major waterways and fertile Loess plains towards central Europe, extending as far as the Paris Basin in the West and Ukraine in the East8,9.
Starčevo in Hungary: Alsónyék-Bátaszék, Mérnöki telep
The Alsónyék-Bátaszék, Mérnöki telep site was excavated by the Institute of Archaeology, Hungarian Academy of Sciences, between 2006 and 2009 (excavators: A. Osztás, I. Zalai-Gaál). This site is a part of the site complex “Alsónyék”, which forms the largest agglomeration of Neolithic settlements and cemeteries in Hungary. Alsónyék is situated along the Tolna county part of the M6 motorway track in south Hungary. At Mérnöki telep, traces of the Early Neolithic Starčevo culture and other Neolithic features, including LBK and the Late Neolithic Lengyel culture, came to light across a total of 80 hectares. Out of the 1568 archaeological features, more than 400 could be associated with the Starčevo culture including 26 burials. The majority of features were pits, in various shapes and sizes; however, ditches and various oven types were also found. The skeletal remains were all found inside ovens or in pits, and were all in flexed positions. Only one burial contained grave goods10. All 26 Starčevo skeletons were investigated as part of the German Research Foundation (DFG) funded bioarchaeological (ancient DNA and stable isotopes) project entitled “The population history of the Carpathian Basin in the Neolithic period and its influence on the colonization of central Europe”. Skeletons in uncertain stratigraphic positions, and buried without any grave goods, were radiocarbon dated at the CEZA laboratory in Mannheim, Germany. Of these, individual
BAM25a/I0174 (feature 1532, 5710-5550 calBCE, MAMS 11939) was included in this study.
Transdanubian LBK in Hungary: Szemely-Hegyes
The south-Hungarian site Szemely-Hegyes is located in Baranya county along the M6 motorway. The site was excavated between 2006 and 2007 by a commercial archaeology contractor (Ásatárs Ltd; excavators T. Paluch and K. Somogyi). Approximately 1400 archaeological features were documented across 4 hectares, including finds from the LBK, Sopot culture, Copper Age Balaton-Lasinja, and Furchenstich groups11 (T. Paluch, K. Somogyi, J. Jakucs personal communication), including ten Neolithic graves. An LBK settlement with 20 houses, several pits, ditches and ovens was also uncovered. Six out of the ten Neolithic graves could be assigned to either the Transdanubian LBK or to the Sopot period, and the skeletons were radiocarbon dated at Beta Analytic in Miami, USA. Sample
SZEH4b/I0176 (feature 1001, 5210-4940 calBCE, Beta - 310038) associated with the Transdanubian LBK was analyzed here.
LBK in Germany: Halberstadt-Sonntagsfeld
The most intensively studied region in this study is Mittelelbe-Saale (MES) in Saxony-Anhalt, Germany. MES is situated within a tight network of waterways along ancient established trade routes. The region is climatically and geologically well characterized and benefits from its location in the rain-shadow of the nearby Harz Mountains, its deposits of ores and salts, and its fertile black and para-brown soils12–⇓14. The earliest MES culture sampled in this study is the LBK.
The LBK Halberstadt-Sonntagsfeld site was discovered during construction of a new development and was excavated between 1999 and 2002, uncovering 1324 archaeological features across an area of 9947m2. The majority of the finds could be attributed to the LBK, with contours and remnants of seven long houses, several pits and 42 graves15. The remaining finds could be attributed to the Middle Neolithic Bernburg culture as well as the Unetice and Urnfield cultures of the Bronze Age. The site is a classical example of LBK settlement burials, where the majority of graves were grouped around five long houses, and one group of six graves located inside the central yard area of the settlement. Skeletal material from the site is well preserved and led to mitochondrial DNA results for 39 individuals1. We included seven individuals clearly associated with an LBK house:
HAL5/I0046 (grave 2, feature 241.1, 5206-5004 calBCE, MAMS 21479)
HAL25/I0048 (grave 28, feature 861, 5206-5052 calBCE, MAMS 21482)
HAL14/I0056 (grave 15, feature 430, 5206-5052 calBCE, MAMS 21480)
HAL34/I0057 (grave 38, feature 992, 5207-5067 calBCE, MAMS 21483)
HAL4/I0100 (grave 1, feature 139, 5032-4946 calBCE, KIA40341)
HAL2/I0659 (grave 35, feature 999, 5079-4997 calBCE, KIA40350 and 5066-4979 calBCE, KIA30408; Fig. S3.1)
HAL24/I0821 (grave 27, feature 867, 5034-4942 calBCE, KIA40348)
Radiocarbon dates obtained from these samples confirmed the attribution to the older and intermediate phase of the LBK period.
(Photo: J. Lipták, LDA Sachsen-Anhalt, Germany).
LBK in Germany: Oberwiederstedt-Unterwiederstedt
Several sites around Wiederstedt, Mansfeld-Südharz, and Saxony-Anhalt were excavated in the late 20th century. Promontory areas above the Wipper River in the Harz region in Hettstedt were repeatedly used during LBK times, as reflected in traces of settlements. In addition, excavations in 2000 revealed a mass grave of 12 individuals of which individual
UWS4/I0054 (grave 6, feature 1 14, 5209-5070 calBCE, MAMS 21485) was included in this study16.
LBK in Germany: Karsdorf
The site of Karsdorf is located in the valley of Unstrut, Burgenlandkreis, Saxony-Anhalt, Germany. The slope on which Karsdorf is located is characterized by alluvial loess. The place itself was settled intensively since the earliest phase of the LBK in the region. The settlement area is at least 50 acres in size and nearly 30 houses have been excavated. So-called ‘settlement burials’ were regularly found in pits in the center of the settlement area, of which individual
KAR6/I0795 (feature 170, 5207-5070 calBCE, MAMS 22823) was sampled for this study.
LBK in Germany: Stuttgart-Mühlhausen
The site Stuttgart-Mühlhausen “Viesenhäuser Hof” was excavated in several campaigns between 1931 and 1993. It reflects a long period of habitation starting from the Early Neolithic to the Iron Age. The Early Neolithic is represented by a large number of well-preserved burials from the LBK, separated into two areas including burials from the early phases of the LBK (area-2; 5500-5300 BCE) and the middle and later phases (area-1). Three individuals from area-2 are newly reported in this study (a fourth LBK sample from the Viesenhäuser Hof site was previously sequenced to high coverage and is co-analyzed with the newly sequenced LBK in this study2). Based on morphology, individual
LBK1976/I0022 (grave II-106, area-2, 5500-5300 BCE) is a female who died at an estimated age of 50-65 years. The skeleton derives from a grave that was excavated among 90 others from area-2 of the cemetery. The woman was buried in the characteristic way of the LBK, lying in flexed position on the right side, but lacks any grave goods. The burial was oriented from SW-NE with the skull facing NW. Most of the body parts were represented and in anatomical order.
The second well-preserved skeleton
LBK1992/I0025 (grave II-113, area-2, 5500-5300 BCE) is a male who died at an estimated age of 50-70 years. The skeleton was found prone in the grave with the position of the left leg indicated a flexed position. The burial was oriented from SE-NW with the skull facing south. The grave only contained a single pottery sherd found in the filling of the grave.
The third skeleton is a female
LBK2155/I0026 (grave II-125, area-2, 5500-5300 BCE) who died at 35-50 years. This individual was found prone with flexed legs imitating a seated position. The grave was oriented from W-E with the skull facing north. The only associated finding was a pottery fragment from the filling.
Epicardial Neolithic in Spain: Els Trocs
The Pyrenean cave site of Els Trocs (close to San Feliu de Veri, Bisaurri (Huesca), Spain) is located in the Upper Ribagorza of Aragón at 1561 meters above sea level, inside a conical hill, which dominates the surrounding ‘Selvaplana’ plain, traditionally used for pasture and cultivation. The entrance to the cave (UTM coordinates: x-298.198, y-4.702.955, z-1561) is oriented to the south and hidden by abundant vegetation and huge stone boulders, which partially close the entrance (currently 2.3 m high and 1.8 m wide). After a steep access ramp, a chamber of 15 m of length and 6 m of maximum width opens up, in which the temperature remains stable at 6 and 8°C throughout the year. The excavations carried out inside the cave have uncovered a stratigraphic sequence that in combination with a series of radiocarbon dates characterize the following four phases of human occupation17:
TROCS I: the first occupation of the cave, dated to 5300-5000 BCE.
TROCS II: the Middle Neolithic horizon, dated around 4500 BCE.
TROCS III: intensive use spanning >1000 years between 4000-2900 BCE.
TROCS IV: upper layer of the cave from historical times (100 BCE) to today.
Human bones were discovered from all four phases, but especially from Trocs I during which they appear to be associated with a marked and complex ritual behavior, which involved manipulation of skeletal elements and the deposition of abundant faunal remains (mainly young lambs) that had been consumed.
The first occupation phase of the cave (Trocs I) is related to the so-called Epicardial tradition. However, this attribution is currently under re-consideration18 since both the incised-impressed-grooved decorative techniques and the Cardial pottery types are assumed to be two variants of the same archaeological culture responsible of the initial Neolithization of Iberia. The samples successfully analyzed for this study are
Troc1/I0409 (5311-5218 cal BCE, MAMS 16159)
Troc3/I0410 (5178-5066 cal BCE, MAMS 16161)
Troc4/I0411 (5177-5068 cal BCE, MAMS 16162)
Troc5/I0412 (5310-5206 cal BCE, MAMS 16164)
Troc7/I0413 (5303-5204 cal BCE, MAMS 16166)
The genetic data indicates that Troc4 was a close relative of Troc3. We excluded the data from Troc4 from many genome-wide analyses since Troc3 was higher coverage.
Middle Neolithic
Baalberge, Salzmuende, and Bernburg in Germany: Esperstedt
Esperstedt is located in the Merseburg-Querfurt district, Saxony-Anhalt, and is situated in the centre of the Querfurter Platte, a plain that is primarily loess.
The single grave of individual
ESP30/I0807 (feature 6220; 3887-3797 calBCE, Er7784)
discovered in 2004 during investigations in advance to major roadworks could be assigned to the Baalberge group.
Site 4b is located east of the Weida near Esperstedt and forms part of large-scale excavations that were initiated in 2005 in the context of major infrastructural roadworks in Saxony-Anhalt, Germany. Site 4b revealed mostly finds from the Middle Neolithic Salzmünde and Bernburg cultures, viewed as regional forms of the Funnel Beaker tradition (or Trichterbecherkultur TRB), which defines the Middle Neolithic in central Europe. The genetic results from individual
ESP24/I0172 (3360-3086 calBCE, Erl8699)
were retrieved from skeletal remains found in a settlement pit that was attributed to the Bernburg culture, but had no associated grave goods. Radiocarbon dating from the >60 year-old male individual matched both the temporally overlapping phases of the Salzmünde (3400-3025 BCE) and Bernburg (3100-2650 BCE) cultures19.
Baalberge in Germany: Quedlinburg
The site Quedlinburg, Harzkreis, is situated in the fertile foothills of the northern Harz, a region characterized by rich loess soils. The specific site Lehofsberg (Reference site 9) was excavated during major roadworks for highway B6n, at which archaeological excavations revealed a small graveyard with a dozen graves (without trapezoidal enclosure) from the Baalberge culture and a total 20 burials spread over a length of 200 meters. The individuals
QLB15/I0559 (feature 21033, 3645-3537 cal BCE, MAMS 22818)
QLB18/I0560 (feature 21039, 3640-3510 calBCE, Er7856)
that were sampled for this study are likely to be burials of commoners as more elaborate grave architectural elements such as cists are missing.
Middle Neolithic in Spain: La Mina
The site La Mina (Alcubilla de las Peñas, Soria, Spain) includes a classic passage grave with a corridor longer than five meters oriented south-southeast. The site suffered from a systematic process of closure and dismantling of its stone structure with all orthostats of the chamber and the passage being removed. However, the burial chamber, from which the remains of approximately 20 buried individuals have been recovered, remained intact. At the time of the burials, the appearance of the tomb was modified by increasing the diameter and height of the original mound, placing a menhir higher than three meters possibly on top of the monument, and building a wall of orthostats parallel to the old burial surrounding the entire perimeter wall. As a result, the original collective grave had been transformed into a ceremonial center, and given its unique topographic location the mound became a territorial landmark. A radiocarbon date from a human bone from the ossuary (3780-3700 calBCE, Beta 316132) places the tomb at the beginning of Megalithism in the inner Iberian Peninsula, at the early 4th millennium.
The samples of La Mina originate from two seasons. The samples of Mina1-10 are from season 2010 and Mina 11-18 from 2012. In both seasons, samples were taken directly at the excavation with gloves and facemask and immediately stored under cool conditions. In 2010, samples were taken by geneticist Sarah Lang and in 2011 by archaeologist Manuel Rojo-Guerra. The samples that produced 390k data and that are included in our analysis are:
Mina3/I0405 (3900-3600 BCE)
Mina5/I0406 (3900-3600 BCE)
Mina6/I0407 (3900-3600 BCE)
Mina18/I0408 (3900-3600 BCE)
Late Copper/Early Bronze Age steppe chronology
The Yamnaya horizon was the first cultural complex that spread across all of the Pontic-Caspian-Ural steppes, beginning about 3300 BCE. It is divided into at least six regional groups and at least two chronological stages (up to four phases have been suggested), and the earliest ceramics included at least two distinct types (originating on the lower Don and lower Volga), both of which were decorated with cord impressions and usually were round-bottomed. The unifying traits were: a mobile pastoral economy that operated from newly invented wheeled vehicles using horseback-mounted herders; the introduction of new metal types (shafthole axes, tanged daggers, and spiral hair-rings or Lockenringe), new metal techniques, and copper mining on a significant scale, perhaps inspired by the Maikop culture; the burial of a select few adult males (who made up 75-80% of the graves under kurgans in the Samara region) under kurgans that usually covered 1 to 3 graves (although in the Kuban and NW Pontic steppes a wider range of people was buried under kurgans); the burial of wheels or whole wagons above or beside elite graves; deposition of animal sacrifices (usually sheep-goat) in about 15% of graves; and erection of grave-associated stone stelae, in the Ukrainian steppes. A few stratified settlements are found in the Ukrainian steppes west of the Don but none are known east of the Don in the Volga-Ural region. The Mikhailovka settlement on the Dnieper River is the standard stratigraphic guide to Yamnaya chronology20,21. Pre-Yamnaya level 1 began about 3800 BCE, early Yamnaya level 2 began about 3500 BCE, and late Yamnaya level 3 began about 2800 BCE. At Mikhailovka the Yamnaya phase (early level 2) began about 3500-3300 BCE22, but the majority of early Yamnaya kurgans began later, closer to 3300 BCE, from the Volga to the Dnieper, so 3300 BCE is a safer date for the general beginning of the Yamnaya era23. EBA Yamnaya evolved into the MBA Poltavka culture in the Volga-Ural steppes and into the MBA Catacomb culture in the Dnieper-Don-Caucasus steppes beginning about 2800 BCE. In the Dniester-Bug steppes, graves of the late Yamnaya style continued as late as 2200 BCE.
Yamnaya in Russia: Ekaterinovka
The site Ekaterinovka is located in the Southern Steppe on the left bank of the Volga 128 km southwest of the city of Samara. The >45 years old mature male individual included in this study is
SVP3/I0231 (Ekaterinovka, grave 1, 2910-2875 calBCE, Beta 392487).
Yamnaya in Russia: Lopatino I
A large cemetery of 39 kurgans was located on a low terrace beside the Sok River, Samara oblast, Russia (N53°38’24”/E50°39’18”). Eight kurgans were excavated in different years by various teams. Five were constructed in the Yamnaya period and three were added by the MBA Poltavka culture. We included three individuals from this site:
SVP5/I0357 (kurgan 35, central grave 1, 3090-2910 calBCE, Beta 39248) was from a grave that contained the remains of two Yamnaya individuals, including an adult woman and a child, an unusual pair, because 75-80% of individuals in Yamnaya kurgans in the Samara region were adult males.
SVP38/I0429 (kurgan 31, central grave 1, 3339-2917 calBCE, AA47804) was from a grave of an adult male 35-45 years old, 175 cm tall, supine, with a triangular flint projectile point beside him.
SVP52/I0439 (kurgan 1, central grave 1, 3305-2925 calBCE, Beta 392491) was from an adult male 25-35 years old, 178.5 cm tall.
Yamnaya in Russia: Ishkinovka I
Ishkinovka (or Ishkinino) is a kurgan cemetery located 30 km north of Novotroitsk on a right-bank tributary of the Ural River, among the easternmost Yamnaya sites, 570 km southeast of Lopatino I. The region contains copper ores exploited by miners who were active throughout the Bronze Age beginning in the EBA, which might explain the Yamnaya occupation of this eastern region, 250 km east of the Yamnaya mines at the Kargaly copper ore field24. The male individual included in this study is
SVP10/I0370 (Kurgan 3, grave 7, 3300-2700 BCE)
Yamnaya in Russia: Luzhki I
This unique cemetery, 1-2 km distant from Lopatino I, might represent the undocumented majority of the Yamnaya population that was not buried under kurgans. Individual,
SVP50/I0438 (3021-2635 calBCE, AA47807)
an adult male 25-35 years old was found in a cemetery with no obvious kurgan, surrounded by the graves of 5 children. He was buried face down with his hands behind his back. He was crippled by a blow with a heavy blunt instrument to his right hip that crushed his femur just below the trochanter, with no healing evident, and his skull was gouged pre-mortem in six places with a serrated tool. His wounds and burial position suggest that he was tortured and executed.
Yamnaya in Russia: Kurmanaevka III
This cemetery of three Yamnaya kurgans was erected on the first terrace overlooking the floodplain of the Buzuluk River, a left-bank tributary of the upper Samara River, in western Orenburg oblast, Russia, 170 km southeast of Lopatino I.
SVP54/I0441 (Kurgan 3, 3010-2622 calBCE, AA47805) The sample is taken from the femur of an adult woman 35-45 years old whose bones were heavily stained with red ochre. Her grave is a peripheral grave (burial 2), and is located above the central grave of a male (burial 1).
Yamnaya in Russia: Lopatino II
A cemetery of three kurgans was located on the first terrace above the floodplain 2 km northeast of the village of Lopatino on the Sok River in Samara oblast, Russia. One kurgan (3) was assigned to the EBA Yamnaya culture, the second (kurgan 1) to the MBA Poltavka culture, and the third (kurgan 2) to the final MBA II Potapovka culture, related culturally to Sintashta east of the Urals.
SVP57/I0443 (Grave 1, kurgan 3, 3300-2700 BCE) contained a male aged 15-17, apparently killed by an unhealed blunt-force trauma to his right parietal by a hammer-like weapon. Unusually for Yamnaya kurgans, none of the three graves under kurgan 3 were located under the center of the mound, but grave 1 was farthest from the center, near the northern margin.
Yamnaya in Russia: Kutuluk
Kutuluk kurgan cemetery I, located 60 km east of the city of Samara, contained:
SVP58/I0444 (central grave 1, kurgan 4, 3335-2881 calBCE, AA12570)
The remains are of male aged 25-35 years (Fig. S3.2), estimated height 176 cm, with no obvious injury or disease, and buried with the largest metal object found in a Yamnaya grave anywhere26. The object was a blunt mace 48 cm long, 767 g in weight, cast/annealed and made of pure copper, like most Yamnaya metal objects.
Late Neolithic
Since most of our samples were collected in present-day Germany, we follow the German/north-central European chronology for the Late Neolithic. Here, the Late Neolithic horizon in central Europe is defined as 2725–2200 BCE, even though earliest signs of the Corded Ware culture can be found around 2800 BCE, whereas remains of Bell Beaker cultures prevail until 2050 BCE. The Bell Beaker horizon is evident in the archaeological record from 2500 BCE onward in the southern Mittelelbe-Saale (MES), when it starts to move into settlement areas previously occupied by Corded Ware people, the latter of which have cultural affinities to archaeological groups further east such as the Globular Amphora and the Yamnaya culture in the North Pontic and Russian steppes. The settlement density increases during later Bell Beaker phases (2300-2050 BCE), when the Corded Ware is superseded by Bell Beaker elements, often at the same sites. This Late Neolithic Bell Beaker phenomenon is of interest, since the earliest archaeological evidence has been described in the Tagus region of Western Iberia around 2800-2700 BCE and it has been traced archaeologically over large parts of Western Europe but is also found as far as Hungary, Ireland, and southern Scandinavia, and in smaller enclaves in North Africa27. Here, earlier Neolithic cultures were overlain by discernable Bell Beaker elements, which became visible in rich grave goods (including the eponymous bell-shaped ceramic beakers). During the transition to the Bronze Age, Early Bronze Age cultural elements of the Únětice culture appear contemporaneously to late elements of the Bell Beaker culture, again sometimes also at the same site. A number of sites, such as Eulau and Quedlinburg show the presence of both Corded Ware and Bell Beaker cultures, and later of Únětice culture, and highlight the cultural diversity and dynamic at the time.
Corded Ware in Germany: Esperstedt
The site Esperstedt forms part of large-scale excavations initiated in 2005 in the context of major infrastructural roadworks in Saxony-Anhalt, Germany, to build motorway A38. Individuals from Esperstedt reference site 4 could be unambiguously assigned to the Corded Ware, both by accompanying pottery and characteristic orientation of the burials28. Males were usually buried in a right-hand side flexed position with head in the west and facing south, while females were buried on their left-hand side with their head in the east. We studied four individuals from this site:
ESP11/I0104 (feature 6216, 2473-2348 calBCE, MAMS 21487)
ESP16/I0103 (feature 6236, 2566-2477 calBCE, MAMS 21488)
ESP22/I0049 (feature 6233.1, 2454-2291 calBCE, MAMS 21489)
ESP 26/I0106 (feature 6216, 2454-2291 calBCE, MAMS 21490)
Bell Beaker in Germany: Rothenschirmbach
The site Rothenschirmbach in the district Merseburg-Querfurt is also located on the loess plain of the ‘Querfurter Platte’. The site features a densely occupied cemetery from the Bell Beaker period including 14 burials. Females of the Bell Beaker culture were usually buried on their right side with the head in the south and facing east, while males were buried with their heads in the north, facing west. Key features of this cemetery are a high proportion of children’s burials and the large number of rich and embellished graves with partially complex architecture (cists, menhirs, e.g. Fig. S3.3), and the rare finding of gold in a man’s grave (the oldest gold find in central Germany)29. We sampled three individuals from this site: Individual
ROT3/I0060 (feature 10011, 2294-2206 calBCE, MAMS 22819) is a 4-6 year old child (infans I) buried in a stone cist.
ROT4/I0111 (feature 10142, 2414-2333 calBCE, Er8712) featured remnants of a strong wooden cist covered by a 400kg menhir stone of 1.5 x 0.55m in size.
ROT6/I0108 (feature 10044, 2497-2436 calBCE, Er8710) is an adult buried in a open stone cist.
Bell Beaker and Late Neolithic in Germany: Benzingerode-Heimburgxs
BZH6/I0059 (grave 2, feature 1287, 2286-2153 calBCE, MAMS 21486) was attributed to the Bell Beaker period by excavator Tanja Autze as the pit overlaps with a neighbouring grave harboring BBC features. The orientation matches BBC customs, and the radiocarbon date supports assignment to this period.
BZH4/I0058 (grave 7, feature 4607, 2283-2146 calBCE, MAMS 21491) had no grave goods, and the skeleton was oriented SE-NW, rightside-flexed position, facing north.
BZH12/I0171 (grave 3, feature 2350, 2204-2136 calBCE, KIA27952) was buried in SE-NW orientation, in a right-sided flexed position, facing west. While the burial customs do not match standard Bell Beaker or the Corded Ware rites, the radiocarbon date supports a Late Neolithic assignment.
Interestingly, radiocarbon dates of all three individuals fall in the overlapping period of the late Corded Ware, Bell Beaker and early Únětice period in this region30.
Bell Beaker and Late Neolithic in Germany: Quedlinburg VII and XIIa
Reference site XII-West is located northeast of Quedlinburg, Saxony-Anhalt, and forms part of a small graveyard with mixed grave goods from Late Neolithic cultures31. The grave
QUEXII6/I0112 (feature 6256, 2340-2190 calBCE, Er7038) was archaeologically assigned to Bell Beaker based on the orientation, however grave goods include both a Bell Beaker ‘Füsschenschale’ and a ‘Corded Ware’ cup with ornaments characteristic for the single grave culture. The individual was an outlier from other Bell Beaker samples in PCA, and so we marked it as such for genome-wide analysis.
QUEXII4/I0113 (feature 6255.1, 2290-2130 cal BCE (Er7283) was also a female whose single grave good, a small cup, was uninformative. The orientation of her body is typical for the Bell beaker period.
Another group of six graves was discovered at Quedlinburg reference site VII and attributed to the Bell beaker culture based on form and orientation of the burials32. We included a >50 year old male individual buried in an extreme flexed position.
QLB28/I0806 (feature 19617, 2296-2206 calBCE, MAMS 22820)
Other Late Neolithic samples in Germany: Karsdorf
We also included a sample from an unusual burial at the Karsdorf site in Germany
KAR22/I0550 (feature 00191, date unknown)
The original archaeological attribution to the Baalberge culture is ambiguous and while the grave appears prehistoric, the determination was entirely based its position inside the eastern gate of a burial complex from the Baalberge culture. The mitochondrial genome of KAR22 could be determined as T1a1, matching previous HVS-I based results1. By analyzing genomic data KAR22 clusters with Late Neolithic Corded Ware individuals (Figure 2a), while the admixture proportions in Figure 3 reveal a complete absence of an Early Neolithic component (even less than other Corded Ware individuals). Given the large proportion of Yamnaya-related ancestry, a broader group assignment to the Late Neolithic appears likely. Radiocarbon dating results are pending.
Other Late Neolithic samples in Germany: Alberstedt
The site Alberstedt in Merseburg-Querfurt, Saxony-Anhalt, on the loess-bearing Querfurter Platte, is located on a hilltop. This promontory site was used by the Corded Ware and Bell Beaker people as a burial ground. The single grave of individual
ALB3/I0118 (feature 7144.2, 2459-2345 calBCE, MAMS 21492)
was uncovered within a strip of 20m width in preparation for major roadworks. The grave was initially ascribed to the Bell Beaker culture but is rather an unusual burial complex strewn with cattle bones as well as a few sherds of Corded Ware-like pottery in the back filling28. The radiocarbon date falls in line with both the Bell Beaker and Corded Ware occupation phases of this region. Given the ambiguous archaeological classification we have decided to use the location and date to classify this sample (Late Neolithic Alberstedt). The intermediate position of this sample on the PCA plot (Figure 2a) between unambiguously assigned Corded Ware (Esperstedt) and Bell Beaker (Rothenschirmbach) individuals, and >50% Yamnaya ancestry are consistent with an individual who has mixed Corded Ware and Bell Beaker ancestry.
Early Bronze Age
Únětice in Germany: Esperstedt
The reference site 4e at Esperstedt in Merseburg-Querfurt, Saxony-Anhalt/Germany includes a small graveyard of 10 burials that could be assigned to the Early Bronze Age Únětice culture. All individuals are buried on the their right-hand side in flexed position, which is typical for this time period, with the head in the south and facing west. The graves at Esperstedt are slightly tilted towards SE, facing NE. Individuals
ESP2/I0114 (feature 3340.1, 2131-1979 calBCE, MAMS 21493) and
ESP29/I0117 (feature 3332/3333, 2199-2064 calBCE, MAMS 21496) form a small group and appear to be genetically closely related.
ESP4/I0116 (feature 3322/3323, 2118-1961 calBCE, MAMS 21495) was also buried as part of a small group of three graves nearby, while individual
ESP3/I0115 (feature 1559.1, 1931-1780 calBCE, MAMS 21494), a female adult from a more recent phase at reference site 4b, formed part of a double inhumation with a ~10 year-old child at approximately 100 meters distance from the older graveyard33.
Únětice in Germany: Quedlinburg VIII
The site in Quedlinburg, located in the northern Harz region, in the foreland of the Harz mountains, harbors several graves from the Early Bronze Age. Three adjacent grave groups were found next to the stream Sülze and could be assigned to the Únětice culture. From these, we sampled individual
QUEVIII6/I0164 (feature 3580, 2012-1919 calBCE, MAMS 21497) whose otherwise ordinary grave was sealed with a stone cover.
Únětice in Germany: Eulau
The site Eulau, Burgenlandkreis, is located in the valley of the Saale river. The promontory that forms "Eulau" is a loess above-gravel formation and was intensively populated during the Early Bronze Age. Individual
EUL41A/I0803 (feature 882, 2115-1966 calBCE, MAMS 22822) was buried on the eastern edge of the promontory in a small burial ground of less than 10 burials attributed to the the Early Bronze Age. Individual
EUL57B/I0804 (feature 1911.5, 2131-1982 calBCE, MAMS 22821) was buried 200 meters distance away on the western edge of the promontory in another burial ground with approximately 20 features. Feature 1911 is an unusual grave context, including skeletal remains from three juvenile males in the bottom, and a cist in which a child and a neonate had been laid down. The samples taken here are from one of the three male individuals. The graves have been dated to 2200-1550 BCE based on archaeological context.
Bronze Age in Germany: Halberstadt-Sonntagsfeld, please see also N.B. below
I0047/HAL16 (grave 19, feature 613.1, 2022-1937 calBCE, MAMS 21481) features a skeleton buried in right-handed flexed position, with head in the west, facing south. The grave contained one bone artifact, but no pottery. This individual was originally attributed to the LBK based on the presence of an LBK settlement with associated burials nearby15, but direct radiocarbon dating newly generated for this study revealed a younger date overlapping with the Bronze Age Únětice culture.
I0099/HAL36C (grave 40, feature 1114, 1113-1021 calBCE, MAMS 21484) was buried in right-handed flexed position, head SSW, facing SE. Two decorated LBK pots and two undecorated globular pots were found above the grave but it was not clear whether they were part of the burial or the back filling. Thus, the skeleton was also originally thought to be part of the LBK burial series found at the same site, but subsequent radiocarbon dating performed for this study indicated a much younger date, placing this individual within the Late Bronze Age Urnfield culture of the Mittelelbe-Saale region.
Nota bene: To our knowledge, these results are the first published case in which ancient DNA data have been used to identify outlier individuals: individuals who are genetically distinct from others at the same site that have been classified as being from the same archaeological culture (in this case, the Early Neolithic LBK). The fact that this genetic outlier status is consistent with the recent radiocarbon dates and the position of the individuals at the periphery of the site – not directly associated with one of the grave groups that accompany each of the LBK houses (Figure S3.4) – indicates that the genetic analysis is likely to be accurately identifying individuals that are outliers. These results suggest that in the future, genetic analysis may be useful for classifying burials when grave goods and other context are missing or unclear.
The graves dating to the Bronze Age (features 1114 and 0613) are clearly marked as outliers with 20-30m distances to the LBK graves (red rectangles) (source: LDA Sachsen-Anhalt, Germany).
Supplementary Information 4 Sex determination and Y chromosome analysis
Sex determination
We determined the sex of the 69 individuals newly analyzed in this study by examining the number of reads overlapping targets on 390k capture reagent on the X chromosome (n=1,829) and Y chromosome (n=2,258) SNPs targeted by the 390k capture reagent.
We applied the read mapping and filtering procedure described in the Online Methods section. Table S4.1 gives the number of non-duplicated reads mapping to the X and Y chromosome targets for each individual.
The ratio of Y/(Y+X) reads1 (Fig. S4.1) shows a bimodal distribution, with 35 individuals having a ratio that is 0.0011 ± 0.0012 (1 standard deviation) who we interpret as female, and 34 individuals having a ratio of 0.508 ± 0.025 who we interpret as male. The remainder of this note focuses on Y chromosome haplogroups for the 34 individuals determined to be male.
Samples are sorted on the value of this ratio, demonstrating a discontinuity between females (where the ratio is ~0) and males (where it is ~0.5).
Y chromosome haplogroup determination
The 390k capture reagent targeted all SNPs present in the Y-DNA SNP index of the International Society of Genetic Genealogy (ISOGG) version 8.22 as of April 22, 2013 (http://isogg.org/tree/ISOGG_YDNA_SNP_Index.html). At each SNP, we represent the individual using the majority allele (breaking ties randomly) for all non-duplicated reads overlapping the SNP, requiring MAPQ≥30, base quality≥30, and trimming 2 bases at the ends of reads.
We performed Y-haplogroup determination by examining the state of SNPs present in ISOGG version 9.129 (accessed Dec 08, 2014); we used this later version—even though it includes many more SNPs than were present in version 8.22 used during the design of the 390k capture reagent—in order to obtain up-to-date Y-haplogroup nomenclature. We determined Y chromosome haplogroups by identifying the most derived Y chromosome SNP in each individual.
We discuss the specific results for individual samples below.
I0559 (Baalberge_MN)
An assignment to haplogroup R is possible based on P224:17285993C→T. This may represent ancient DNA damage, so the assignment should be viewed with caution. The sample can be assigned to the upstream haplogroup P1 (the newly defined parent node of haplogroups Q and R) based on P230:17470112G→A. Downstream haplogroups that could be excluded were R1a1a (M515:14054623T→A), and R1b1a2a1a (L151:16492547C→T), so it is R*(xR1a1a, R1b1a2a1a).
I0807 (Baalberge_MN)
The assignment to haplogroup F* is based on a single mutation P316:16839641A→T. We could exclude haplogroups G (Page94:2846401C→T, PF2956:14993358A→G, PF3134:15275200C→G), H1a2a (Z14683:17431284A→C), H1b1 (Z14006:7786084G→T), H1b2a (Z14385:22458616G→C), H3a1 (Z13118:17493503T→C, Z13181:23121729G→A), H3a2 (Z12794:22191274A→C), I1a3 (S243:14401486C→T), I2a1b (M423:19096091G→A), J1a2b3a (L818:19136821A→G), J2a1h2 (L25:19136822T→C), J2b2a1a1 (Z631:2819161G→A), L (M11:21730647A→G), NO (P194:16202980C→G) and P1 (P237:8334875A→G, P240:14598808T→C, P282:18028661A→G).
I0806 (Bell_Beaker_LN)
The individual was assigned to haplogroup R1b1a2a1a2 based on mutation P312:22157311C→A. Two Bell Beaker individuals from Kromsdorf, Germany were previously determined2 to belong to haplogroup R1b.
The individual also has upstream mutations for R1 (P236:17782178C→G), R1b1 (L278:18914441C→T), R1b1a2 (F1794:14522828G→A), and R1b1a2a1 (L51:8502236G→A). Its haplotype is ancestral for R1b1a2a1a2a1a1a (S1217:7193830C→G, Z262:16320197C→T), R1b1a2a1a2c1a (DF49:22735599G→A), R1b1a2a1a2c1a1 (DF23:17774409G→A), R1b1a2a1a2c1f1 (L554:15022777A→G), R1b1a2a1a2c1f2 (S868:19033817T→C), R1b1a2a1a2c1i (CTS6581:16992602T→C) and R1b1a2a1a2c1l1a1 (CTS2457.2:14313081C→T).
I0104 (Corded_Ware_LN)
This individual was assigned to haplogroup R1a1a1 based on mutation M417:8533735G→A, and also had six upstream mutations placing it in haplogroup R1a1a (M515:14054623T→A, M198:15030752C→T, L168:16202177A→G, M512:16315153C→T, M514:19375294C→T, and L449:22966756C→T) and four mutations placing it in haplogroup R1a1 (L145:14138745C→A, L62:17891241A→G and L63:18162834T→C, L146:23473201T→A).
We could further exclude lineages R1a1a1a (CTS7083:17275047T→G) and R1a1a1b (S441:7683058G→A, S224:8245045C→T), so I0104 could be more precisely described as R1a1a1*(x R1a1a1a, R1a1a1b).
Three related individuals belonging to haplogroup R1a were previously described from Corded Ware individuals from Eulau, Germany3. The R-M417 haplogroup has an estimated TMRCA of ~5,800 years ago4, which indicates that I0104 lived about 1.5ky after the foundation of this lineage from which the vast majority of modern R1a-related chromosomes from across Eurasia are descended. More than 96% of modern European R-M417 Y-chromosomes belong4 to lineage R1a1a1b1a-Z282 which can be excluded for individual I0104, both indirectly, based on its ancestral state for the upstream mutation defining haplogroup R1a1a1b, and directly, as the individual was ancestral for the Z282 polymorphism itself (15588401T→C). Thus, I0104 was related to the modern European set of R1a Y-chromosomes but did not belong to the more dominant group within this set.
I0172 (Esperstedt_MN)
This individual was assigned to haplogroup I2a1b1a based on mutation L1498:18668472C→T, and could also be assigned to upstream haplogroups I2a1b1 (L161.1:22513718C→T) and I2a1b (CTS1293:7317227G→A, CTS1802:14074218A→T, L178:15574052G→A, CTS8239:17893806A→G, M423:19096091G→A, CTS11030:22905944G→C).
Thus, I0172 belonged to a more derived clade than the ~8,000-year old Loschbour male5 from Luxembourg, and may represent a hunter-gatherer Y-chromosomal lineage that was incorporated in the population of Middle Neolithic farmers from Germany. Haplogroup I2a lineages were also detected in Swedish hunter-gatherers5,6 from 7-5 thousand years ago, an early Hungarian individual (~5,700 years cal BC) with a “hunter-gatherer” autosomal makeup that belonged to an early farmer community7, as well as later ~5,000 year old individuals from Treilles, France8, while haplogroup I lineages were observed in two early Neolithic farmers from Hungary belonging to the early Neolithic Trans-Danubian Linear Pottery (LBKT) and Starcevo cultures9. It thus appears that there was gene flow from male hunter-gatherers into the Early and Middle Neolithic farmers across Europe.
I0099 (Halberstadt_LBA)
This single individual from the Late Bronze Age could be assigned to haplogroup R1a1a1b1a2 based on S204:16474793G→A, and to upstream R1a1a1b1a (S198:15588401T→C), and R1a1a1b1 (PF6217:21976303T→A), and R1a1a1b (S224:8245045C→T, S441:7683058G→A). It is thus more derived than the earlier Corded Ware I0104 individual and belongs firmly within the present-day European variation of R1a Y-chromosomes.
I0061 (Karelia_HG)
In contrast to I0104 and I0099, the hunter-gatherer from Karelia could only be assigned to haplogroup R1a1 (M459:6906074A→G, Page65.2:2657176C→T) and the upstream haplogroup R1a (L145:14138745C→A, L62:17891241A→G, L63:18162834T→C, L146:23473201T→A). It was ancestral for the downstream clade R1a1a (M515:14054623T→A, M198:15030752C→T, M512:16315153C→T, M514:19375294C→T, L449:22966756C→T). Thus, it can be designated as belonging to haplogroup R1a1*(xR1a1a) and it occupied a basal position to the vast majority of modern Eurasian R1a-related Y-chromosomes4, although more basal (R1a-M420*) Y-chromosomes have been detected in Iran and eastern Turkey4. Overall, our detection of haplogroup R1a1 in a northwest Russian hunter-gatherer establishes the early presence of this lineage in eastern Europe, and is consistent with a later migration from eastern Europe into central Europe which contributed such haplogroups to the Corded Ware population.
I0048 (LBK_EN)
This individual could be assigned to haplogroup G2a2a (PF3185:22894488C→T) and to upstream haplogroup G2a (L31:14028148C→A). It was ancestral for haplogroup G2a2a1b (L91:21645555G→C), so it could be designated G2a2a*(xG2a2a1b).
Haplogroup G2a has been found in early Neolithic farmers from Germany10, Hungary9, the Tyrolean Iceman11, ~5,000 year old farmers from France8, and ~7,000 year old ones from Spain12. It it thus a link between Early Neolithic farmers of central Europe and the Mediterranean, as its presence13 in modern Sardinians, a population with known links to the early European farmers5,11,14,15 also suggests.
I0056 (LBK_EN)
This individual also belonged to haplogroup G2a2a (PF3147:7738069G→A, PF3175:18962113C→T, PF3181:21808944C→A), but not to G2a2a1a (M286:22741799G→A) or G2a2a1b1 (FGC5668:22467833A→G), so it could be designated G2a2a*(xG2a2a1a, G2a2a1b1).
I0659 (LBK_EN)
This individual also belonged to haplogroup G2a2a1 (PF3170:18090604G→A), and to upstream haplogroup G2a2a (PF3151:9785736A→G, PF3161:15702713A→C, PF3175:18962113C→T, PF3184:22576860C→T, PF3185:22894488C→T), but not to downstream haplogroup G2a2a1 (PF3177:21327198C→T). Thus, this individual carried the derived state for one of the SNPs defining haplogroup G2a2a1 (PF3170), and the ancestral state for another (PF3177), suggesting that the first of these mutations occurred before the second.
I0795 (LBK_EN)
This individual belonged to haplogroup T1a (PF5604:7890461C→T, M70:21893881A→C). This is the first instance of this haplogroup in an ancient individual that we are aware of and strengthens the case for the early Neolithic origin of this lineage in modern Europeans16, rather than a more recent introduction from the Near East where it is more abundant today.
I0821 (LBK_EN)
This individual belonged to haplogroup G2a2a1 (PF3155:14006343T→C) and also to upstream haplogroup G2a2a (PF3166:16735582T→G), and G2a2 (CTS4367:15615340C→G). We could exclude downstream haplogroup G2a2a1b (L91:21645555G→C), so it could be designated G2a2a1*(xG2a2a1b).
I0012 (Motala_HG)
This is the Motala2 individual whose shotgun data was previously analyzed5. It belonged to haplogroup I2c2 (PF3827:22444389T→A), with the upstream haplogroup I2c (L597:18887888T→A) also supported. Our higher coverage capture data defines its haplogroup more precisely than the I haplogroup previously reported5.
I0013 (Motala_HG)
This is the Motala3 individual whose shotgun data was previous analyzed5. It belonged to haplogroup I2a1b (M423:19096091G→A), consistent with the previous analysis. Haplogroup I2a1b1 (L161.1:22513718C→T) could be excluded, and it could thus be designated I2a1b*(xI2a1b1). The analysis of the shotgun data5 could reject M359.2 (previously listed as I2a1b1, but currently designated as under “Investigation” by ISOGG), and I2a1b2-L621 (previously listed as I2a1b3).
I0015 (Motala_HG)
This is the Motala6 individual whose shotgun data was previous analyzed5. It belonged to haplogroup I2a1 (P37.2:14491684T→C) and the upstream haplogroup I2a (L460:7879415A→C). Its phylogenetic position could not be determined in the previous analysis of the shotgun data. We also find that it was ancestral for I2a1a (L159.1:15810964T→G, M26:21865821G→A and L158:23496560G→A), I2a1b (M423:19096091G→A), I2a1c (L233:14487362G→A), and I2a1e (L1294:2887401T→C), so it could be designated I2a1*(xI2a1a, I2a1b, I2a1c, I2a1e).
I0016 (Motala_HG)
This is the Motala9 individual whose shotgun data was previous analyzed5. It belonged to haplogroup I2a1a1a (L672:22228628T→A) and the upstream haplogroups I2a1 (P37.2:14491684T→C) and I2a (L460:7879415A→C), so it could be better resolved than in the analysis of the shotgun data which could only designated it as I*(xI1). It was also ancestral for I2a1a1a1a1b (Z118:20834727A→G), and could be designated I2a1a1a*(xI2a1a1a1a1b).
I0017 (Motala_HG)
This is the Motala12 individual whose shotgun data was previous analyzed5. It could be assigned to haplogroup I2a1b2a1 (L147.2:6753258T→C) and also the upstream haplogroup I2a1b (L178:15574052G→A, M423:19096091G→A). However, in the shotgun data haplogroup I2a1b2-L621 (previously known as I2a1b3) could be excluded based on mutation L621:1876008G→A, which is inconsistent with this individual carrying the derived state for haplogroup I2a1b2a1. We do not have a call for L621 in the capture data, so we are certain only of this individual’s assignment to haplogroup I2a1b.
To summarize the data from the five Motala males, we could assign 4 of 5 males to haplogroup I2a1 and its subclades and one (Motala2/I0012) to haplogroup I2c2.
I0124 (Samara_HG)
The hunter-gatherer from Samara belonged to haplogroup R1b1 (L278:18914441C→T), with upstream haplogroup R1b (M343:2887824C→A) also supported. However, he was ancestral for both the downstream haplogroup R1b1a1 (M478:23444054T→C) and R1b1a2 (M269:22739367T→C) and could be designated as R1b1*(xR1b1a1, R1b1a2). Thus, this individual was basal to most west Eurasian R1b individuals which belong to the R-M269 lineage as well as to the related R-M73/M478 lineage that has a predominantly non-European distribution17. The occurrence of chromosomes basal to the most prevalent lineages within haplogroups R1a and R1b in eastern European hunter-gatherers, together with the finding of basal haplogroup R* in the ~24,000-year old Mal’ta (MA1) boy18 suggests the possibility that some of the differentiation of lineages within haplogroup R occurred in north Eurasia, although we note that we do not have ancient DNA data from more southern regions of Eurasia. Irrespective of the more ancient origins of this group of lineages, the occurrence of basal forms of R1a and R1b in eastern European hunter-gatherers provide a geographically plausible source for these lineages in later Europeans where both lineages are prevalent4,17,19.
I0410 (Spain_EN)
We determined that this individual belonged to haplogroup R1b1 (M415:9170545C→A), with upstream haplogroup R1b (M343:2887824C→A) also supported. However, the individual was ancestral for R1b1a1 (M478:23444054T→C), R1b1a2 (PF6399:2668456C→T, L265:8149348A→G, L150.1:10008791C→T and M269:22739367T→C), R1b1c2 (V35:6812012T→A), and R1b1c3 (V69:18099054C→T), and could thus be designated R1b1*(xR1b1a1, R1b1a2, R1b1c2, R1b1c3).
The occurrence of a basal form of haplogroup R1b1 in both western Europe and R1b1a in eastern Europe (I0124 hunter-gatherer from Samara) complicates the interpretation of the origin of this lineage. We are not aware of any other western European R1b lineages reported in the literature before the Bell Beaker period (ref. 2 and this study). It is possible that either (i) the Early Neolithic Spanish individual was a descendant of a Neolithic migrant from the Near East that introduced this lineage to western Europe, or (ii) there was a very sparse distribution of haplogroup R1b in European hunter-gatherers and early farmers, so the lack of its detection in the published literature may reflect its occurrence at very low frequency.
The occurrence of a basal form of R1b1 in western Europe logically raises the possibility that present-day western Europeans (who belong predominantly to haplogroup R1b1a2-M269) may trace their origin to early Neolithic farmers of western Europe. However, we think this is not likely given the existence of R1b1a2-M269 not only in western Europe but also in the Near East; such a distribution implies migrations of M269 males from western Europe to the Near East which do not seem archaeologically plausible. We prefer the explanation that R-M269 originated in the eastern end of its distribution, given its first appearance in the Yamnaya males (below) and in the Near East17.
I0412 (Spain_EN)
We determined that this individual belonged to haplogroup I2a1b1 (L161.1:22513718C→T), with upstream haplogroup I2a1b also supported (CTS1293:7317227G→A, L178:15574052G→A, M423:19096091G→A). Haplogroup I-L161.1 has not been studied in representative samples of modern Europeans to our knowledge. A project devoted to this haplogroup in the genetic genealogy community suggests a relatively high (but not exclusive) occurrence in the present-day British Isles (https://www.familytreedna.com/public/I2a-L161/; administered by Robert Gabel (Ulrich); accessed Dec. 09, 2014). This may be a hunter-gatherer lineage that was absorbed by early farmers of western Europe, as its present-day distribution and discovery in an early Neolithic Iberian suggest.
I0411 (Spain_EN_relative_of_I0410)
This individual is not included in the Spain_EN sample as we that determined it was a relative to individual I0410 and we retained I0410 because of the latter’s better quality. We could only assign it to haplogroup F based on mutation P135:21618856C→T. Of the known subclades of F, it was found to be ancestral for haplogroup G (F1551:9448354A→G), I1 (M450:7548915G→A), I2a (S247:15224591G→A), J (CTS26:2675457A→T, YSC0000228:22172960G→T), L1b2 (M274:22737801C→T), T (PF5607:8459278G→A, CTS5268:16174116C→T, CTS7749:17644174C→T), O2b (M176:2655180G→A), Q1a2a (L475:18146921G→A), Q1b1 (FGC1861:21365952G→A), R1a1a (L449:22966756C→T) and R1b1c2 (V35:6812012T→A).
I0405 (Spain_MN)
This individual was assigned to haplogroup I2a1a1 (L672:22228628T→A). We note, however, that haplogroup H2 is also supported (L279:6932824G→T, L285:21869856C→T). Given the occurrence of haplogroup I2a chromosomes in many individuals from prehistoric Europe (this study and ref.5–⇓⇓⇓9), assignment to I2a1a1 seems plausible. Haplogroup I-L672 is nested within haplogroup I-M26, which is rare in Europe today except in Sardinians (40.9%) and other populations from Southwestern Europe20. Haplogroup H2 is detected in an early Neolithic Starcevo individual (I0174, below), so we cannot determine this individual’s haplogroup with certainty.
I0406 (Spain_MN)
This individual was assigned to haplogroup I2a2a1 (CTS9183:18732197A→G) with upstream haplogroup I2a2a also supported (L368:6931594C→T, L34:7716262A→C, P221:8353707C→A, P223:16699334C→G, P222:18888200C→G, M223:21717307G→A and P220:24475669G→T). We could exclude downstream haplogroups I2a2a1a1a (L1195:18865320G→A), I2a2a1b1 (L702:7629205C→T), I2a2a1b2a (L801:21763755A→C), and I2a2a1b2b (L147.3:6753258T→C), so this individual could be designated I2a2a1*(x I2a2a1a1a, I2a2a1b1, I2a2a1b2a, I2a2a1b2b).
Haplogroup I2a2a1 is nested within haplogroup I2a2a-M223, previously designated I1c, which occurs at low frequency throughout Europe20, and represents another European hunter-gatherer lineage in the Middle Neolithic farmers of Spain.
I0174 (Starcevo_EN)
This individual was assigned to haplogroup H2 (L281:8353840T→G). Upstream haplogroup F was also supported (P142:7218079G→A, P145:8424089G→A, P138:14199284T→C, P316:16839641A→T, P14:17398598C→T, P159:18097251C→A). An individual bearing mutation P96 which also defines haplogroup H2 was found in the Netherlands21; while haplogroup H is rare in present-day Europeans, its discovery in I0174 suggests that it was present in Neolithic Europe.
I0116 (Unetice_EBA)
This individual was assigned to haplogroup I2c2 (PF3827:22444389T→A) and upstream haplogroups I2c (L597:18887888T→A), I2 (M438:16638804A→G) were also supported.
I0804 (Unetice_EBA)
This individual was assigned to haplogroup I2 (L68:18700150C→T) with upstream haplogroup F also supported (P158:17493513C→T).
I0114 (Unetice_EBA_relative_of_I0117)
This individual was initially assigned to haplogroup I2a2a (L368:6931594C→T) with upstream haplogroups I2a2 (L181:19077754G→T, P218:17493630T→G, P217:7628484C→T) and I2 (M438:16638804A→G) also supported. However, the sample was ancestral for other mutations defining haplogroup I2a2a (L34:7716262A→C, P223:16699334C→G, M223:21717307G→A), so it is possible that either it represents a branch of the Y-chromosome phylogeny that possessed the L368 but not the L34, P223, and M223 mutations, or that the derived L368C→T represents ancient DNA damage. We thus assign it only to haplogroup I2a2.
I0231 (Yamnaya)
This individuals was assigned to haplogroup R1b1a2a2 (CTS1078:7186135G→C, Z2105:15747432C→A) with upstream haplogroups R1b1a2a (L23:6753511G→A), and R1b1a2 (PF6399:2668456C→T, L265:8149348A→G, PF6434:8411202A→G, L150.1:10008791C→T, PF6482:18381735A→G, M269:22739367T→C) also supported. It was ancestral for R1b1a2a2a (L584:28731917C→T), so it could be designated R1b1a2a2*(xR1b1a2a2a).
Notably, the individual did not belong to haplogroup R1b1a2a1-M412 (8502236G→A), and it has been observed that R-L23*(xM412) chromosomes “often exceed 10% frequency in the Caucasus, Turkey and some SE Europe and Circum-Uralic populations” but “they typically display frequencies ≤5% in Western Europe (except for an instance of 27% in Switzerland’s Upper Rhone Valley) in contrast to the prominent spread of derived M412 varieties in West Europe (Figure 1f).” (ref. 17). A study of modern Armenians22 reports a frequency of ~28% of L23 in modern Armenians while noting that “The derived M412 allele, which is found in nearly all haplogroup R1b1b1*-L23 chromosomes in Europe, is absent in the sampled Armenians, which also exhibit a scarcity of haplotype sharing with Europeans, suggesting a limited role for Armenians in the introduction of R1b into Europe.” Moreover, results from the Armenian DNA Project (https://www.familytreedna.com/public/ArmeniaDNAProject/default.aspx?section=ysnp; administered by Hovann Simonian, Mark Arslan, and Peter Hrechdakian; accessed Dec 09, 2014) indicate the presence of the Z2103 derived state in many modern Armenians.
It is not possible to determine whether the appearance of R-Z2103 in the Yamnaya individual is due to (i) gene flow from the south to the steppe and related to the autosomal signal of “dilution” of Eastern European hunter-gatherers, or (ii) gene flow from the steppe to the south. Modern Armenians have a signal of admixture from the Yamnaya, as when we test f3-statistics of the form f3(Armenian; Yamnaya, X) we find the lowest Z-score for f3(Armenian; Yamnaya, BedouinB) = -0.00296 (Z=-7.1). However, the lowest Z-score of statistics of the form f3(Armenian; X, Y) involves the (X, Y) = (LBK_EN, Sindhi) pair (value -0.00575, Z=-15.3), so the signal of admixture from the Yamnaya is not the strongest one for Armenians. Moreover, as shown in SI 7, the Yamnaya have a negative f3-statistic with (X, Y) = (Karelia_HG, Armenian). A negative statistic for both Armenians and Yamnaya with each other as a reference population may suggest that a third (unsampled) population admixed into both the Yamnaya and to Armenians. The question of directionality can only be furthered elucidated by the study of additional ancient samples from the Caucasus, Near East and the steppe.
I0370 (Yamnaya)
This individual was assigned to haplogroup R1b1a2a2 (CTS1078/Z2103:7186135G→C), with upstream haplogroups R1b1a2 (M269:22739367T→C, L150.1:10008791C→T), R1b1a (L320:4357591C→T) also supported.
I0429 (Yamnaya)
This individual was assigned to haplogroup R1b1a2a2 (Z2105:15747432C→A) and to the upstream haplogroups R1b1a2a (L23:6753511G→A) and R1b1a2 (L150.1:10008791C→T, M269:22739367T→C). It was ancestral for R1b1a2a2a (L584:28731917C→T) and so could be designated R1b1a2a2*(x R1b1a2a2a).
I0438 (Yamnaya)
This individual could also be assigned to haplogroup R1b1a2a2 (Z2105:15747432C→A). It could also be assigned to the upstream haplogroups R1b1a2a (L23:6753511G→A), R1b1a (L320:4357591C→T). It was ancestral for R1b1a2a2a (L584:28731917C→T), and R1b1a2a2c (CTS7822:17684699A→T), so it could be designated R1b1a2a2*(xR1b1a2a2a, R1b1a2a2c).
I0439 (Yamnaya)
This individual could be assigned to haplogroup R1b1a (P297:18656508G→C), with upstream haplogroup R1 (M173:15026424A→C, M306:22750583C→A) also supported. It was ancestral for haplogroup R1b1a2a1 (L51:8502236G→A) and so could be designated R1b1a*(xR1b1a2a1).
I0443 (Yamnaya)
This individual could only be assigned to haplogroup R1b1a2a (L49.1:2842212T→A, L23:6753511G→A). It could also be assigned to the upstream haplogroups R1b1a2 (PF6399:2668456C→T, L150.1:10008791C→T, L1353:19179540G→A, PF6509:22190371A→G, M269:22739367T→C, CTS12478:28590278G→A). The individual was ancestral for haplogroup R1b1a2a1 (L51/M412:8502236G→A) and, unlike I0231, I0370 and I0438 also for R1b1a2a2 (Z2105:15747432C→A). Thus, it could be designated as R1b1a2a*(xR1b1a2a1, R1b1a2a2).
I0444 (Yamnaya)
The individual could be assigned to haplogroup R1b1a2a2 (CTS1078/Z2103:7186135G→C) and also to the upstream haplogroups R1b1a2 (L150.1:10008791C→T) and R1b1 (M415:9170545C→A).
Summarizing the results from the Yamnaya males, all seven belonged to haplogroup R1b1a. Six of these could be further assigned to haplogroup R1b1a2a, and five of these to haplogroup R1b1a2a2. The uniformity of R1b Y-chromosomes in this sample suggests a patrilineal organization of the Yamnaya, or at least of the people who were given expensive Kurgan burials. We cannot exclude the presence of other haplogroups in the general population, or in other individuals located elsewhere in the expansive Yamnaya horizon23. We also emphasize the absence of M412 (the dominant lineage within haplogroup R-M269 in Europe) in this sample, as well as the absence of the R1a haplogroup which was detected in the Corded Ware and Late Bronze Age Halberstadt individual from central Europe. A survey of other European steppe groups may reveal the more immediate patrilineal kin of the major founding lineages of modern European R1a and R1b chromosomes.
Discussion
In Table S4.3 we summarize results from previouslys of 61 ancient European and 25 ancient Siberian/Central Asian Y-chromosomes >1,000BCE. In combination with our own results (Table S4.2), this summary makes it clear that only a single R1b Y-chromosome has been found in 70 ancient Europeans outside Russia (1.4%) before the Late Neolithic period. In contrast, all 9 ancient individuals (100%) from Russia belonged to haplogroups R1a and R1b, and 18/23 (78%) Bronze Age individuals from Central Asia/Siberia belonged to haplogroup R1a. In Europe except Russia, both R1a and R1b have been found in the Late Neolithic and Bronze Age periods for a combined frequency of 6/10 (60%). Present-day Europeans have high frequencies4,17 of haplogroups R1a and R1b.
Thus, it appears that before ~4,500 years ago, the frequency of R1a and R1b in Europe outside Russia was very low, and it rose in the Late Neolithic/Bronze Age period. The young, star-like phylogenies of these two haplogroups24 also suggest relatively recent expansions. The ubiquity of these haplogroups in Russia, Siberia, and Central Asia suggest that their rise in Europe was likely to have been due to a migration from the east, although more work is needed to trace these migrations and also to correlate them with regions of the world that have not yet been studied with ancient DNA (such as southern Europe, the Caucasus, the Near East, Iran, and Central and South Asia). Nonetheless, the Y-chromosome results suggest the same east-to-west migration as our analysis of autosomal DNA.
We do not include the Motala individuals from ref.5 in this tabulation as they are included in Table S4.2. We use haplogroup nomenclature from ISOGG version 9.129 and note the terminal derived mutation assessed for each sample.
Acknowledgments
We thank Peter Bellwood, Joachim Burger, Paul Heggarty, Mark Lipson, Colin Renfrew, Jared Diamond, Svante Pääbo, Ron Pinhasi and Pontus Skoglund for critical comments. We thank Svante Pääbo for support for establishing the ancient DNA facilities in Boston, and Pontus Skoglund for detecting the presence of two related individuals in our dataset. We thank Ludovic Orlando, Thorfinn S. Korneliussen, and Cristina Gamba for help in obtaining data. We thank Agilent Technologies and Götz Frommer for help in developing the capture reagents. We thank Clio Der Sarkissian, Guido Valverde, Luka Papac, and Birgit Nickel for wet lab support. We thank archaeologists Veit Dresely, Robert Ganslmeier, Oleg Balanvosky, José Ignacio Royo Guillén, Anett Osztás, Vera Majerik, Tibor Paluch, Krisztina Somogyi and Vanda Voicsek for sharing samples and discussion about archaeological context. This research was supported by an Australian Research Council grant to W.H. and B.L. (DP130102158), and German Research Foundation grants to K.W.A. (Al 287/7-1 and 7-3, Al 287/10-1 and Al 287/14-1) and to H.M. (Me 3245/1-1 and 1-3). D.R. was supported by U.S. National Science Foundation HOMINID grant BCS-1032255, U.S. National Institutes of Health grant GM100233, and the Howard Hughes Medical Institute.
Author contributions
WH, NP, NR, JK, KWA and DR supervised the study. WH, EB, CE, MF, SF, RGP, FH, VK, AK, MK, PK, HM, OM, VM, NN, SP, RR, MARG, CR, ASN, JW, JKr, DB, DA, AC, KWA and DR assembled archaeological material, WH, IL, NP, NR, SM, AM and DR analyzed genetic data. WH, NR, BL, GB, SN, EH, KS and AM performed wet laboratory ancient DNA work. NR, QF, MM and DR developed the 390k capture reagent. WH, IL and DR wrote the manuscript with help from all co-authors.
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