The Neolithic transition in the Baltic and Ukraine thus had a different tempo to that of central Europe, and it is unclear how this may have shaped the genetic composition of these regions. To investigate this, we sampled three Mesolithic and three Neolithic individuals from the archaeological site of Zvejnieki (Latvia), which is one of the richest Stone Age cemeteries in Northern Europe for number of inhumations, as well as duration of use [] (see the Supplemental Experimental Procedures for site details). We also sampled a Mesolithic and a Neolithic individual from cemeteries found along the Dnieper River in Ukraine (Vasilyevka 3 and Vovnigi 2, respectively). DNA was extracted from the petrous portion of the temporal bone (see the Experimental Procedures ), which yielded between 4.30% and 55.99% endogenous DNA for all samples. Samples were shotgun sequenced using Illumina sequencing technology to between 0.22- and 4.37-fold coverage ( Figure 1 ). The authenticity of the data was assessed in silico by examining the data for signatures of post-mortem DNA damage and evaluating the mitochondrial contamination rate in all samples along with the X chromosome contamination rate in males (see the Supplemental Experimental Procedures ). All samples had degradation patterns typical of ancient DNA ( Figure S1 ) and low contamination estimates of ∼1% or less ( Table 1 ).

MT, mitochondria; c+md, percentage contamination including sites with potentially damaged bases; c−md, percentage contamination excluding sites with potentially damaged bases. For X chromosome contamination estimation, two tests were performed as described by Rasmussen et al. []. Test 1 used all high-quality reads provided per sample, whereas only a single read was sampled per site for test 2, thereby removing the assumption of independent error rates. The associated p values calculated using Fisher’s exact test [] were ≤0.05 for all tests. See also Table S1 for imputed genotypes probabilities for selected loci.

Radiocarbon dates (in cal BP) are shown under the sample name. Mean genome coverage is shown in yellow squares, mitochondrial haplogroups in blue squares, and Y chromosome haplogroups for male samples (where discernible) in magenta squares. The chronology of the Latvian site of Zvejnieki is adapted from []. The Ukrainian chronology is taken from [].

In Europe, the Neolithic transition marked the beginning of a period of innovations that saw communities shift from a mobile lifestyle, dependent on hunting and gathering for survival, to a more sedentary way of life based on food production. This new lifeway, which originated in the Near East ∼11,500 calibrated years before present (cal BP) [], had arrived in southeast Europe by ∼8,500 cal BP [], from where it spread quickly across the continental interior of Europe and introduced animal husbandry, cultivated cereals, pottery, and ground stone tools to the region. There is a long-standing debate among archaeologists whether this spread was due to the dispersal of farmers into new lands (i.e., demic diffusion) or horizontal cultural transmission []. Genetic evidence suggests that these cultural and technological changes were accompanied by profound genomic transformation, consistent with the migration of people of most likely Anatolian origin []. In contrast to central Europe, the adoption of agriculture in northern and eastern parts of this continent, in the areas which encompass modern-day Latvia and Ukraine, was slow and relatively recent []. Although some features of the Neolithic package, such as ceramics, appeared as early as 8,500–7,500 cal BP [], agriculture was not adopted as a primary subsistence economy until the Late Neolithic/Bronze Age [].

Material culture, landscapes of action, and emergent causation: a new model for the origins of the European Neolithic.

A new look at the introduction of the Neolithic way of life in Southeastern Europe. Changing paradigms of the expansion of the Neolithic way of life.

Episodes of Continuity and Change during the Mesolithic and Neolithic in the Baltic

17 Meadows J.

Bērziņš V.

Legzdiņa D.

Lübke H.

Schmölcke U.

Zagorska I.

Zariņa G. Stone-age subsistence strategies at Lake Burtnieks, Latvia. 21 21 25 Alexander D.H.

Novembre J.

Lange K. Fast model-based estimation of ancestry in unrelated individuals. 27 Green R.E.

Krause J.

Briggs A.W.

Maricic T.

Stenzel U.

Kircher M.

Patterson N.

Li H.

Zhai W.

Fritz M.H.-Y.

et al. A draft sequence of the Neandertal genome. Figure 2 PCA and ADMIXTURE Analysis for Ancient Latvian and Ukrainian Samples Show full caption (A) Ancient data presented in this study as well as published ancient data (see Data S1 for sample details) were projected onto the first two principal components defined by selected modern Eurasians from the Human Origins dataset (see the Supplemental Experimental Procedures ). Our Latvian Mesolithic samples cluster tightly together between western and eastern hunter-gatherers in PCA space, whereas the Latvian Neolithic samples are more variable in their position, suggesting impacts from exogenous populations. The Ukrainian Mesolithic and Neolithic samples fall close together between western and eastern hunter-gatherers, suggesting a degree of continuity across the Mesolithic-Neolithic boundary in this region. 25 Alexander D.H.

Novembre J.

Lange K. Fast model-based estimation of ancestry in unrelated individuals. 26 Allentoft M.E.

Sikora M.

Sjögren K.-G.

Rasmussen S.

Rasmussen M.

Stenderup J.

Damgaard P.B.

Schroeder H.

Ahlström T.

Vinner L.

et al. Population genomics of Bronze Age Eurasia. (B) ADMIXTURE ancestry components (K = 17) [] for ancient samples showing that the Latvian Neolithic samples do not have the yellow component that dominates in Anatolian and early European farmers. The Latvian and Ukrainian samples presented in this study are displayed in a gray box and at twice the height of the other ancient samples for ease of visualization. The arrow shows an Estonian Bronze Age sample (RISE00) [] that has a yellow component, suggesting that an early European farmer genetic influence had arrived in the Baltic by the Bronze Age. HG, hunter-gatherer; BA, Bronze Age; W, western; C, Central. See also Figures S1–S4 Table 2 Key D Statistics of the Form D(A,B; X,Y) for Latvian Samples A B X Y D Z Score Loci Latvian Mesolithic Samples a a The Latvian Mesolithic samples share more affinity to WHG than to EHG, but they do not belong entirely to either group. Mbuti Latvia_HG EHG WHG 0.0787 13.064 103,420 Mbuti EHG Latvia_HG WHG −0.0281 −4.797 103,420 Mbuti WHG Latvia_HG EHG −0.1065 −17.418 103,420 Mbuti Latvia_HG Karelia (EHG) Bichon (WHG) 0.0801 11.725 3,188,010 Mbuti Latvia_HG Karelia (EHG) Loschbour (WHG) 0.0928 13.521 3,204,237 Mbuti Karelia (EHG) Latvia_HG Bichon (WHG) −0.0432 −6.494 3,188,010 Mbuti Karelia (EHG) Latvia_HG Loschbour (WHG) −0.0354 −5.367 3,204,237 Mbuti Bichon (WHG) Karelia (EHG) Latvia_HG 0.1228 17.680 3,188,010 Mbuti Loschbour (WHG) Karelia (EHG) Latvia_HG 0.1277 18.643 3,204,237 Latvia_MN1 b b There is no evidence for admixture in our Latvian Neolithic sample, Latvia_MN1 (the three largest positive statistics are shown). Mbuti_AF Iran_LN Latvia_HG Latvia_MN1 0.0427 1.459 7,261 Mbuti_AF Anatolia_ChL Latvia_HG Latvia_MN1 0.0249 0.907 8,289 Mbuti_AF Kennewick Latvia_HG Latvia_MN1 0.0181 0.685 9,026 Latvia_MN2 c c There is an eastern influence in the Latvian Middle Neolithic sample, Latvia_MN2, as compared to the Latvian Mesolithic samples (the three most significantly positive results with ancient and modern populations/individuals from the Human Origins SNP panel dataset are shown). Mbuti Zapotec Latvia_HG Latvia_MN2 0.0295 4.171 74,461 Mbuti Guarani Latvia_HG Latvia_MN2 0.0303 4.027 74,461 Mbuti Aymara Latvia_HG Latvia_MN2 0.0282 3.926 74,461 Mbuti EHG Latvia_HG Latvia_MN2 0.0346 3.734 70,000 Mbuti AfontovaGora3 Latvia_HG Latvia_MN2 0.0658 3.519 19,541 Mbuti MA1 Latvia_HG Latvia_MN2 0.0381 3.441 53,389 Mbuti Karelia (EHG) Latvia_HG Latvia_MN2 0.0386 5.742 2,165,498 Mbuti MA1 Latvia_HG Latvia_MN2 0.0553 7.355 1,799,638 Mbuti Karitiana Latvia_HG Latvia_MN2 0.0366 5.307 2,693,045 Latvia_LN1 d d Largest positive results for the test D(Mbuti, X; Latvia_HG, Latvia_LN1). ADMIXTURE results suggest that there may have been a CHG-related influence in Latvia during the Late Neolithic period; however, although D statistics to test this are positive, they do not reach significance. Mbuti CHG Latvia_HG Latvia_LN1 0.0312 2.081 20,994 Mbuti Iran_N Latvia_HG Latvia_LN1 0.0275 1.483 16,000 Mbuti Iran_ChL Latvia_HG Latvia_LN1 0.0203 1.457 19,027 Mbuti Iran_LN Latvia_HG Latvia_LN1 0.0319 1.320 9,481 Mbuti Iranian_Jew_WA Latvia_HG Latvia_LN1 0.0106 1.091 20,998 Mbuti Kotias (CHG) Latvia_HG Latvia_LN1 0.0182 2.138 715,061 Latvian Neolithic Samples e e We do not find evidence for early European/Anatolian farmer admixture in our Latvian Neolithic samples. Mbuti Anatolia_N Latvia_HG Latvia_MN1 −0.0013 −0.108 16,255 Mbuti Europe_EN Latvia_HG Latvia_MN1 −0.0055 −0.476 16,272 Mbuti Anatolia_N Latvia_HG Latvia_MN2 −0.0246 −3.607 74,355 Mbuti Europe_EN Latvia_HG Latvia_MN2 −0.0335 −5.055 74,460 Mbuti Anatolia_N Latvia_HG Latvia_LN1 −0.0221 −2.136 20,969 Mbuti Europe_EN Latvia_HG Latvia_LN1 −0.0308 −2.952 20,998 Mbuti Stuttgart Latvia_HG Latvia_MN1 0.0168 1.804 581,303 Mbuti NE1 Latvia_HG Latvia_MN1 0.0161 1.704 581,758 Mbuti Stuttgart Latvia_HG Latvia_MN2 −0.0300 −4.527 2,722,280 Mbuti NE1 Latvia_HG Latvia_MN2 −0.0272 −3.871 2,724,548 Mbuti Stuttgart Latvia_HG Latvia_LN1 −0.0246 −2.955 715,090 Mbuti NE1 Latvia_HG Latvia_LN1 −0.0215 −2.373 715618 Latvia_HG, Latvian hunter-gatherers; WHG, western hunter-gatherers; EHG, eastern hunter-gatherers; Iran_LN, Iranian Late Neolithic; Anatolia_ChL, Anatolian Chalcolithic; CHG, Caucasus hunter-gatherers; Iran_N, Iranian Neolithic; Iran_ChL, Iranian Chalcolithic; Anatolian_N, Anatolian Neolithic; Europe_EN, European Early Neolithic. Tests performed using the whole genome panel are italicized; otherwise, tests were performed using the Human Origin transversion SNP panel. Ancient samples are shown in bold. Samples include in each ancient group can be found in Data S1 . See Table S1 for key D statistics for Ukrainian samples. The two earliest samples in our Baltic time series, Latvia_HG1 (8,417–8,199 cal BP), associated with the Kunda culture, and Latvia_HG2 (7,791–7,586 cal BP), associated with the Narva culture, derive from the Late Mesolithic period []. A third sample, Latvia_HG3 (7,252–6,802 cal BP), dates to the Late Mesolithic/Early Neolithic period, with the burial showing no major departures from the preceding Mesolithic traditions []. Principal component analysis (PCA) with ancient samples projected onto modern Eurasian genetic variation (see the Supplemental Experimental Procedures ) shows that these three hunter-gatherer samples group together in a PCA plot (first two components, Figures 2 A and S1 A). In keeping with their geographical origins, they are in an intermediate position between Western European hunter-gatherer samples (WHG; from Luxembourg, Hungary, Italy, France, and Switzerland) and Eastern European hunter-gatherer samples (EHG; from Russia). They are composed of the same (blue) major component as these other hunter-gatherer groups in an ancestry coefficient decomposition analysis performed using ADMIXTURE [] ( Figure 2 B), suggesting a close relationship between these groups. We found that although the Latvian Mesolithic samples share closer affinity to WHG than to EHG, the Latvian Mesolithic samples do not belong entirely to either hunter-gatherer group (tested using D statistics [], which offer a formal test of admixture; Table 2 ). This suggests that they may be a previously unsampled component of a hunter-gatherer meta-population that stretched across Northern Europe during the early Holocene.

21 28 Malmström H.

Gilbert M.T.P.

Thomas M.G.

Brandström M.

Storå J.

Molnar P.

Andersen P.K.

Bendixen C.

Holmlund G.

Götherström A.

Willerslev E. Ancient DNA reveals lack of continuity between neolithic hunter-gatherers and contemporary Scandinavians. 1 Haak W.

Lazaridis I.

Patterson N.

Rohland N.

Mallick S.

Llamas B.

Brandt G.

Nordenfelt S.

Harney E.

Stewardson K.

et al. Massive migration from the steppe was a source for Indo-European languages in Europe. 29 Raghavan M.

Skoglund P.

Graf K.E.

Metspalu M.

Albrechtsen A.

Moltke I.

Rasmussen S.

Stafford Jr., T.W.

Orlando L.

Metspalu E.

et al. Upper Palaeolithic Siberian genome reveals dual ancestry of Native Americans. 21 30 Zagorska I. Amber graves of Zvejnieki burial ground. Next we sampled two Middle Neolithic individuals, Latvia_MN1 (6,201–5,926 cal BP), from an isolated grave located among burials from earlier periods, and Latvia_MN2 (6,179–5,750 cal BP), who was interred in a collective burial with five other individuals. During the Middle Neolithic at Zvejnieki, mortuary practices from the preceding periods were partially maintained, but some new features appeared, including collective burials and votive deposits, which are associated with the Comb Ware culture or its influences in the Baltic []. Despite having been roughly contemporaneous, these Middle Neolithic samples cluster in different regions of our PCA plot ( Figure 2 A) and have distinct profiles in ADMIXTURE analysis ( Figure 2 B). In both analyses, Latvia_MN1 groups with the Mesolithic Latvian samples, suggesting a degree of continuity across the Mesolithic-Neolithic transition in this region and consistent with suggestions that the eastern Baltic was a genetic refugium for hunter-gatherer populations during the Neolithic period []. The persistence of hunter-gatherer ancestry in the Baltic until at least the Middle Neolithic also provides a possible source for the resurgence of hunter-gatherer ancestry that is proposed to have occurred in central Europe from 7,000–5,000 cal BP []. In contrast, Latvia_MN2 is placed toward EHG in PCA space and has several components in ADMIXTURE analysis that are found in Native Americans, Siberians, and hunter-gatherer samples from the Caucasus. In keeping with these results, we found that there has been a northern Eurasian influence in the Baltic region since the Mesolithic period, as suggested by significantly positive statistics for the test D(Mbuti, X; Latvia Mesolithic, Latvia_MN2) when X was an EHG, modern and ancient Siberian (including the Upper Palaeolithic Mal’ta genome []), or Native American ( Table 2 ). This influence is supported archaeologically by the appearance of copper rings and amber jewelry in Middle Neolithic collective burials that bear similarities to artifacts found in Estonia, Finland, and northwestern Russia [].

21 1 Haak W.

Lazaridis I.

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Llamas B.

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et al. Massive migration from the steppe was a source for Indo-European languages in Europe. 31 Jones E.R.

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Connell S.

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et al. Upper Palaeolithic genomes reveal deep roots of modern Eurasians. 3 Anthony D.W. The Horse, the Wheel, and Language: How Bronze-Age Riders from the Eurasian Steppes Shaped the Modern World. 4 Bouckaert R.

Lemey P.

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Atkinson Q.D. Mapping the origins and expansion of the Indo-European language family. 32 Renfrew C. Archaeology and Language. 33 Gray R.D.

Atkinson Q.D. Language-tree divergence times support the Anatolian theory of Indo-European origin. 1 Haak W.

Lazaridis I.

Patterson N.

Rohland N.

Mallick S.

Llamas B.

Brandt G.

Nordenfelt S.

Harney E.

Stewardson K.

et al. Massive migration from the steppe was a source for Indo-European languages in Europe. 3 Anthony D.W. The Horse, the Wheel, and Language: How Bronze-Age Riders from the Eurasian Steppes Shaped the Modern World. 34 Mallory J.P.

Adams D.Q. Encyclopedia of Indo-European Culture. The latest Neolithic sample in our Baltic time series, Latvia_LN1 (5,039–4,626 cal BP), which was found in a crouched burial of the type associated with the Late Neolithic Corded Ware culture [], falls near other Late Neolithic and Bronze Age European and Steppe samples in PCA analysis ( Figure 2 A). In ADMIXTURE analysis, it is composed of the blue component ( Figure 2 B), which is predominant in all of the older Latvian samples, but also a green component, which is maximized in hunter-gatherer samples from the Caucasus. A Caucasus-related influence in this sample is also suggested by positive results (although without formal significance, Z > 2) for tests of the form D(Mbuti, Caucasus hunter-gatherer; Latvian Mesolithic, Latvia_LN1). Ancestry related to hunter-gatherers from the Caucasus has previously been postulated to have arrived in Europe through herders from the Pontic Steppe [], and these migrations could potentially be the source of this ancestry in our sample. Interestingly, this individual lived around the time of later date estimates (∼4,500–7,000 cal BP) proposed for the split of Proto-Balto-Slavic from other Indo-European languages []. There are two major theories to explain the distribution of Indo-European languages that constitute the most widely spoken language family in the world: (1) they have an Anatolian origin and were spread by Neolithic agriculturalists [] and (2) they developed in the Pontic Steppe and proliferated through Late Neolithic/Bronze Age migrations []. The presence of a Steppe-related component in Latvia_LN1 in the absence of an Anatolian farmer-related genetic input supports a Steppe rather than an Anatolian origin for the Balto-Slavic branch of the Indo-European language family.