White Jews are not of lineage by Nyansapo

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: m.b.richards@hud.ac.uk).

NATURE COMMUNICATIONS | 4:2543 | DOI: 10.1038/ncomms3543 | www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3543

he origins of Ashkenazi Jews—the great majority of living Jews—remain highly contested and enigmatic to this day1–11. The Ashkenazim are Jews with a recent ancestry in central and Eastern Europe, in contrast to Sephardim (with an ancestry in Iberia, followed by exile after 1492), Mizrahim (who have always resided in the Near East) and North African Jews (comprising both Sephardim and Mizrahim). There is consensus that all Jewish Diaspora groups, including the Ashkenazim, trace their ancestry, at least in part, to the Levant, B2,000–3,000 years ago5,12–14. There were Diaspora communities throughout Mediterranean Europe and the Near East for several centuries prior to the destruction of the Second Temple in Jerusalem in 70 CE (Common Era), and some scholars suggest that their scale implies proselytism and wide-scale conversion, although this view is very controversial9,15. The Ashkenazim are thought to have emerged from dispersals north into the Rhineland of Mediterranean Jews in the early Middle Ages, although there is little evidence before the twelfth century5,15. After expulsions from Western Europe between the thirteenth and fifteenth centuries, the communities are thought to have expanded eastwards, especially in Poland, Lithuania and then Russia. The implied scale of this expansion has led some to argue, again very controversially, for mass conversions in the Khazar kingdom, in the North Caucasus region to the north and east of the Black Sea, following the Khazar leadership’s adoption of Judaism between the ninth and tenth centuries CE8,9. We are then faced with several competing models for Ashkenazi origins: a Levantine ancestry; a Mediterranean/west European ancestry; a North Caucasian ancestry; or, of course, a blend of these. This seems an ideal problem to tackle with genetic analysis, but after decades of intensive study a definitive answer remains elusive. Although we might imagine that such an apparently straightforward admixture question might be readily addressed using genome-wide autosomal markers, recent studies have proposed contradictory conclusions. Several suggest a primarily Levantine ancestry with south/west European admixture3,4, but another concludes that the ancestry is largely Caucasian16, implying a major source from converts in the Khazar kingdom17. An important reason for disagreement is that the Ashkenazim have undergone severe founder effects during their history, drastically altering the frequencies of genetic markers and distorting the relationship with their ancestral populations. This problem can be resolved by reconstructing the relationships genealogically, rather than relying on allele frequencies, using the non-recombining marker systems: the paternally inherited male-specific part of the Y chromosome (MSY) and 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 region allows us to pinpoint the source for those lineages, by applying the parsimony principle. This has indeed been attempted, with the MSY results interpreted plausibly to suggest an overwhelming majority of Near Eastern ancestry on the Ashkenazi male line of descent11,18–21, albeit with much higher levels (450%) of European (potentially east European) lineages in Ashkenazi Levites22, suggesting a possible Khazar source in that particular case. The maternal line has also been studied, and indeed Ashkenazi mtDNAs are highly distinctive, but they have proved difficult to assign to a source population1,2,11. Some progress has been made by targeting whole-mtDNA genomes or mitogenomes, which provide much higher genealogical (and therefore geographical) and chronological resolution than the control-region sequences used previously—although the far larger control-region database remains an invaluable guide to their geographic distribution. 2

Using this approach, Behar et al.2 identified four major founder clusters, three within haplogroup K—amounting to 32% of sampled Ashkenazi lineages—and one within haplogroup N1b, amounting to another 9%. These lineages are extremely infrequent across the Near East and Europe, making the identification of potential source populations very challenging. Nevertheless, they concluded that all four most likely arose in the Near East and were markers of a migration to Europe of people ancestral to the Ashkenazim only B2,000 years ago1,2. The remaining B60% of mtDNA lineages in the Ashkenazim remained unassigned to any source, with the exception of the minor haplogroup U5 and V lineages (B6% in total), which implied European ancestry1,23. Here we focus on both major and minor founders, with a much larger database from potential source populations. We first analyse 956 (72 newly generated) mitogenomes from haplogroup U8 (including 909 from haplogroup K, U8’s major subclade): 477 of these are from Europe and 106 from the Near East/Caucasus. We show that European and Near Eastern lineages largely fall into discrete, ancient clusters, with minor episodes of gene flow, suggesting that haplogroup K diversified separately in Europe and the Near East during the last glacial period. Of the three Ashkenazi founders, K1a1b1a and K1a9 were most likely assimilated in west (perhaps Mediterranean) Europe and K2a2a1 in west/central Europe. Most surprisingly, by analysing two new N1b2 sequences selected from a database of 278 N1b HVS-I sequences, in the context of 44 published N1b sequences24, we show that the highly distinctive N1b2 subclade, making up another 9% of Ashkenazi lineages, was likely assimilated in Mediterranean Europe, rather than in the Near East as previously proposed2. Moreover, from a survey of another 42,500 complete mtDNA genomes and 428,000 control-region sequences from Europe, the Near East and the Caucasus, in comparison with the available database of 836 Ashkenazi control-region sequences and a handful of published mitogenomes, we also evaluate the minor founders. Overall, we estimate that most (480%) Ashkenazi mtDNAs were assimilated within Europe. Few derive from a Near Eastern source, and despite the recent revival of the ‘Khazar hypothesis’16, virtually none are likely to have ancestry in the North Caucasus. Therefore, whereas on the male side there may have been a significant Near Eastern (and possibly east European/ Caucasian) component in Ashkenazi ancestry, the maternal lineages mainly trace back to prehistoric Western Europe. These results emphasize the importance of recruitment of local women and conversion in the formation of Ashkenazi communities, and represent a significant step in the detailed reconstruction of Ashkenazi genealogical history. Results Four major founder lineages within haplogroup K and N1b. Haplogroup K arose within haplogroup U8B36 ka, in Europe or the Near East, with the minor subclades K1b, K1c and K2 all most likely arising in Europe, between the last glacial period and the Neolithic (Fig. 1; Supplementary Note 1; Supplementary Data 1–3; Supplementary Figs S1–S3; Supplementary Tables S1–S3). K1a expanded from B20 ka onwards, both in the Near East and Europe, with its major subclade, K1a1b1 (Fig. 2), mainly restricted to Europe (with a few instances in North Africa), arriving from the Near East by B11.5 ka, the beginning of the Holocene (Supplementary Note 1). Almost half of mtDNAs in west/central European Ashkenazi Jews belong to haplogroup K, declining to B15% in east European Jews1,11, with almost all falling into three subclades: K1a1b1a, K1a9 and K2a2a12,25 (Figs 1–4; Supplementary Fig. S4). These three founder clusters show a strong expansion signal

NATURE COMMUNICATIONS | 4:2543 | DOI: 10.1038/ncomms3543 | www.nature.com/naturecommunications

ARTICLE Effective population size (Nef)

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3543

Likely near eastern origin Undetermined origin Likely European origin Europeans and Ashkenazim

1,000,000 100,000 10,000 1,000 100 10

10 0

Time (ka)

50 U8b

U8b1

40 K

35 30

K2 K1b

K1d′e′f

K2c

25 K1b1

U8a

K1a K1a1

K1a1a

K1a2

K1a8

K1c K2b K1b1a

K1a3a

K1a28 K1a1b1

10 5

K1a4

K1a3

K1a1b

K1a12

K1a4a1

K1b2

K1a13′16′31

K1a9′10′15′26′30

K2a

K1b1c K1c1

K1a1b1a

K1c2 K1a9

K1a1b1a1

K2a2a1

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

K1a1b1 2,628 9,644 13,443 16,093 5,742 15,074 16,224! 16,278

114 200 1,717G 16,092 16,184A

593 2,483 K1a1b1b 9,932 K1a1b1e

14,388 16,092 16,223 K1a1b1d

4,823 6,528 8,842C K1a1b1f 3,796 13,050 16,09311,765A 827 6,284 10,609

114 146 3,705

10,978 16,234

8,291 15,047 11,204

6 195

114 13,851 16,093

K1a1b1a

12,954 477

K1a1b1a1

16,223 11,005 152 14,249 195 8,787 9,921 8,023 14,118

189

3,316 16,278

8,462 189 16,355 5,460 11,020 152 16,093 1,393 15,355

723 6,366 8,047

9,214 16,288

114!

1,709

8,521

114

14,203C 16,234! 7,927

14,517

14,569

789 5,746 11,620

16,093

513 9,861 12,189

5,876

5,583 12,007

390 14,279 5,585 16,222 16,362 K1a1b1c

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 these lineages increasing 13-fold by 275 years ago (Fig.1). K1a1b1a (slightly re-deﬁned, due to the improved resolution of the new tree) (Fig. 2) accounts for 63% of Ashkenazi K lineages (or B20% of total Ashkenazi lineages) and dates to B4.4 ka with maximum likelihood (ML); however, all of the samples within it, except for one, nest within a further subclade, K1a1b1a1, dating to B2.3 ka (Supplementary Data 2). K1a1b1a1 is also present in non-Ashkenazi samples, mostly from central/east Europe. As they are nested by Ashkenazi lineages, these are likely due to gene ﬂow

from Ashkenazi communities into the wider population. The pattern of gene ﬂow out into the neighbouring communities is seen in the other two major K founders, and also in haplogroups H and J; it is especially clear when the nesting and nested populations are more distinct, for example in the case of haplogroup HV1b, which has a deep ancestry in the Near East (Fig. 5; Supplementary Table S4). The K1a1b1 lineages within which the K1a1b1a sequences nest (including 19 lineages of known ancestry) are solely European, pointing to an ancient European ancestry. The closest nesting

NATURE COMMUNICATIONS | 4:2543 | DOI: 10.1038/ncomms3543 | www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3543

Europe Unknown Ashkenazi Jew USA North Africa

195

K1a9′10′15′26′30 14,440

8,400 8,521 16,214A 16,354

1,958 5,655 6,227 15,204

5,240 12,696 15,226

310 12,063

5,054 16,223

16,048 152

2,483 4,452 16,249

8,155

K1a10

146

4,113 K1a26

4,739 5,563 5,964 11,989 12,711 15,758 16,124

16,291 K1a10a 16,524

14,947 16,192 13,651

316 15,431 8,764 16,201 16,047 16,093!

K1a9

K1a30 16,527 11,453

67,25A

11,287

2,258

3,338 16,093! 6,515 14,831 15,758

152

5,300 9,477 9,698! 9,951

16,093!

338

14,160

230T 16,051

9,629 12,403 14,564

K1a15

16,093!

Figure 3 | Phylogenetic tree of haplogroup K1a9 in the context of the putative clade K1a90 100 150 260 30. Time scale (ka) based on ML estimations for mitogenome sequences.

Europe 8,697

Unknown

K2a2

Ashkenazi Jew USA

7.5 11,348 K2a2a 195 9,263T 16,390 5

64 15,520

8,697! 63

512C 9,254 11,914

2.5

K2a2a1 9,214 11,719! 9,461 153 14,599 4,325

Figure 4 | Phylogenetic tree of haplogroup K2a2. Time scale (ka) based on ML estimations for mitogenome sequences.

lineages are from Italy, Germany and the British Isles, with other subclades of K1a1b1 including lineages from west and Mediterranean Europe and one Hutterite (Hutterites trace their ancestry to sixteenth-century Tyrol)26. Typing/HVS-I results have also indicated several from Northwest Africa, matching European HVS-I types2, likely the result of gene ﬂow from Mediterranean Europe. K1a1b1a is also present at low frequencies in 4

Spanish-exile Sephardic Jews, but absent from non-European Jews, including a database of 289 North African Jews2,25. Notably, it is not seen in Libyan Jews25, who are known to have a distinct Near Eastern ancestry, with no known influx from Spanish-exile 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 K1a1b1 most likely had a west European source. K1a9 (Fig. 3; Supplementary Fig. S4), accounting for another 20% of Ashkenazi K lineages (or 6% of total Ashkenazi lineages) and also dating to B2.3 ka with ML (Supplementary Data 2) again includes both Ashkenazi and non-Ashkenazi lineages solely from east Europeans (again suggesting gene flow out into the wider communities). Like K1a1b1a, it is also found, at much 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 the putative clade K1a90 100 150 260 30, dating to B9.8 ka, which 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 K lineages (or B5% of total Ashkenazi lineages) and dates to B8.4 ka (Supplementary Data 2). Ashkenazi lineages are once more found in a shallow subclade, K2a2a1, dating to B1.5 ka, that otherwise again includes only east Europeans, suggesting gene flow from the Ashkenazim. Conversely, the nesting clades, K2a2 and K2a2a, although poorly sampled, include only French and German lineages. K2a2a is not found in non-European Jews25. Haplogroup K is rarer in the North Caucasus than in Europe or the Near East (o4% (ref. 23)) and the three Ashkenazi founder 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, K1a4 and K1a12. Haplogroup K is more common in Chuvashia, but those sampled belong to K1a4, K1a5 and pre-K2a8. The fourth major Ashkenazi founder mtDNA falls within haplogroup N1b (ref. 2). The distribution of N1b is much more focused on the Near East than that of haplogroup K (ref. 24), and the distinctive Ashkenazi N1b2 subclade has accordingly being assigned to a Levantine source2. N1b2 has until now been

NATURE COMMUNICATIONS | 4:2543 | DOI: 10.1038/ncomms3543 | www.nature.com/naturecommunications

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3543

Europe

12,696

Anatolia, South Caucasus and the Near East

HV1b

Unknown

150 3,290 5,134 6,263 9,585

Ashkenazi Jew Eastern Africa North Africa

152 4,047 10,095 16,526

2,626 4,739 7,598 16,274

195 14,305 5,250 16,158 HV1b3 16,234

HV1b1 3,687

3,547 6,023 16,189

HV1b2

9,438 13,434 11,081T 15,463 16,129 5,327 131 1,694 5,033

151 183 7,664 15,172 15,236 15,519 16,178 16,399

11,314 14,861

5,656

8,020 8,715 10,295 10,750 12,879 14,161 16,311

961

HV1b1a

HV1b1b

2,755 9,117 13,708 3,591 7,912 8,027

16,158 5,460 14,464 16,129

152

709 4,856

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 B2.3 ka, it diverged from other N1b lineages B20 ka (ref. 24) (Supplementary Table S5). N1b2 can be recognized in the HVS-I database by the variant 16176A, but Behar et al.2 tested 14 Near Eastern samples (and some east Europeans) with this motif and identified it as a parallel mutation. Therefore, despite the long branch leading to N1b2, no Near Eastern samples are known to belong to it. In our unpublished database of 6991 HVS-I sequences, however, we identified two Italian samples with the 16176A marker, which we completely sequenced. We confirmed that they belong to N1b2 but diverge before the Ashkenazi lineages B5 ka, nesting the Ashkenazi cluster (Fig. 6; Supplementary Table S5). This striking result suggests that the Italian lineages may be relicts of a dispersal from the Near East into Europe before 5 ka, and that N1b2 was assimilated into the ancestral Ashkenazi population on the north Mediterranean B2 ka. Although we found only two samples suggesting an Italian ancestry for N1b2, the controlregion database available for inspection is very large (28,418 HVS-I sequences from Europe, the Near East and the Caucasus, of which 278, or B1%, were N1b). Moreover, the conclusion is supported by our previous founder analysis of N1b HVS-I sequences, which dated the dispersal into Europe to the late Pleistocene/early Holocene24. Minor Ashkenazi mtDNA lineages. There is now a large number of mitogenomes from Europe, the Caucasus and the Near East (B3,500, with 470 Ashkenazim), and a substantial Ashkenazi mtDNA control-region database of 836 samples1,2,11 (Supplementary Table S6). We therefore endeavoured to crossreference the two in order to pinpoint most of the control-region data within the mitogenome phylogeny. Besides the four haplogroup K and N1b founders, the major haplogroup in Ashkenazi Jews is haplogroup H, at 23% of Ashkenazi lineages, which is also the major haplogroup in

Europeans (40–50% in Europe, B25% in the North Caucasus and B19% in the Near East)28. There are 29 Ashkenazi H mitogenomes available (Supplementary Table S7), 26 (90%) of which nest comfortably within European subclades dating to the early Holocene (Supplementary Note 3, Figs 7 and 8; Supplementary Figs S5–S10; Supplementary Table S8). Most, in fact, nest more speciﬁcally within west/central European subclades, with closely matching sequences in east Europe, as with the pattern for the K founder clades. The Ashkenazi mitogenomes from haplogroup H include 39% belonging to H1 or H3, which are most frequent in west Europe and rare outside Europe. The nesting relationships in some cases point (albeit tentatively) to a central European source, but in many cases comparison with the HVS-I database indicates matches in west Europe. The phylogeographic conclusions based on the nesting relationships are strongly supported for haplogroup H by evidence from the study of prehistoric remains, showing in almost all cases that the lineages concerned were present in Europe since at least the early Bronze Age, B3.5 ka (Supplementary Table S7)29. There is no suggestion of assimilation from the North Caucasus, where most H lineages differ from those of Europe23 (Supplementary Note 2). Haplogroup J comprises 7% of the Ashkenazi control-region 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 Ashkenazi J lineages have a European origin, with B7% (J1b and J2b) less clearly associated. Many have a probable west/central European source, despite (like H) being most frequent in eastern Ashkenazim. The four Ashkenazi J mitogenomes, in J1c5, J1c7a1a and J1c7d, once again show a striking pattern of Mediterranean, west and central European lineages enclosing Ashkenazi/east European ones (Fig. 9). Haplogroups U5, U4 and HV0 (6.3% between them overall) arose within Europe. Some of these lineages, which are again more frequent in the eastern than western Ashkenazi, may have

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3543

Europe Anatolia, South Caucasus and the Near East

1,703 3,921A 4,960 8,472 12,822 16,145

Ashkenazi Jew North Caucasus North Africa

20 N1b 5,480 7,142 9,185G 13,350 16,124 16,256 16,400

9,335 11,362

N1b1 16,129

N1b2 681 789 14,118

14,581 151 11,719! 551d 13,708 379 8,676 13,635 563 7,526T 16,390! 13,114A

4,904 N1b1b

2,263 10,497

15,944d 16,271 16,343

4,967

150 4,55.1T 235 1,703! 16,311 16,126 185 4,136 1,593! 8,084 8,472! 9,957 4,227 3,221 8,443 12,372 5,987 16,311 N1b1a 4,820 16,180 N1b1c 9,921 195 199 N1b1d 5,291 16,093 6,752 16,256 8,261 7,010 5,553 8,410 8,155 9,861 16,037 16,291 7,337 8,290 8,309 8,251! 93.1A 10,373 10,688 8,020 16,075 8,469 13,967 11,050 8,264 769 13,419 16,257 9,438 16,311 9,133 16,223! 8,888 4,461 15,317 12,891 16,569iGATC 3,571.1C 9,230 9,335! 16,362 9,116 16,209 13,768 271 14,053 9,335! 14,690 12,771 15,071 16,093 10,909 9,882 146 15,813G 15,079 146 195 16,297 13,608 12,297 16,093 150 1,406 5,528 15,043 5,237 16,176A 320 8,950 6,045 15,883 6,272 16,223! 961 15,924 8,020 16,390! 152! 9,921 9,65.2C 16,297 12,797 15,790G 3,083 16,311 14,470 8,962 9,093C 185 188 8,763

4,735A 4,917 11,928 12,092 13,129 13,710 16,176A

867 3,308 8,477 13,851 14,560 16,249

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 (5% overall) are more difficult to assign, but at least 60% (in T2a1b, T2b, T2e1 and T2e4) are likely of European and B10% (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 DNA analyses31. Haplogroup W3 may have originated in the Near East but spread to Europe as early as the Late Glacial31. The M1a1b lineage is characteristic of the north Mediterranean and was most likely assimilated there32, but the U6a and L2a1l lineages are more difficult to pin down. The main lineages with a potentially Near Eastern source include HV1, R0a1a and U7a5 (B8.3% in all). HV1b2 mitogenomes, in particular, date to B2 ka and nest within a cluster of Near Eastern HV1b lineages dating to B18 ka (Fig. 5; Supplementary Table S4). Others such as U1a and U1b have an ultimately Near Eastern origin but, like N1b, have been subsequently distributed around the north Mediterranean. In general, it is more difficult to assign lineages to a Near Eastern source with confidence, as the much larger control-region database indicates that (as with N1b2) many lineages with deep Near Eastern ancestry became widely dispersed along the north Mediterranean during the Holocene, and may alternatively have been assimilated there. If we allow for the possibility that K1a9 and N1b2 might have a Near Eastern source, then we can estimate the overall fraction of European maternal ancestry at B65%. Given the strength of the case for even these founders having a European source, however, our best estimate is to assign B81% of Ashkenazi lineages to a European source, B8% to the Near East and B1% further to the east in Asia, with B10% remaining ambiguous (Fig. 10; 6

Supplementary Table S9). Thus at least two-thirds and most likely more than four-ﬁfths of Ashkenazi maternal lineages have a European ancestry. Discussion The extent to which Ashkenazi Jewry trace their ancestry to the Levant or to Europe is a long-standing question5, which remains highly controversial3,4,6,12–14,16,17. Our results, primarily from the detailed analysis of the four major haplogroup K and N1b founders, but corroborated with the remaining Ashkenazi mtDNAs, suggest that most Ashkenazi maternal lineages trace their ancestry to prehistoric Europe. Previous researchers proposed a Levantine origin for the three Ashkenazi K founders from several indirect lines of evidence: shared ancestry with non-Ashkenazi Jews, shared recent ancestry with Mediterranean samples, and their absence from amongst non-Jews2, and this suggestion has been widely accepted4. However, our much more detailed analyses show that two of the major Ashkenazi haplogroup K lineages, K1a1b1a and K2a2a1 have a deep European ancestry, tracing back at least as far as the early and mid-Holocene respectively. They both belong to ancient European clades (K1a1b1 and K2) that include primarily European mtDNAs, to the virtual exclusion of any from the Near East. Despite some uncertainty in its ancestral branching relationships, a European ancestry seems likely for the third founder clade, K1a9. The heavy concentration of Near Eastern haplogroup K lineages within particular, distinct subclades of the tree, and indeed the lack of haplogroup K lineages in Samaritans, who might be expected to have shared an ancestral gene pool with ancient Israelites, both strongly imply that we are unlikely to have

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3543

Brotherton et al. 29

Europe North Caucasus Anatolia/South Caucasus/Near East North Africa America/Canada/Australia Ashkenazi Jew

3,010

H1 73

16,356

477

5,460

H1b

H1c

H1e

16,162

15,817

8,512 8,251 16,189 16,080

H1a

16,129

H1e2

H1e1 H1e1a

183 8,286 16,183C 16,360

9,986

9,923

H1s

H1t

H1u

H1i

H1n

150 14,053

15,323

H1m

H1v

327 11,428

15,047 16,189

H1z

H1ab

H1x

4,131

H1t2 H1t1

H1r

H1o

7,765 10,410A 16,037 16,256

10,314

5,978 14,129

9,356

8,966

H1aa

15,299 16,189

14,224

H1w

H1t1a

H1e4a

14,259

H1e1b

8,572

H1q

H1f1

H1e3

146 16,311 4,859 2,098 16,355

H1j

H1h

7,309

16,114

152 4,733 6,237A

7,013

H1f

H1e4

93 960.1C 1,462 4,883

14,902 453

H1b2

3,316

14,212 4,452 16,189 9,066s 16,093 16,189 H1g

H1y

8,951

16,311Y

H1b2a 7,691

H1b2a1 8,285.2C

15,553

H1ah

6,272 14,869

14,133

3,666

H1aj

H1ak

207 8,618 9,621 16,172 16,192 16,456

6,722 15,088

H1ae H1ag

H1ai

14 1

152 15,394

8,410

183 16,468

H1ar

146 11,809 16,278

4,763A

H1ap

H1an

H1am

152 5,780 16,189

H1ao

EU148452

H1ac

84 1 1

JQ703137

H1af

JQ705236

8,950 12,507

16,357

11,893

EU262984

46 1 1

JQ704894

JQ703268

2 5

2 1 6 2

4,688

15,758

7762s

H1as

H1at

H1av

H1as2

H1au

16,270

10,325

H1ba

H1bc

8,308

H1az

12 7

10,750 13,035

152 11,864

9,966

H1bb

H1bd

16,239

H1be

5,054 7,849

13,768

6 1

1 6 3

3,745

10003 16,126

H1bi

H1bp

H1bx

8,478 9,921

H1bt H1bw

10,454

789 8,740

H1bs H1bv 16,527

H1by

H1br

H1bh

H1bf

6 1

H1bq

H1bk

11,377

16,220C 12,681

14,467

522dCA 11,084

H1bg

H1as1

H1aq

H1ax 8,701

150 6,216

13,386 10,006R

5,054C 7,471 8,429

11,515A 16,148

980

H1ap1

460 16,129

H1aw

6 1

1 11

JQ704370

JQ703655

JQ703788

1 3

5 3 1

1 2 1 2

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 8,047 2,352 5,237 16,218 568 7,813 16,192

13,953 152

16,311 14,944

60 73,16C 9,948 14,094 16,356

7,080 10,370

4,580

9,055 15,884

H6a1a8

H6a1a8a 16,482!

146 16189

6,185 16,145

9,068 3,397

41 16,482! H6a1a2a 1,598 7,001 7,364

5785

827

7,094 7,269

H6a1a3a

16,311

11,923 9,773 11,662 H6a1a2b 146 16,482!

150 3,705 H6a1a2b1

195

41 152

239! 16,218 16,297

297

H6a1a3

H6a1a6 709 16,140

5,460 H6a1a1

10,586 16,278

288

16,298

249d 6,548 14,029

6,260 9,545 13,191

204 6,182 12,369 13,020 14,560 16,295

7,202 H6a1a2

3,548 5,048 10,166 14,211

93 9,055 10,187 15,226C 16,168 16,172

11,978A 8,978 11,914 14,527 12,501 2,581 16,526 16,527

10,237

H6a1a5

195 16,148 11,204 16,482! 3,944

748 2,010 4,947

93 6,468 13,105

150 7,805 H6a1a7 14,182A 16,319 7,325 9,362 11,611 16,311

2,361 9,025 15,287 14,970 16,092 537G 980

H6a1a4 10,936

5,302

H6a1a1a 4,991 0

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. NATURE COMMUNICATIONS | 4:2543 | DOI: 10.1038/ncomms3543 | www.nature.com/naturecommunications

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Europe North Caucasus Anatolia/South Caucasus/Near East North Africa America/Canada/Australia Ashkenazi Jew Asia

20 185 228 14,798

J1c 12,453

16,319

J1c12

16,261 15,113

J1c7 482 3,394

J1c12b 185! 5,024s

13,934 4,025

J1c1

J1c3

6,554 12,127

J1c6

188

J1c2

9,632 12,083G

16,519

152 4,484 5,460 12,361

2,387 10,192

J1c7a1a

10,084 10,398!

J1c11

J1c9

J1c12a

J1c8

2 15 2 2 11

JF812166

20 2 1 14 1 9 4 1

6,887

6,464A 13,681

HM627319

3 3

J1c7a1

195 178R 5,291 12,630 15,103 16,255

HM159445

4,688s 522-23d 7,364 228! 7,372 13,434s 14,769 4,838 J1c5a 9,100 14,798! J1c5b 4,452 185! 228! 5,339 5,978 522-23d 368 15,916 7,340 11,087 16,309 7,888 16,287 146 16,527 10,598 188 185! 3,592 8,255 16,519

21 6 1 33 18 1 2

15,262 16,224

16,092

J1c5

J1c4

J1c2a

J1c10

J1c7a

5,198

146 8,152 9,117 13,917 14,182 15,514 16,527

2 5 2 1 1 1 1 2 1 3 1

2 3 1 1 1

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: 9.9% Asian: 1.1% Near Eastern: 8.3% H: 20.5% W: 1.6% U5: 2.0% HV0: 4.1% U: 0.2%

T: 3.0%

I: 1.3% J: 6.3%

N1b: 9.2%

European: 81%

M1: 0.7% K: 31.8%

Figure 10 | Estimated contributions of European mtDNA lineages to the Ashkenazi mtDNA pool shown by major haplogroup. The possible overall Near Eastern contribution and fraction of unassigned lineages are also indicated.

missed a hitherto undetected Levantine ‘reservoir’ of haplogroup K variation (Supplementary Note 1). Furthermore, our results suggest that N1b2, for which a Near Eastern ancestry was proposed (with much greater conﬁdence than for K) by Behar et al.2, is more likely to have been assimilated into the ancestors of the Ashkenazi in the north Mediterranean. Finally, our cross-comparison of control-region and mitogenome databases shows that the great majority of the remaining B60% of Ashkenazi lineages, belonging to haplogroups H, J, T, HV0, U4/U5, I, W and M1 also have a predominantly European ancestry. Overall, it seems that at least 80% of Ashkenazi maternal ancestry is due to the assimilation of mtDNAs indigenous to 8

Europe, most likely through conversion. The phylogenetic nesting patterns suggest that the most frequent of the Ashkenazi mtDNA lineages were assimilated in Western Europe, B2 ka or slightly earlier. Some in particular, including N1b2, M1a1b, K1a9 and perhaps even the major K1a1b1, point to a north Mediterranean source. It seems likely that the major founders were the result of the earliest and presumably most profound wave of founder effects, from the Mediterranean northwards into central Europe, and that most of the minor founders were assimilated in west/central Europe within the last 1,500 years. The sharing of rarer lineages with Eastern European populations may indicate further assimilation in some cases, but can often be explained by exchange via intermarriage in the reverse direction. The Ashkenazim therefore resemble Jewish communities in Eastern Africa and India, and possibly also others across the Near 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 results in fact fit well with genome-wide studies, which imply a significant European component, with particularly close relationships to Italians3,4,6,7. As might be expected from the 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 ancestry seen in Ashkenazi Levites22. Evidence for haplotype sharing with non-Ashkenazi Jews for each of the three main haplogroup K founders may imply a partial common ancestry in Mediterranean Europe for Ashkenazi and Spanish-exile Sephardic Jews, but may also, at least in part, be due to subsequent gene flow, especially into Bulgaria and Turkey, both of which witnessed substantial immigration from Ashkenazi communities in the fourteenth and fifteenth centuries. Gene flow could have been substantial in some cases—ongoing intermarriage is likely when these communities began living in closer proximity after the Spanish exile6. A partial common ancestry for all European Jews—both Ashkenazi and Sephardic—is again strongly supported by the autosomal results3,4.

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Jewish communities were already spread across the GraecoRoman and Persian world 42,000 years ago. It is thought that a substantial Jewish community was present in Rome from at least the mid-second century BCE, maintaining links to Jerusalem and numbering 30,000–50,000 by the first half of the first century CE15. By the end of the first millennium CE, Ashkenazi communities were historically visible along the Rhine valley in Germany33. After the wave of expulsions in Western Europe during the fifteenth century, they began to disperse once more, into Eastern Europe33. These analyses suggest that the first major wave of assimilation probably took place in Mediterranean Europe, most likely in the Italian peninsula B2 ka, with substantial further assimilation of minor founders in west/central Europe. There is less evidence for assimilation in Eastern Europe, and almost none for a source in the North Caucasus/Chuvashia, as would be predicted by the Khazar hypothesis8,9—rather, the results show strong genetic continuities between west and east European Ashkenazi communities10, albeit with gradual clines of frequency of founders between east and west1,2 (Supplementary Note 2). There is surprisingly little evidence for any significant founder event from the Near East. Fewer than 10% of the Ashkenazi mtDNAs can be assigned to a Near Eastern source with any confidence, and these are found at very low frequencies (Fig. 2). The most frequent, belonging to HV1b2, R0a1a and U7, are found at only B3, 2 and 1% respectively. All are widespread across Ashkenazi communities, and might conceivably be relicts of early Levantine founders, but it seems likely that other more minor Near Eastern lineages are the result of more recent gene flow into the Ashkenazim. The age estimates for the European founders might suggest (very tentatively, given the imprecision with present data) that these ancestral Jewish populations harboring haplogroup K and especially N1b2 may have had an origin in the first millennium BCE, rather than in the wake of the destruction of the Jerusalem Temple in 70 CE. In fact, some scholars have argued from historical evidence that the large-scale expansion of Judaism throughout the Mediterranean in the Hellenistic period was primarily the result of proselytism and mass-conversion, especially amongst women9. We anticipate that a combination of large-scale mitogenome and whole Y-chromosome analysis, complementing full human genome sequencing, will be able to address this question in much finer detail in the near future. Despite the potential of genomic studies, the particular value of full-mitogenome sequencing should be stressed, as some studies dismissed the value of uniparental markers because of the impact of drift in the Ashkenazim6. In fact, the reverse may be the case: autosomal studies may be confounded by drift whereas the fine genealogical resolution of full mitogenomes, given sufficient sampling, can provide a detailed reconstruction of the history of Ashkenazi women. The mtDNA genealogy may even be considered to have particular relevance due to the matrilineal inheritance found in Judaism since at least B200 CE and possibly several centuries earlier, helping to ‘fix’ incoming lineages from converts within the Ashkenazi community after this time. With sufficient resolution, a detailed genealogical history for every maternal lineage in the Ashkenazim is now within reach; in fact, it should soon be possible to reconstruct the outlines of the entire dispersal history of each community. Methods Samples and analysis of mtDNA sequence variation. Although there is a growing database of whole mitogenomes, almost all those from haplogroup U8 are from Europeans or individuals of European (predominantly west European) ancestry. Yet evidence from the Near East is critical in drawing up a meaningful picture of European (and wider west Eurasian) demographic prehistory. We

therefore selected 67 predominantly Near Eastern haplogroup K samples (identified by full control-region sequencing of 111 haplogroup K samples) for mitogenome 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 appropriate informed consent of the subjects and the work was approved by the University of Leeds, Faculty of Biological Sciences Ethics Committee, the Ethics Committee for Clinical Experimentation at the University of Pavia, and the Western Institution Review Board (WIRB), Olympia, WA, USA. We sequenced them using Sanger sequencing30,34 and, to maximize the number of samples, we performed a phylogenetic analysis alongside 884 published U8 sequences (a total of 909 belonging to haplogroup K) (Supplementary Data 1) and four haplogroup N outgroup sequences, using Network 4.6 software and the reduced-median algorithm35. We then constructed a putative most-parsimonious tree of the 956 U8 sequences by hand from the network, following PhyloTree36 for known subclades. We used mtDNA-GeneSyn37 to convert files. As there are a number of extremely variable sites in K1 (positions 195 and 16,093 in particular), we confirmed the 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 previously published tree24. Age estimates and phylogeographic distribution. We estimated coalescence times of clades, using the r statistic and ML38,39, with Bayesian estimations for mitogenomes using BEAST40. For the r statistic and ML, we corrected for purifying selection using the calculator we developed previously (Supplementary Data 4)38. We defined some sub-haplogroups to be a priori monophyletic in the analysis (U8, U8a, U8b, K, K1, K1a, K1b, K1c, K2, K2a and K2b) and assumed a generation time of 25 years41. We also obtained Bayesian skyline plots42–44 to estimate ‘haplogroup-effective’ population sizes associated with U8 over time, and estimated the period of maximum growth39. For a broader overview of the diversity and geographic distribution of lineages, we also compiled 1,917 haplogroup K HVS-I (hypervariable sequence I) sequences (in the range 16,051–16,400), 87 from U8a and 52 from U8b1 (from Europe, the Near East and North Africa, from a total database of 33,127 HVS-I sequences) (Supplementary Tables S1 and S2). We displayed frequency and diversity distributions of haplogroups K, U8a1 and U8b1 sequences, identified from their motifs in the HVS-I database, on interpolation maps using Surfer. For the frequency analyses, we analysed the data at the level of published regional populations; for the diversity analyses we aggregated them into broader areas, as described in Supplementary Table S2. For the analyses of other Ashkenazi lineages we compared 836 published control-region sequences1,2,11 with available Ashkenazi whole mitogenomes and the global mitogenome database available on GenBank, in order to assign the Ashkenazi control-region lineages to subclades. For geographic distributions, we supplemented and checked this information against a database of control-region data (38,244 records from west Eurasia, Central Asia and North Africa).

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Acknowledgements We thank Doron Behar for discussions and suggestions, and Pierre-Marie Danze, Mukaddes Go¨lge, Anne Cambon-Thomsen, CEPH, Steve Jones, Ariella Oppenheim, Gheorghe Stefanescu, Mark Thomas and the donors themselves for generously providing DNA samples. FCT, the Portuguese Foundation for Science and Technology, supported this work through the research project PTDC/CS–ANT/113832/2009 and the personal grants to M.D.C. (SFRH/BD/48372/2008), J.B.P. (SFRH/BD/45657/2008), V.F. (SFRH/ BD/61342/2009) and P.S. (SFRH/BPD/64233/2009). We also received support from the Italian Ministry of Education, University and Research: Progetti Futuro in Ricerca 2008 (RBFR08U07M) and 2012 (RBFR126B8I) (to A.O. and A.A.) and Progetti Ricerca Interesse Nazionale 2009 and 2012 (to A.A.), the Sorenson Molecular Genealogy Foundation (to U.A.P. and S.R.W.), the Leverhulme Trust (research project grant 10 105/ D) (to MBR) and the DeLaszlo Foundation (to M.B.R./P.S.). IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by FCT.

Author contributions M.B.R., L.P. and P.S. devised and supervised the project, M.D.C., J.B.P., M.C. and A.O. carried out the laboratory work, M.D.C., J.B.P., M.P., V.F., P.S., L.P. and M.B.R. carried 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., 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 helped to revise the text.

Additional information Data access: Sequence data have been deposited in GenBank nucleotide core database under accession numbers JX273243 to JX273297, KC878709 to KC878725 and KF297808 to KF297809. Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ How to cite this article: Costa, M.D. et al. A substantial prehistoric European ancestry amongst Ashkenazi maternal lineages. Nat. Commun. 4:2543 doi: 10.1038/ncomms3543 (2013). This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. To view a copy of this licence visit http:// creativecommons.org/licenses/by/3.0/.

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