A substantial prehistoric European ancestry amongst Ashkenazi maternal lineages

Abstract

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, ~40% 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.

ARTICLE

Received 11 Jul 2013 | Accepted 4 Sep 2013 | Published 8 Oct 2013

Marta D. Costa1,2,*, Joana B. Pereira1,2,*, Maria Pala3, Vernica Fernandes1,2, Anna Olivieri4, Alessandro Achilli5, Ugo A. Perego4,6, Sergei Rychkov7, Oksana Naumova7, Jii Hatina8, Scott R. Woodward6,9, Ken Khong Eng1,10, Vincent Macaulay11, Martin Carr3, Pedro Soares2, Lusa 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 difcult 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 signicant 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 Hudderseld, Queensgate, Hudderseld HD1 3DH, UK. 4 Dipartimento di Biologia e Biotecnologie, Universit di Pavia, Pavia 27100, Italy. 5 Dipartimento di Chimica, Biologia e Biotecnologie, Universit 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: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms3543 OPEN

A substantial prehistoric European ancestry amongst Ashkenazi maternal lineages

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3543

The origins of Ashkenazi Jewsthe great majority of living Jewsremain highly contested and enigmatic to this day111. 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, B2,0003,000 years ago5,1214. 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 fteenth 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 leaderships 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 denitive 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-specic 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 descent11,1821, 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 difcult 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 previouslyalthough the far larger control-region database remains an invaluable guide to their geographic distribution.

Using this approach, Behar et al.2 identied four major founder clusters, three within haplogroup Kamounting to 32% of sampled Ashkenazi lineagesand one within haplogroup N1b, amounting to another 9%. These lineages are extremely infrequent across the Near East and Europe, making the identication 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 rst analyse 956 (72 newly generated) mitogenomes from haplogroup U8 (including 909 from haplogroup K, U8s 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 ow, suggesting that haplogroup K diversied 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 hypothesis16, virtually none are likely to have ancestry in the North Caucasus. Therefore, whereas on the male side there may have been a signicant 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 signicant step in the detailed reconstruction of Ashkenazi genealogical history.

ResultsFour 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 13; Supplementary Figs S1S3; Supplementary Tables S1S3). 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 14; Supplementary Fig. S4). These three founder clusters show a strong expansion signal

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3543 ARTICLE

1,000,000

Likely near eastern origin

Undetermined origin

Likely European origin

Europeans and Ashkenazim

Effective population size (Nef)

100,000

10,000

1,000

100

10

0 5 10

10

55

15 20

Time (ka)

U8

50

45

40

35

30

25

20

15

10

5

0

U8b

U8b1

K

K2

K1def K1b

K1b1

K1b1a K1b2

K1b1c

K2c

U8a

K1a1b

K1a1 K1a1a K1a2

K1a910152630

K1a

K1a3 K1a4

K1a8 K1a12

K1

K1c

K1a1b1

K1a1b1a

K1a1b1a1 K1a9

K1a28 K1a3a

K1a4a1

K1a131631

K2a

K2b

K1c1

K1c2

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

12

9

6

3

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 these lineages increasing 13-fold by 275 years ago (Fig.1).

K1a1b1a (slightly re-dened, 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 ow

from Ashkenazi communities into the wider population. The pattern of gene ow 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

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Europe

Unknown

Ashkenazi Jew

USANorth Africa

195

8

6

4

2

0

K1a910152630

14,440

16,527 11,453

11,287 2,258

310

12,063

5,240

12,696

15,226

1,958

5,655

6,227

15,204

5,054

16,223 2,483

4,452

16,249

8,155

16,048

K1a10

4,113

8,400

8,521

16,214A

16,354

152

146 4,739

5,563

5,964

11,989

12,711

15,758

16,124

K1a26

16,291

K1a10a

14,947 K1a30

16,524 K1a9

15,431

316 16,201

5,300

9,477

9,698!

9,951 16,093!

3,338

6,515

16,093!

14,831

15,758

230T

16,051

9,629

12,403

14,564

13,651

16,192

67,25A

152

K1a15

14,160

338

93 16,093!

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.

Europe

8,697

K2a2

Unknown

Ashkenazi Jew

USA

10

7.5

5

2.5

0

11,348

K2a2a

195 9,263T 16,390

64 15,520

8,697! 63 512C

9,254 11,914

K2a2a1

9,214

153 14,599

11,719! 9,461

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 ow from Mediterranean Europe. K1a1b1a is also present at low frequencies in

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 inux 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 ow 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 K1a9010015026030, 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 ow 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

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3543 ARTICLE

Europe

20

15

10

5

0

12,696

HV1b

Anatolia, South Caucasus and the Near East

Unknown

Ashkenazi Jew

Eastern Africa

North Africa

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

152

4,047 195

5,250

14,305 16,158

16,234

3,687

2,626

4,739 7,598 16,274

10,095 16,526

HV1b3

HV1b1

11,314

2,755 9,117 13,708

3,591 7,912 8,027

152

5,656

14,861

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

151 183 7,664 15,172 15,236 15,519

16,178

9,438 16,129 1,694

131 5,033

HV1b1b HV1b1a

3,547 6,023 16,189

13,434 5,327

961

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

709 4,856

HV1b2

11,081T 15,463

16,399

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 identied 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 identied two Italian samples with the 16176A marker, which we completely sequenced. We conrmed 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 control-region 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 cross-reference 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 (4050% 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 S5S10; Supplementary Table S8). Most, in fact, nest more specically 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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Europe

Anatolia, South Caucasus and the Near East

Ashkenazi Jew

North Caucasus

North Africa

1,703 3,921A

4,960 8,472 12,822 16,145

N1b

20

15

10

5,480 7,142 9,185G

13,350 16,124 16,256 16,400

9,335 11,362

N1b1

16,129

185 188 8,763

8,261

8,410

16,291

4,967

150 235 16,311

8,472! 12,372 16,180

2,263 10,497

4,55.1T 1,703!

4,136 8,084 4,22716,311 N1b1a

15,944d 16,271 16,343

4,904

N1b1b

9,957

16,256

N1b1d

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

185

1,593! 8,443

3,221 4,820 5,291 5,553 8,309 11,050

199 6,752 8,155 8,251!

8,264 8,888 16,362

5,987 9,921 16,093

93.1A 769 4,461 9,116 12,771

10,373 13,419 15,317 16,209

16,126

195

N1b1c

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

N1b2

9,861 10,688 16,257

3,571.1C 14,053 15,813G

16,297

5,528 6,045 8,020

8,290 13,967 16,223!

271 10,909

9,230 9,335!

9,882 13,608 15,043 15,883 16,390!

16,037 16,075 16,311 16,569iGATC

8,020 9,438 12,891 13,768 15,071 15,079 16,093

7,010 7,337 8,469 9,133 9,335! 14,690

146 150 320 961 9,65.2C 3,083 8,962 9,093C

5

0

681

789

16,093

5,237

6,272

9,921 15,790G

146 195 1,406 8,950 15,924 16,297 16,311

12,297 16,176A

16,223!

14,118

152! 12,797 14,470

13,708

14,581

551d

151 11,719! 8,676

379 13,635

563 16,390!

7,526T 13,114A

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 difcult 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 difcult 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 difcult to assign lineages to a Near Eastern source with condence, 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;

Supplementary Table S9). Thus at least two-thirds and most likely more than four-fths of Ashkenazi maternal lineages have a European ancestry.

DiscussionThe extent to which Ashkenazi Jewry trace their ancestry to the Levant or to Europe is a long-standing question5, which remains highly controversial3,4,6,1214,16,17. 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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Europe

North Caucasus

North Africa America/Canada/Australia Ashkenazi Jew

Anatolia/South Caucasus/Near East

Brotherton et al. 29

3,010

H1

73

16,162

477 5,460

8,512

14,902

8,572 9,986

5,978 14,129

14,224

16,356

14,212

16,189

152

6,237A

4,733

146

2,098

4,859

9,923

10,314

4,452

9,066s

16,093

16,189

7,765

10,410A

16,037

16,256

327

11,428

15,047

16,189

H1b

H1c

H1e

H1j

16,311

16,355

150

14,053

H1q

H1s

H1t

H1u

H1v

H1g

H1i

H1n

H1z

15,817 16,129

93

960.1C

1,462

4,883

3,316

16,114

H1

ab

3

15,323

9,356

7,013

1

H1a

8,251

16,189

16,080

183

8,286

16,183C

16,360

7,691

8,285.2C

H1e2

H1f

4,131

H1m

H1r

H1t2

H1t1

H1e1

H1e1a

H1h

H1o

7,309

8,966

15,299

16,189

H1

aa

453

H1b2

H1f1

H1w

H1t1a

1

H1e3

H1e4

H1e4a

H1y

14,259

8,951

H1e1b

16,311Y

JQ703268

JQ704894

EU262984

JQ705236

JQ703137

EU148452

1

76

46 1 1

1

H1b2a

H1b2a1

2

84 1 1

44 3

3

14

1

1

7

2

5

2 1

10 6 2 73

6 3 33 6 1

14 1 4 12 7 1 3

H1x

1 6 3 3

6 1 3 1 5

93

146

11,809

16,278

3,666

11,893

5,054C

7,471

8,429

16,357

8,950

12,507

152

15,394

8,410

152

5,780

16,189

183

16,468 4,688

980

150

6,216

15,758

7762s

16,270

10,325

10,750

13,035

3,745

10003 16,126

14,467

16,220C

12,681

789

8,740

5

ak

H1

at

H1

16,239

5,054

7,849

13,768

H1ac

H1

af

H1aj H1

H1

H1

ap

ar H1as H1

av

H1

ba

H1

be

H1

H1

H1

H1

ah

an

H1

H1

bi

H1

bp

H1

H1

6,722

15,088

14,133

207

8,618

9,621

16,172

16,192

16,456

4,763A

11,515A

16,148

H1

ax

6,272

14,869

152

11,864

bc

H1

15,553

9,966

522dCA

11,084

bs

H1

16,527

H1

bq

bv

H1

bg

H1

bx

H1

8,478

H1

9,921

ae

am

8,308

H1as2

8,701

au

bd

H1

bk

10,454

bt

bw

H1

ag

H1

ai

H1

bb

11,377

H1

ao

H1

ap1

H1

az

H1

br

by

1

H1

bf

H1

bh

10,006R

13,386

H1

aq

H1

as1

460

16,129

H1

aw

JQ703655

JQ704370

1 9 1

4 6 1 4

JQ703788

1 1 11 1 6

2

2 3

4 3

1

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

1

2 1 1 3 1 1 2 1 2 2 3 1 4 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.

EuropeUnknownAshkenazi JewUSAIran JewNorth CaucasusNear East, South Caucasus and Anatolia

8

6

11,253

H6a1a

11,978A

8,047

2,352

5,237

16,218

568

11,914 12,501

16,527

8,978

7,202

H6a1a2

14,527

16,526

2,581

93

9,055

10,187

15,226C

3,548

5,048

10,166

14,211

10,586

16,278

249d 6,548

14,029

5,460

16,168

16,172

H6a1a1 239!

16,218

13,953

15,884

7,813

16,192

6,260

9,545

13,191

41

16,482!

H6a1a2a

16,297

11,923

152

13,020

12,369 16,295

14,560 60

73,16C

9,948

14,094 16,356

204

6,182 4,580

1,598

7,001

7,364

297

4

2

0

5785

288

709

16,140

9,773

11,662

16,482!

748

2,010

10,237

16,148

9,055

H6a1a4

H6a1a2b

10,936

16,311

14,944

10,370

7,080

H6a1a6

146 H6a1a5

195

11,204 16,482!

93

6,468

13,105 150

7,805

14,182A

16,319

7,325

9,362

11,611

16,311

4,991

H6a1a8

H6a1a8a

16,298

6,185

16,145

9,068

3,397

146

16189

41

152

827

195

H6a1a3

7,269

3,944

7,094

16,311

H6a1a7

H6a1a1a

150 3,705 4,947

2,361 9,025

15,287

14,970

537G

16,092 980

H6a1a2b1

16,482!

5,302

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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EuropeNorth Caucasus

North Africa America/Canada/Australia Ashkenazi Jew

Anatolia/South Caucasus/Near East

Asia

20

0

185 228 14,798

J1c

16,319

12,453

J1c12

15

16,261

15,113

J1c7

J1c12b

482 3,394

13,934

185! 5,024s

4,025

J1c1

6,554 12,127

J1c10

5,198

J1c7a

15,262 16,224

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

10

J1c2

9,632 12,083G

J1c5

J1c5a

16,092

6,464A

13,681

16,519

188

J1c7a1

6,887

J1c11

J1c4

152 4,484 5,460 12,361

J1c9

J1c12a

J1c2a

522-23d

228!

7,364 7,372 14,769

2,38710,192 4,688s

13,434s

10,084 10,398!

J1c7a1a

4,838 9,100

J1c8

14,798!

J1c5b

5

4,452 5,339 15,916 16,309

185!

195 5,291 15,103

228!

368 11,087 16,287 16,527

5,978 7,340 7,888

178R 12,630 16,255

10,598

522-23d

188 8,255

146

3,592

185! 16,519

HM159445

J1c6

HM627319

JF812166

21 6 1 3318 2

1 3 3

J1c3

20 2 1 14 1 9 4 1

2

15 2

2

11

2 1 1 1 2 1 3 1 3

2 2 3 1 1 1

5 2 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.

H: 20.5%

HV0: 4.1%

I: 1.3%

J: 6.3%

Asian: 1.1%

Unassigned:9.9%

Near Eastern:8.3%

W: 1.6%

U5: 2.0%

U: 0.2%

T: 3.0%

N1b: 9.2%

M1: 0.7%

European: 81%

K: 31.8%

Figure 10 | Estimated contributions of European mtDNA lineagesto the Ashkenazi mtDNA pool shown by major haplogroup. The possible overall Near Eastern contribution and fraction of unassigned lineagesare 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 condence 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

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 t well with genome-wide studies, which imply a signicant 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 ow, especially into Bulgaria and Turkey, both of which witnessed substantial immigration from Ashkenazi communities in the fourteenth and fteenth centuries. Gene ow could have been substantial in some casesongoing intermarriage is likely when these communities began living in closer proximity after the Spanish exile6. A partial common ancestry for all European Jewsboth Ashkenazi and Sephardicis again strongly supported by the autosomal results3,4.

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Jewish communities were already spread across the Graeco-Roman 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 30,00050,000 by the rst half of the rst century CE15. By the end of the rst millennium CE, Ashkenazi communities were historically visible along the Rhine valley in Germany33. After the wave of expulsions in Western Europe during the fteenth century, they began to disperse once more, into Eastern Europe33.

These analyses suggest that the rst 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 hypothesis8,9rather, 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 signicant founder event from the Near East. Fewer than 10% of the Ashkenazi mtDNAs can be assigned to a Near Eastern source with any condence, 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 ow 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 rst 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 ner 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 ne genealogical resolution of full mitogenomes, given sufcient 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 x incoming lineages from converts within the Ashkenazi community after this time. With sufcient 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 (identied by full control-region sequencing of 111 haplogroup K samples) for mitogenome sequencing, plus ve 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 les. As there are a number of extremely variable sites in K1 (positions 195 and 16,093 in particular), we conrmed 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 dened 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 plots4244 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 16,05116,400), 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, identied 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 Glge, 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/CSANT/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.

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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).

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

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