A Substantial Prehistoric European Ancestry Amongst Ashkenazi Maternal Lineages

ARTICLE

Received 11 Jul 2013 | Accepted 4 Sep 2013 | Published 8 Oct 2013 DOI: 10.1038/ncomms3543 OPEN A substantial prehistoric European ancestry amongst Ashkenazi maternal lineages

Marta D. Costa1,2,*, Joana B. Pereira1,2,*, Maria Pala3, Vero´nica Fernandes1,2, Anna Olivieri4, Alessandro Achilli5, Ugo A. Perego4,6, Sergei Rychkov7, Oksana Naumova7, Jirˇi Hatina8, Scott R. Woodward6,9, Ken Khong Eng1,10, Vincent Macaulay11, Martin Carr3, Pedro Soares2, Luı´sa Pereira2,12 & Martin B. Richards1,3

The origins of Ashkenazi Jews remain highly controversial. Like Judaism, mitochondrial DNA is passed along the maternal line. Its variation in the Ashkenazim is highly distinctive, with four major and numerous minor founders. However, due to their rarity in the general population, these founders have been difﬁcult to trace to a source. Here we show that all four major founders, B40% of Ashkenazi mtDNA variation, have ancestry in prehistoric Europe, rather than the Near East or Caucasus. Furthermore, most of the remaining minor founders share a similar deep European ancestry. Thus the great majority of Ashkenazi maternal lineages were not brought from the Levant, as commonly supposed, nor recruited in the Caucasus, as sometimes suggested, but assimilated within Europe. These results point to a signiﬁcant role for the conversion of women in the formation of Ashkenazi communities, and provide the foundation for a detailed reconstruction of Ashkenazi genealogical history.

1 Institute of Integrative and Comparative Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK. 2 IPATIMUP (Instituto de Patologia e Imunologia Molecular da Universidade do Porto), Porto 4200-465, Portugal. 3 School of Applied Sciences, University of Huddersﬁeld, Queensgate, Huddersﬁeld HD1 3DH, UK. 4 Dipartimento di Biologia e Biotecnologie, Universita` di Pavia, Pavia 27100, Italy. 5 Dipartimento di Chimica, Biologia e Biotecnologie, Universita` di Perugia, Perugia 06123, Italy. 6 Sorenson Molecular Genealogy Foundation, Salt Lake City, Utah 84115, USA. 7 Vavilov Institute of General Genetics, Moscow 119991, Russia. 8 Charles University, Medical Faculty in Pilsen, Institute of Biology, CZ-301 66 Pilsen, Czech Republic. 9 Ancestry, Provo, Utah 84604, USA. 10 Centre for Global Archaeological Research, Universiti Sains Malaysia, 11800 USM Penang, Malaysia. 11 School of Mathematics and Statistics, University of Glasgow, Glasgow G12 8QQ, UK. 12 Faculdade de Medicina da Universidade do Porto, Porto 4200-319, Portugal. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.B.R. (email: [email protected]).

NATURE COMMUNICATIONS | 4:2543 | DOI: 10.1038/ncomms3543 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3543

he origins of Ashkenazi Jews—the great majority of Using this approach, Behar et al.2 identified four major founder living Jews—remain highly contested and enigmatic to clusters, three within haplogroup K—amounting to 32% of Tthis day1–11. The Ashkenazim are Jews with a recent sampled Ashkenazi lineages—and one within haplogroup N1b, ancestry in central and Eastern Europe, in contrast to Sephardim amounting to another 9%. These lineages are extremely (with an ancestry in Iberia, followed by exile after 1492), infrequent across the Near East and Europe, making the Mizrahim (who have always resided in the Near East) and identification of potential source populations very challenging. North African Jews (comprising both Sephardim and Mizrahim). Nevertheless, they concluded that all four most likely arose in the There is consensus that all Jewish Diaspora groups, including Near East and were markers of a migration to Europe of people the Ashkenazim, trace their ancestry, at least in part, to the ancestral to the Ashkenazim only B2,000 years ago1,2. The Levant, B2,000–3,000 years ago5,12–14. There were Diaspora remaining B60% of mtDNA lineages in the Ashkenazim communities throughout Mediterranean Europe and the Near remained unassigned to any source, with the exception of the East for several centuries prior to the destruction of the Second minor haplogroup U5 and V lineages (B6% in total), which Temple in Jerusalem in 70 CE (Common Era), and some scholars implied European ancestry1,23. suggest that their scale implies proselytism and wide-scale Here we focus on both major and minor founders, with a much conversion, although this view is very controversial9,15. larger database from potential source populations. We first The Ashkenazim are thought to have emerged from dispersals analyse 956 (72 newly generated) mitogenomes from haplogroup north into the Rhineland of Mediterranean Jews in the early U8 (including 909 from haplogroup K, U8’s major subclade): 477 Middle Ages, although there is little evidence before the twelfth of these are from Europe and 106 from the Near East/Caucasus. century5,15. After expulsions from Western Europe between the We show that European and Near Eastern lineages largely fall thirteenth and fifteenth centuries, the communities are thought to into discrete, ancient clusters, with minor episodes of gene flow, have expanded eastwards, especially in Poland, Lithuania and suggesting that haplogroup K diversified separately in Europe then Russia. The implied scale of this expansion has led some to and the Near East during the last glacial period. Of the three argue, again very controversially, for mass conversions in the Ashkenazi founders, K1a1b1a and K1a9 were most likely Khazar kingdom, in the North Caucasus region to the north and assimilated in west (perhaps Mediterranean) Europe and east of the Black Sea, following the Khazar leadership’s adoption K2a2a1 in west/central Europe. Most surprisingly, by analysing of Judaism between the ninth and tenth centuries CE8,9. two new N1b2 sequences selected from a database of 278 N1b We are then faced with several competing models for HVS-I sequences, in the context of 44 published N1b sequences24, Ashkenazi origins: a Levantine ancestry; a Mediterranean/west we show that the highly distinctive N1b2 subclade, making up European ancestry; a North Caucasian ancestry; or, of course, a another 9% of Ashkenazi lineages, was likely assimilated in blend of these. This seems an ideal problem to tackle with genetic Mediterranean Europe, rather than in the Near East as previously analysis, but after decades of intensive study a definitive answer proposed2. Moreover, from a survey of another 42,500 complete remains elusive. Although we might imagine that such an mtDNA genomes and 428,000 control-region sequences from apparently straightforward admixture question might be readily Europe, the Near East and the Caucasus, in comparison with the addressed using genome-wide autosomal markers, recent studies available database of 836 Ashkenazi control-region sequences and have proposed contradictory conclusions. Several suggest a a handful of published mitogenomes, we also evaluate the minor primarily Levantine ancestry with south/west European admix- founders. Overall, we estimate that most (480%) Ashkenazi ture3,4, but another concludes that the ancestry is largely mtDNAs were assimilated within Europe. Few derive from a Near Caucasian16, implying a major source from converts in the Eastern source, and despite the recent revival of the ‘Khazar Khazar kingdom17. An important reason for disagreement is that hypothesis’16, virtually none are likely to have ancestry in the the Ashkenazim have undergone severe founder effects during North Caucasus. Therefore, whereas on the male side there may their history, drastically altering the frequencies of genetic have been a significant Near Eastern (and possibly east European/ markers and distorting the relationship with their ancestral Caucasian) component in Ashkenazi ancestry, the maternal populations. lineages mainly trace back to prehistoric Western Europe. These This problem can be resolved by reconstructing the relation- results emphasize the importance of recruitment of local women ships genealogically, rather than relying on allele frequencies, and conversion in the formation of Ashkenazi communities, and using the non-recombining marker systems: the paternally represent a significant step in the detailed reconstruction of inherited male-specific part of the Y chromosome (MSY) and Ashkenazi genealogical history. the maternally inherited mitochondrial DNA (mtDNA). This kind of analysis can be very powerful, because nesting of particular lineages within clusters from a particular geographical Results region allows us to pinpoint the source for those lineages, by Four major founder lineages within haplogroup K and N1b. applying the parsimony principle. This has indeed been Haplogroup K arose within haplogroup U8B36 ka, in Europe or attempted, with the MSY results interpreted plausibly to suggest the Near East, with the minor subclades K1b, K1c and K2 all most an overwhelming majority of Near Eastern ancestry on the likely arising in Europe, between the last glacial period and the Ashkenazi male line of descent11,18–21, albeit with much higher Neolithic (Fig. 1; Supplementary Note 1; Supplementary Data 1–3; levels (450%) of European (potentially east European) lineages Supplementary Figs S1–S3; Supplementary Tables S1–S3). K1a in Ashkenazi Levites22, suggesting a possible Khazar source in expanded from B20 ka onwards, both in the Near East and that particular case. Europe, with its major subclade, K1a1b1 (Fig. 2), mainly restricted The maternal line has also been studied, and indeed Ashkenazi to Europe (with a few instances in North Africa), arriving from mtDNAs are highly distinctive, but they have proved difficult to the Near East by B11.5 ka, the beginning of the Holocene assign to a source population1,2,11. Some progress has been made (Supplementary Note 1). by targeting whole-mtDNA genomes or mitogenomes, which Almost half of mtDNAs in west/central European Ashkenazi provide much higher genealogical (and therefore geographical) Jews belong to haplogroup K, declining to B15% in east and chronological resolution than the control-region sequences European Jews1,11, with almost all falling into three subclades: used previously—although the far larger control-region database K1a1b1a, K1a9 and K2a2a12,25 (Figs 1–4; Supplementary Fig. S4). remains an invaluable guide to their geographic distribution. These three founder clusters show a strong expansion signal

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1,000,000

100,000 Likely near eastern origin 10,000 Undetermined origin Likely European origin 1,000

Europeans and Ashkenazim 100

10 10 55 Effective population size (Nef) population size Effective 0510 15 20 U8 Time (ka) 50

U8b 45 U8b1

35 K

30 K1 K2 K1d′e′f K1b K2c 25

K1b1 U8a 20 K1a K1c K1a1 K1a12 K1a1a K1a2 K1a8 K2b K1a3 K1a4 15 K1a1b K1a3a K1b1a K1b2 K1a28 K1a1b1 10 K1a4a1 K1a13′16′31 K1a9′10′15′26′30 K1b1c K2a K1c1 5 K1a1b1a K1c2 K1a9 K1a1b1a1 K2a2a1 0

Figure 1 | Inferred ancestry of the main subclades within haplogroup U8. The timescale (ka) is based on ML estimations for mitogenomes. Inset:

Bayesian skyline plot of 34 Ashkenazi haplogroup K lineages, showing growth in effective population size (Nef) over time.

Europe Unknown Ashkenazi Jew USA 12 K1a1b1 2,628 9,644 13,443 16,093

5,742 15,074 16,224! 16,278

9 114

200 1,717G 16,092 593 16,184A 2,483 K1a1b1b

9,932 789 K1a1b1e 5,746 11,620 6 114 16,093 14,388 4,823 146 8,291 195 16,092 6,528 3,705 15,047 16,223 8,842C 10,978 16,234 11,204 K1a1b1f K1a1b1d 114 K1a1b1a 3,796 13,851 13,050 16,093 16,09311,765A 827 6,284 12,954 10,609

K1a1b1a1 477 5,583 3 16,223 189 12,007 11,005 152 14,249 195 3,316 9,214 8,787 9,921 8,023 8,462 189 16,355 16,278 723 6,366 16,288 14,118 5,460 390 11,020 152 16,093 8,047 8,521 114 14,279 1,393 15,355 14,203C 16,234! 7,927 5,585 14,517 5,876 16,222 513 16,362 9,861 14,569 1,709 12,189 114! 114! K1a1b1c 0

Figure 2 | Phylogenetic tree of haplogroup K1a1b1. Time scale (ka) based on ML estimations for mitogenome sequences. beginning B2.3 ka, with the overall effective population size for from Ashkenazi communities into the wider population. The these lineages increasing 13-fold by 275 years ago (Fig.1). pattern of gene flow out into the neighbouring communities is K1a1b1a (slightly re-defined, due to the improved resolution of seen in the other two major K founders, and also in haplogroups the new tree) (Fig. 2) accounts for 63% of Ashkenazi K lineages H and J; it is especially clear when the nesting and nested (or B20% of total Ashkenazi lineages) and dates to B4.4 ka with populations are more distinct, for example in the case of maximum likelihood (ML); however, all of the samples within it, haplogroup HV1b, which has a deep ancestry in the Near East except for one, nest within a further subclade, K1a1b1a1, dating (Fig. 5; Supplementary Table S4). to B2.3 ka (Supplementary Data 2). K1a1b1a1 is also present in The K1a1b1 lineages within which the K1a1b1a sequences nest non-Ashkenazi samples, mostly from central/east Europe. As they (including 19 lineages of known ancestry) are solely European, are nested by Ashkenazi lineages, these are likely due to gene flow pointing to an ancient European ancestry. The closest nesting

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Europe Unknown Ashkenazi Jew

195 USA North Africa 8

K1a9′10′15′26′30

1,958 8,400 14,440 5,240 5,054 5,655 16,223 8,521 310 12,696 16,214A 15,226 6,227 12,063 2,483 8,155 16,354 15,204 4,452 6 16,048 16,249

152 K1a10

146 4,739 5,563 5,964 4 11,989 12,711 4,113 15,758 K1a26 16,124 16,291

K1a10a 14,947 16,524 316 15,431 5,300 2 16,192 K1a9 8,764 16,201 9,477 9,629 K1a30 16,047 16,527 11,453 9,698! 12,403 67,25A 3,338 16,093! 16,093! 230T 13,651 9,951 14,564 11,287 2,258 6,515 14,831 152 16,093! 16,051 K1a15 15,758

93 16,093! 14,160 338 0

Figure 3 | Phylogenetic tree of haplogroup K1a9 in the context of the putative clade K1a9010015026030. Time scale (ka) based on ML estimations for mitogenome sequences.

10 Europe Spanish-exile Sephardic Jews, but absent from non-European Jews, including a database of 289 North African Jews2,25. Notably, Unknown 8,697 it is not seen in Libyan Jews25, who are known to have a distinct K2a2 Ashkenazi Jew Near Eastern ancestry, with no known influx from Spanish-exile USA immigrants (although Djerban Jews, with a similar history, have not been tested to date for mtDNA, they closely resemble Libyan Jews in autosomal analyses27). Thus the Ashkenazi subclade of 7.5 K1a1b1 most likely had a west European source. K1a9 (Fig. 3; Supplementary Fig. S4), accounting for another 11,348 20% of Ashkenazi K lineages (or 6% of total Ashkenazi lineages) K2a2a and also dating to B2.3 ka with ML (Supplementary Data 2) again includes both Ashkenazi and non-Ashkenazi lineages solely 195 from east Europeans (again suggesting gene flow out into the 9,263T 16,390 wider communities). Like K1a1b1a, it is also found, at much 5 lower frequencies, in Sephardim. Here the ancestral branching relationships are less clear (Supplementary Note 1 and Supplementary Fig. S4), but K1a9 is most plausibly nested within 0 0 0 0 B 64 the putative clade K1a9 10 15 26 30, dating to 9.8 ka, which 15,520 otherwise includes solely west European (and one Tunisian) lineages, again pointing to a west European source. K2a2 (Fig. 4) accounts for another 16% of Ashkenazi 8,697! 63 512C 9,254 K lineages (or B5% of total Ashkenazi lineages) and dates to 11,914 2.5 B8.4 ka (Supplementary Data 2). Ashkenazi lineages are once more found in a shallow subclade, K2a2a1, dating to B1.5 ka, K2a2a1 that otherwise again includes only east Europeans, suggesting 11,719! 9,214 9,461 153 gene flow from the Ashkenazim. Conversely, the nesting clades, 4,325 14,599 K2a2 and K2a2a, although poorly sampled, include only French and German lineages. K2a2a is not found in non-European Jews25. 0 Haplogroup K is rarer in the North Caucasus than in Europe or Figure 4 | Phylogenetic tree of haplogroup K2a2. Time scale (ka) based the Near East (o4% (ref. 23)) and the three Ashkenazi founder on ML estimations for mitogenome sequences. clades have not been found there (Supplementary Note 2). We tested all eight K lineages out of 208 samples from the North Caucasus, and all belonged to the Near Eastern subclades K1a3, lineages are from Italy, Germany and the British Isles, with other K1a4 and K1a12. Haplogroup K is more common in Chuvashia, subclades of K1a1b1 including lineages from west and Mediter- but those sampled belong to K1a4, K1a5 and pre-K2a8. ranean Europe and one Hutterite (Hutterites trace their ancestry The fourth major Ashkenazi founder mtDNA falls within to sixteenth-century Tyrol)26. Typing/HVS-I results have also haplogroup N1b (ref. 2). The distribution of N1b is much more indicated several from Northwest Africa, matching European focused on the Near East than that of haplogroup K (ref. 24), HVS-I types2, likely the result of gene flow from Mediter- and the distinctive Ashkenazi N1b2 subclade has accordingly ranean Europe. K1a1b1a is also present at low frequencies in being assigned to a Levantine source2. N1b2 has until now been

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Europe 20 12,696 Anatolia, South Caucasus and the Near East HV1b Unknown 150 Ashkenazi Jew 3,290 5,134 Eastern Africa 6,263 9,585 North Africa 152 2,626 15 4,739 7,598 4,047 195 14,305 16,274 10,095 5,250 16,158 16,526 HV1b1 HV1b3 16,234 3,687

11,314 14,861 8,020 5,656 10 8,715 10,295 HV1b1b HV1b1a 10,750 2,755 12,879 9,117 14,161 13,708 16,311 3,591 151 961 7,912 183 8,027 7,664 16,158 5,460 5 15,172 152 3,547 14,464 15,236 6,023 16,129 16,189 15,519

HV1b2 16,178 9,438 709 13,434 15,463 16,399 11,081T 16,129 4,856 5,327 131 1,694 5,033 0

Figure 5 | Phylogenetic tree of haplogroup HV1b. Time scale (ka) based on ML estimations for mitogenome sequences. found exclusively in Ashkenazim, and although it dates to only Europeans (40–50% in Europe, B25% in the North Caucasus and B2.3 ka, it diverged from other N1b lineages B20 ka (ref. 24) B19% in the Near East)28. There are 29 Ashkenazi H (Supplementary Table S5). N1b2 can be recognized in the HVS-I mitogenomes available (Supplementary Table S7), 26 (90%) of database by the variant 16176A, but Behar et al.2 tested 14 Near which nest comfortably within European subclades dating to the Eastern samples (and some east Europeans) with this motif and early Holocene (Supplementary Note 3, Figs 7 and 8; identified it as a parallel mutation. Therefore, despite the long Supplementary Figs S5–S10; Supplementary Table S8). Most, in branch leading to N1b2, no Near Eastern samples are known to fact, nest more specifically within west/central European belong to it. subclades, with closely matching sequences in east Europe, as In our unpublished database of 6991 HVS-I sequences, with the pattern for the K founder clades. The Ashkenazi however, we identified two Italian samples with the 16176A mitogenomes from haplogroup H include 39% belonging to H1 marker, which we completely sequenced. We confirmed that they or H3, which are most frequent in west Europe and rare outside belong to N1b2 but diverge before the Ashkenazi lineages B5 ka, Europe. The nesting relationships in some cases point (albeit nesting the Ashkenazi cluster (Fig. 6; Supplementary Table S5). tentatively) to a central European source, but in many cases This striking result suggests that the Italian lineages may be relicts comparison with the HVS-I database indicates matches in west of a dispersal from the Near East into Europe before 5 ka, and that Europe. The phylogeographic conclusions based on the nesting N1b2 was assimilated into the ancestral Ashkenazi population on relationships are strongly supported for haplogroup H by the north Mediterranean B2 ka. Although we found only two evidence from the study of prehistoric remains, showing in samples suggesting an Italian ancestry for N1b2, the control- almost all cases that the lineages concerned were present in region database available for inspection is very large (28,418 Europe since at least the early Bronze Age, B3.5 ka HVS-I sequences from Europe, the Near East and the Caucasus, (Supplementary Table S7)29. There is no suggestion of of which 278, or B1%, were N1b). Moreover, the conclusion is assimilation from the North Caucasus, where most H lineages supported by our previous founder analysis of N1b HVS-I differ from those of Europe23 (Supplementary Note 2). sequences, which dated the dispersal into Europe to the late Haplogroup J comprises 7% of the Ashkenazi control-region Pleistocene/early Holocene24. database. Around 72% of these can be assigned to J1c, now thought to have arisen within Late Glacial Europe30, and 19% belong to J1b1a1, also restricted to Europe. Thus 490% of the Minor Ashkenazi mtDNA lineages. There is now a large Ashkenazi J lineages have a European origin, with B7% (J1b and number of mitogenomes from Europe, the Caucasus and the J2b) less clearly associated. Many have a probable west/central Near East (B3,500, with 470 Ashkenazim), and a substantial European source, despite (like H) being most frequent in eastern Ashkenazi mtDNA control-region database of 836 samples1,2,11 Ashkenazim. The four Ashkenazi J mitogenomes, in J1c5, J1c7a1a (Supplementary Table S6). We therefore endeavoured to cross- and J1c7d, once again show a striking pattern of Mediterranean, reference the two in order to pinpoint most of the control-region west and central European lineages enclosing Ashkenazi/east data within the mitogenome phylogeny. European ones (Fig. 9). Besides the four haplogroup K and N1b founders, the major Haplogroups U5, U4 and HV0 (6.3% between them overall) haplogroup in Ashkenazi Jews is haplogroup H, at 23% of arose within Europe. Some of these lineages, which are again Ashkenazi lineages, which is also the major haplogroup in more frequent in the eastern than western Ashkenazi, may have

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Europe

Anatolia, South Caucasus and the Near East 1,703 3,921A Ashkenazi Jew 4,960 8,472 North Caucasus 12,822 North Africa 16,145 20 N1b

5,480 7,142 9,185G 13,350 16,124 16,256 9,335 16,400 15 11,362

N1b1 16,129 2,263 15,944d 4,967 10,497 16,271 4,904 867 16,343 150 4,55.1T 185 3,308 N1b1b 235 8,477 1,703! 16,311 188 185 4,136 16,126 13,851 9,957 1,593! 8,084 8,472! 8,763 8,443 4,227 14,560 3,221 12,372 5,987 10 16,311 N1b1c 4,820 199 N1b1a 16,180 9,921 195 4,735A N1b1d 16,249 16,256 5,291 6,752 16,093 4,917 8,261 5,553 7,010 9,861 8,410 8,155 11,928 16,291 8,309 16,037 7,337 10,688 8,020 8,290 8,251! 93.1A 10,373 12,092 11,050 16,075 8,469 16,257 9,438 13,967 8,264 769 13,419 13,129 16,311 9,133 12,891 16,223! 8,888 4,461 15,317 13,710 3,571.1C 9,230 16,569iGATC 9,335! 13,768 16,362 9,116 16,209 16,176A 14,053 271 9,335! 14,690 12,771 15,813G 15,071 10,909 9,882 16,093 146 N1b2 16,297 15,079 146 13,608 195 16,093 12,297 5 5,528 150 15,043 5,237 1,406 681 16,176A 6,045 320 15,883 8,950 6,272 16,223! 789 961 8,020 16,390! 152! 15,924 14,581 9,65.2C 9,921 14,118 12,797 16,297 3,083 15,790G 14,470 16,311 13,708 379 151 11,719! 551d 8,962 563 13,635 8,676 9,093C 16,390! 7,526T 13,114A 0

Figure 6 | Phylogenetic tree of haplogroup N1b. Time scale (ka) based on ML estimations for mitogenome sequences.

been assimilated in central Europe. The haplogroup T lineages Supplementary Table S9). Thus at least two-thirds and most likely (5% overall) are more difficult to assign, but at least 60% (in more than four-fifths of Ashkenazi maternal lineages have a T2a1b, T2b, T2e1 and T2e4) are likely of European and B10% European ancestry. (T1b3 and T2a2) Near Eastern origin30. The haplogroup I lineages have evidently been present in Europe at least since the Neolithic, as indicated by both phylogeographic and ancient Discussion DNA analyses31. Haplogroup W3 may have originated in the The extent to which Ashkenazi Jewry trace their ancestry to the Near East but spread to Europe as early as the Late Glacial31. The Levant or to Europe is a long-standing question5, which remains M1a1b lineage is characteristic of the north Mediterranean and highly controversial3,4,6,12–14,16,17. Our results, primarily from the was most likely assimilated there32, but the U6a and L2a1l detailed analysis of the four major haplogroup K and N1b lineages are more difficult to pin down. founders, but corroborated with the remaining Ashkenazi The main lineages with a potentially Near Eastern source mtDNAs, suggest that most Ashkenazi maternal lineages trace include HV1, R0a1a and U7a5 (B8.3% in all). HV1b2 mito- their ancestry to prehistoric Europe. genomes, in particular, date to B2 ka and nest within a cluster Previous researchers proposed a Levantine origin for the three of Near Eastern HV1b lineages dating to B18 ka (Fig. 5; Ashkenazi K founders from several indirect lines of evidence: Supplementary Table S4). Others such as U1a and U1b have an shared ancestry with non-Ashkenazi Jews, shared recent ancestry ultimately Near Eastern origin but, like N1b, have been with Mediterranean samples, and their absence from amongst subsequently distributed around the north Mediterranean. In non-Jews2, and this suggestion has been widely accepted4. general, it is more difficult to assign lineages to a Near Eastern However, our much more detailed analyses show that two of source with confidence, as the much larger control-region the major Ashkenazi haplogroup K lineages, K1a1b1a and K2a2a1 database indicates that (as with N1b2) many lineages with deep have a deep European ancestry, tracing back at least as far as the Near Eastern ancestry became widely dispersed along the north early and mid-Holocene respectively. They both belong to ancient Mediterranean during the Holocene, and may alternatively have European clades (K1a1b1 and K2) that include primarily been assimilated there. European mtDNAs, to the virtual exclusion of any from the If we allow for the possibility that K1a9 and N1b2 might have a Near East. Despite some uncertainty in its ancestral branching Near Eastern source, then we can estimate the overall fraction of relationships, a European ancestry seems likely for the third European maternal ancestry at B65%. Given the strength of the founder clade, K1a9. The heavy concentration of Near Eastern case for even these founders having a European source, however, haplogroup K lineages within particular, distinct subclades of the our best estimate is to assign B81% of Ashkenazi lineages to a tree, and indeed the lack of haplogroup K lineages in Samaritans, European source, B8% to the Near East and B1% further to who might be expected to have shared an ancestral gene pool with the east in Asia, with B10% remaining ambiguous (Fig. 10; ancient Israelites, both strongly imply that we are unlikely to have

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Europe Brotherton et al. 29 North Caucasus Anatolia/South Caucasus/Near East North Africa America/Canada/Australia 3,010 Ashkenazi Jew H1 73 5,460 14,212 152 4,733 146 16,311 8,572 9,986 9,923 16,356 477 4,452 4,859 10,314 7,765 327 15,047 16,189 6,237A 2,098 16,355 9,066s 10,410A 11,428 16,189 H1s 16,037 H1b H1c H1e 16,093 H1j H1q H1t H1u H1v 16,162 H1n 16,256 8,512 15,817 16,189 H1g H1i H1z H1ab 16,129 3,316 3 150 5,978 14,129 15,323 9,356 8,251 7,013 1 14,053 H1x H1a 16,189 H1e2 H1f 4,131 H1e1 93 H1e4 H1m H1r H1t2 H1t1 16,080 H1h H1o 960.1C 7,309 8,966 15,299 14,902 453 16,114 H1aa H1b2 1,462 14,224 16,189 H1e1a 4,883 H1f1 H1w 183 H1e4a H1t1a H1y 8,286 1 H1e3 16,183C 14,259 8,951 16,360 H1e1b H1b2a 16,311Y

7,691

H1b2a1

8,285.2C EU262984 EU148452 JQ703268 JQ704894 JQ705236 JQ703137

1 76 46 1 1 1 2 84 1 1 44 3 3 14 1 1 7 2 5 10 2 1 6 2 73 6 14 1 4 12 7 1 3 33 3 6 1 1 6 3 3 6 1 3 1 5

16,357 8,950 3,666 152 93 8,410 7762s 5,054C 16,270 10,325 10,750 3,745 10003 16,126 16,220C 12,681 789 11,893 14,133 183 4,688 15,758 16,239 12,507 15,394 16,468 7,471 13,035 8,740 5 8,429 H1ba H1bi H1af H1aj H1ak H1an H1ap H1as H1at H1av H1bc H1bp H1bs H1bv H1ac H1ah H1ar H1be 5,054 14,467 H1bx 7,849 15,553 207 146 11,515A H1ax 152 522dCA 16,527 6,272 6,722 4,763A 152 980 9,966 8,478 8,618 11,809 5,780 16,148 11,864 11,084 H1bq 14,869 15,088 8,308 H1bg 9,921 9,621 H1am 16,278 16,189 H1ae H1as2 8,701 H1bk 10,454 H1bt H1bw H1ag H1ai 16,172 H1au H1bb H1bd 13,768 11,377 16,192 H1ao H1az H1by 16,456 H1ap1 150 H1br 6,216 1 H1bf H1bh 13,386 10,006R H1as1 H1aq 460 16,129 H1aw JQ704370 JQ703788 JQ703788 JQ703655

1 9 4 6 1 4 1 1 1 11 1 6 2 2 3 4 3 1 2 1 3 3 21 4 2 2 6 11 5 3 1 1 2 2 1 1 3 1 1 1 2 1 2 2 3 1 4 1 73 3 11

Figure 7 | Schematic phylogenetic tree of haplogroup H1. Only the Ashkenazi lineages are shown in full detail; the distribution of other lineages is indicated using small squares by the number present in the full tree for each subclade. Prehistoric European (all Neolithic, except for the H1aw lineage, which dates to the Iron Age) lineages are shown using red circles29.

Europe

Unknown

Ashkenazi Jew

USA

Iran Jew 8 North Caucasus

Near East, South Caucasus and Anatolia 11,253 H6a1a 7,202 8,047 11,978A 8,978 11,914 93 3,548 H6a1a2 249d 14,527 9,055 5,048 5,460 12,501 2,581 6,548 6 2,352 16,526 10,187 10,166 16,527 14,211 14,029 H6a1a1 5,237 15,226C 16,168 16,218 10,586 239! 16,172 16,218 568 16,278 13,953 16,297 7,813 6,260 41 152 16,192 16,482! 9,545 11,923 204 H6a1a2a 6,182 13,191 1,598 9,773 10,237 12,369 13,020 4,580 9,055 7,001 11,662 4 14,560 16,295 15,884 5785 7,364 H6a1a4 16,311 288 H6a1a2b 297 10,936 195 16,148 60 16,298 146 93 73,16C H6a1a6 H6a1a3 11,204 6,468 146 16,482! H6a1a5 150 9,948 H6a1a8 13,105 14,944 14,094 16189 827 7,094 7,269 16,482! 3,944 7,805 16,356 709 150 748 H6a1a3a H6a1a7 7,080 6,185 16,140 16,311 3,705 2,010 10,370 16,145 195 4,947 14,182A H6a1a2b1 H6a1a8a 41 16,319 152 2,361 7,325 2 9,068 9,025 9,362 16,482! 3,397 15,287 11,611 14,970 16,311

H6a1a1a 16,092 537G 5,302 980 4,991

Figure 8 | Phylogenetic tree of Ashkenazi founders within haplogroup H6a1a. Time scale (ka) based on ML estimations for mitogenome sequences. A Late Neolithic Corded Ware lineage from central Europe29 is shown in red emerging directly from the root.

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Europe North Caucasus Anatolia/South Caucasus/Near East North Africa 20 America/Canada/Australia 185 Ashkenazi Jew 228 Asia 14,798 J1c 12,453 16,319 J1c12 16,261 15 15,113 J1c7 J1c12b 482 185! 3,394 13,934 5,024s 4,025 146 J1c1 J1c3 6,554 8,152 12,127 J1c10 9,117 188 J1c6 15,262 13,917 5,198 J1c7a 14,182 10 9,632 16,224 J1c2 16,092 15,514 12,083G J1c5 16,527 16,519 J1c7a1 6,887 J1c11 J1c4 152 4,484 6,464A J1c9 J1c12a 2,387 5,460 13,681 10,084 J1c2a 522-23d 7,364 228! 4,688s 10,192 12,361 10,398! 7,372 13,434s J1c7a1a 14,769 4,838 J1c5a J1c8 14,798! 9,100 J1c5b 4,452 195 185! 178R 5 5,339 228! 5,291 368 5,978 522-23d 12,630 15,916 7,340 15,103 16,309 11,087 16,255 16,287 7,888 16,527 10,598 146 HM627319 HM159445 185! 188 JF812166 3,592 16,519 8,255

21 6 1 3318 1 2 3 3 20 2 1 14 1 9 4 1 2 15 2 2 11 2 5 2 1 1 1 1 2 1 3 1 2 3 2 3 1 1 1 0

Figure 9 | Schematic phylogenetic tree of haplogroup J1c. Only the Ashkenazi lineages are shown in full detail; the distribution of other lineages is indicated using small squares for each subclade with the number present in the full tree given in each case. For the full tree see Pala et al.30 Time scale (ka) based on ML estimations for mitogenome sequences.

Unassigned: Europe, most likely through conversion. The phylogenetic 9.9% nesting patterns suggest that the most frequent of the Ashkenazi B Asian: 1.1% mtDNA lineages were assimilated in Western Europe, 2kaor slightly earlier. Some in particular, including N1b2, M1a1b, K1a9 Near Eastern: and perhaps even the major K1a1b1, point to a north 8.3% H: 20.5% Mediterranean source. It seems likely that the major founders were the result of the earliest and presumably most profound W: 1.6% wave of founder effects, from the Mediterranean northwards U5: 2.0% into central Europe, and that most of the minor founders HV0: 4.1% were assimilated in west/central Europe within the last 1,500 U: 0.2% T: 3.0% I: 1.3% years. The sharing of rarer lineages with Eastern European J: 6.3% populations may indicate further assimilation in some cases, but N1b: 9.2% can often be explained by exchange via intermarriage in the reverse direction. The Ashkenazim therefore resemble Jewish communities in European: M1: 0.7% Eastern Africa and India, and possibly also others across the Near 81% K: 31.8% East, Caucasus and Central Asia, which also carry a substantial fraction of maternal lineages from their ‘host’ communities11,25. Despite widely differing interpretations of autosomal data, these Figure 10 | Estimated contributions of European mtDNA lineages results in fact fit well with genome-wide studies, which imply a to the Ashkenazi mtDNA pool shown by major haplogroup. The possible significant European component, with particularly close overall Near Eastern contribution and fraction of unassigned lineages relationships to Italians3,4,6,7. As might be expected from the are also indicated. autosomal picture, Y-chromosome studies generally show the opposite trend to mtDNA (with a predominantly Near Eastern source) with the exception of the large fraction of European missed a hitherto undetected Levantine ‘reservoir’ of haplogroup ancestry seen in Ashkenazi Levites22. K variation (Supplementary Note 1). Evidence for haplotype sharing with non-Ashkenazi Jews for Furthermore, our results suggest that N1b2, for which a Near each of the three main haplogroup K founders may imply a Eastern ancestry was proposed (with much greater confidence partial common ancestry in Mediterranean Europe for Ashkenazi than for K) by Behar et al.2, is more likely to have been and Spanish-exile Sephardic Jews, but may also, at least in part, be assimilated into the ancestors of the Ashkenazi in the north due to subsequent gene flow, especially into Bulgaria and Turkey, Mediterranean. Finally, our cross-comparison of control-region both of which witnessed substantial immigration from Ashkenazi and mitogenome databases shows that the great majority communities in the fourteenth and fifteenth centuries. Gene flow of the remaining B60% of Ashkenazi lineages, belonging to could have been substantial in some cases—ongoing intermar- haplogroups H, J, T, HV0, U4/U5, I, W and M1 also have a riage is likely when these communities began living in closer predominantly European ancestry. proximity after the Spanish exile6. A partial common ancestry for Overall, it seems that at least 80% of Ashkenazi maternal all European Jews—both Ashkenazi and Sephardic—is again ancestry is due to the assimilation of mtDNAs indigenous to strongly supported by the autosomal results3,4.

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Jewish communities were already spread across the Graeco- therefore selected 67 predominantly Near Eastern haplogroup K samples (identi- Roman and Persian world 42,000 years ago. It is thought that a fied by full control-region sequencing of 111 haplogroup K samples) for mito- substantial Jewish community was present in Rome from at least genome sequencing, plus five samples belonging to non-K U8 and two from Italy potentially belonging to N1b2 (Supplementary Data 1). We collected samples with the mid-second century BCE, maintaining links to Jerusalem the appropriate informed consent of the subjects and the work was approved by the and numbering 30,000–50,000 by the first half of the first century University of Leeds, Faculty of Biological Sciences Ethics Committee, the Ethics CE15. By the end of the first millennium CE, Ashkenazi Committee for Clinical Experimentation at the University of Pavia, and the Western Institution Review Board (WIRB), Olympia, WA, USA. We sequenced communities were historically visible along the Rhine valley in 30,34 33 them using Sanger sequencing and, to maximize the number of samples, we Germany . After the wave of expulsions in Western Europe performed a phylogenetic analysis alongside 884 published U8 sequences (a total of during the fifteenth century, they began to disperse once more, 909 belonging to haplogroup K) (Supplementary Data 1) and four haplogroup N into Eastern Europe33. outgroup sequences, using Network 4.6 software and the reduced-median algo- 35 These analyses suggest that the first major wave of assimilation rithm . We then constructed a putative most-parsimonious tree of the 956 U8 sequences by hand from the network, following PhyloTree36 for known subclades. probably took place in Mediterranean Europe, most likely in the We used mtDNA-GeneSyn37 to convert files. As there are a number of extremely Italian peninsula B2 ka, with substantial further assimilation of variable sites in K1 (positions 195 and 16,093 in particular), we confirmed the minor founders in west/central Europe. There is less evidence for overall topology by running networks of coding-region data only. We performed similar analyses for haplogroups H, J and T, and for N1b we augmented our assimilation in Eastern Europe, and almost none for a source in 24 the North Caucasus/Chuvashia, as would be predicted by the previously published tree . 8,9 Khazar hypothesis —rather, the results show strong genetic Age estimates and phylogeographic distribution. We estimated coalescence continuities between west and east European Ashkenazi times of clades, using the r statistic and ML38,39, with Bayesian estimations for communities10, albeit with gradual clines of frequency of mitogenomes using BEAST40. For the r statistic and ML, we corrected for founders between east and west1,2 (Supplementary Note 2). purifying selection using the calculator we developed previously (Supplementary Data 4)38. We defined some sub-haplogroups to be a priori monophyletic in the There is surprisingly little evidence for any significant founder analysis (U8, U8a, U8b, K, K1, K1a, K1b, K1c, K2, K2a and K2b) and assumed a event from the Near East. Fewer than 10% of the Ashkenazi generation time of 25 years41. We also obtained Bayesian skyline plots42–44 to mtDNAs can be assigned to a Near Eastern source with any estimate ‘haplogroup-effective’ population sizes associated with U8 over time, and 39 confidence, and these are found at very low frequencies (Fig. 2). estimated the period of maximum growth . For a broader overview of the diversity and geographic distribution of lineages, The most frequent, belonging to HV1b2, R0a1a and U7, are we also compiled 1,917 haplogroup K HVS-I (hypervariable sequence I) sequences found at only B3, 2 and 1% respectively. All are widespread (in the range 16,051–16,400), 87 from U8a and 52 from U8b1 (from Europe, the across Ashkenazi communities, and might conceivably be relicts Near East and North Africa, from a total database of 33,127 HVS-I sequences) of early Levantine founders, but it seems likely that other more (Supplementary Tables S1 and S2). We displayed frequency and diversity distributions of haplogroups K, U8a1 and U8b1 sequences, identified from their minor Near Eastern lineages are the result of more recent gene motifs in the HVS-I database, on interpolation maps using Surfer. For the flow into the Ashkenazim. frequency analyses, we analysed the data at the level of published regional The age estimates for the European founders might suggest populations; for the diversity analyses we aggregated them into broader areas, as (very tentatively, given the imprecision with present data) that described in Supplementary Table S2. For the analyses of other Ashkenazi lineages we compared 836 published control-region sequences1,2,11 with available these ancestral Jewish populations harboring haplogroup K and Ashkenazi whole mitogenomes and the global mitogenome database available on especially N1b2 may have had an origin in the first millennium GenBank, in order to assign the Ashkenazi control-region lineages to subclades. BCE, rather than in the wake of the destruction of the Jerusalem For geographic distributions, we supplemented and checked this information Temple in 70 CE. In fact, some scholars have argued from against a database of control-region data (38,244 records from west Eurasia, historical evidence that the large-scale expansion of Judaism Central Asia and North Africa). throughout the Mediterranean in the Hellenistic period was References primarily the result of proselytism and mass-conversion, 9 1. Behar, D. M. et al. Differential bottleneck effects in the mtDNA gene pool of especially amongst women . We anticipate that a combination Ashkenazi Jewish populations. Eur. J. Hum. Genet. 12, 355–364 (2004). of large-scale mitogenome and whole Y-chromosome analysis, 2. Behar, D. M. et al. The matrilineal ancestry of Ashkenazi Jewry: portrait of a complementing full human genome sequencing, will be able to recent founder event. Am. J. Hum. Genet. 78, 487–497 (2006). address this question in much finer detail in the near future. 3. Behar, D. M. et al. The genome-wide structure of the Jewish people. Nature 466, 238–242 (2010). 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Signatures of founder effects, admixture, and selection in the Ashkenazi women. The mtDNA genealogy may even be Ashkenazi Jewish population. Proc. Natl Acad. Sci. USA 107, 16222–16227 considered to have particular relevance due to the matrilineal (2010). inheritance found in Judaism since at least B200 CE and possibly 8. Koestler, A. The Thirteenth Tribe: The Khazar Empire and its Heritage (Random House, 1976). several centuries earlier, helping to ‘fix’ incoming lineages from 9. Sand, S. The Invention of the Jewish People (Verso, 2009). converts within the Ashkenazi community after this time. With 10. Guha, S. et al. Implications for health and disease in the genetic signature of the sufficient resolution, a detailed genealogical history for every Ashkenazi Jewish population. Genome Biol. 13, R2 (2012). maternal lineage in the Ashkenazim is now within reach; in fact, 11. Thomas, M. et al. 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Genet. 67, 1251–1276 (2000). Gheorghe Stefanescu, Mark Thomas and the donors themselves for generously providing 24. Fernandes, V. et al. The Arabian cradle: Mitochondrial relicts of the first steps DNA samples. FCT, the Portuguese Foundation for Science and Technology, supported along the southern route out of Africa. Am. J. Hum. Genet. 90, 347–355 (2012). this work through the research project PTDC/CS–ANT/113832/2009 and the personal 25. Behar, D. M. et al. Counting the founders: The matrilineal genetic ancestry of grants to M.D.C. (SFRH/BD/48372/2008), J.B.P. (SFRH/BD/45657/2008), V.F. (SFRH/ the Jewish Diaspora. PLoS One 3, e2062 (2008). BD/61342/2009) and P.S. (SFRH/BPD/64233/2009). We also received support from the 26. Pichler, I. et al. Drawing the history of the Hutterite population on a genetic Italian Ministry of Education, University and Research: Progetti Futuro in Ricerca 2008 landscape, inference from Y-chromosome and mtDNA genotypes. Eur. J. Hum. (RBFR08U07M) and 2012 (RBFR126B8I) (to A.O. and A.A.) and Progetti Ricerca Genet. 18, 463–470 (2010). Interesse Nazionale 2009 and 2012 (to A.A.), the Sorenson Molecular Genealogy 27. Campbell, C. L. et al. North African Jewish and non-Jewish populations form Foundation (to U.A.P. and S.R.W.), the Leverhulme Trust (research project grant 10 105/ distinctive, orthogonal clusters. Proc. Natl Acad. Sci. USA 109, 13865–13870 D) (to MBR) and the DeLaszlo Foundation (to M.B.R./P.S.). IPATIMUP is an Associate (2012). Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and 28. Soares, P. et al. The archaeogenetics of Europe. Curr. Biol. 20, R174–R183 is partially supported by FCT. (2010). 29. Brotherton, P. et al. Neolithic mitochondrial haplogroup H genomes and the Author contributions genetic origins of Europeans. Nat. Commun. 4, 1764 (2013). M.B.R., L.P. and P.S. devised and supervised the project, M.D.C., J.B.P., M.C. and A.O. 30. Pala, M. et al. Mitochondrial DNA signals of Late Glacial re-colonization of carried out the laboratory work, M.D.C., J.B.P., M.P., V.F., P.S., L.P. and M.B.R. carried Europe from Near Eastern refugia. Am. J. Hum. Genet. 90, 915–924 (2012). out the data analyses, M.D.C., J.B.P., P.S., L.P. and M.B.R. wrote the text, M.P., A.O., 31. Olivieri, A. et al. Mitogenomes from two uncommon haplogroups mark Late A.A., U.A.P., S.R., ON., J.H., S.R.W., K.K.E., M.C. and V.M. discussed the results and Glacial/postglacial expansions from the Near East and Neolithic dispersals helped to revise the text. within Europe. PLoS One 8, e70492 (2013). 32. Olivieri, A. et al. The mtDNA legacy of the Levantine Early Upper Palaeolithic Additional information in Africa. Science 314, 1767–1770 (2006). Data access: Sequence data have been deposited in GenBank nucleotide core database 33. Limor, O. in The Illustrated History of the Jewish People. (ed De Lange, N.) under accession numbers JX273243 to JX273297, KC878709 to KC878725 and KF297808 (Aurum Press, 1997). to KF297809. 34. Torroni, A. et al. Do the four clades of the mtDNA haplogroup L2 evolve at different rates? Am. J. Hum. Genet. 69, 1348–1356 (2001). Supplementary Information accompanies this paper at http://www.nature.com/ 35. Bandelt, H.-J., Forster, P., Sykes, B. C. & Richards, M. B. Mitochondrial portraits naturecommunications of human populations using median networks. Genetics 141, 743–753 (1995). 36. van Oven, M. & Kayser, M. Updated comprehensive phylogenetic tree of global Competing financial interests: The authors declare no competing financial interests. human mitochondrial DNA variation. Hum. Mutat. 30, E386–E394 (2009). Reprints and permission information is available online at http://npg.nature.com/ 37. Pereira, L. et al. The diversity present in 5,140 human mitochondrial genomes. reprintsandpermissions/ Am. J. Hum. Genet. 84, 628–640 (2009). 38. Soares, P. et al. Correcting for purifying selection: an improved human How to cite this article: Costa, M.D. et al. A substantial prehistoric European ancestry mitochondrial molecular clock. Am. J. Hum. Genet. 84, 740–759 (2009). amongst Ashkenazi maternal lineages. Nat. Commun. 4:2543 doi: 10.1038/ncomms3543 39. Soares, P. et al. The expansion of mtDNA haplogroup L3 within and out of (2013). Africa. Mol. Biol. Evol. 29, 915–927 (2012). 40. Pereira, L. et al. Population expansion in the North African Late Pleistocene This article is licensed under a Creative Commons Attribution 3.0 signalled by mitochondrial DNA haplogroup U6. BMC Evol. Biol. 10, 390 Unported Licence. To view a copy of this licence visit http:// (2010). creativecommons.org/licenses/by/3.0/.