A genetic contribution from the Far East into Ashkenazi Jews via the ancient Silk Road

Contemporary Jews retain a genetic imprint from their Near Eastern ancestry, but obtained substantial genetic components from their neighboring populations during their history. Whether they received any genetic contribution from the Far East remains unknown, but frequent communication with the Chinese has been observed since the Silk Road period. To address this issue, mitochondrial DNA (mtDNA) variation from 55,595 Eurasians are analyzed. The existence of some eastern Eurasian haplotypes in eastern Ashkenazi Jews supports an East Asian genetic contribution, likely from Chinese. Further evidence indicates that this connection can be attributed to a gene flow event that occurred less than 1.4 kilo-years ago (kya), which falls within the time frame of the Silk Road scenario and fits well with historical records and archaeological discoveries. This observed genetic contribution from Chinese to Ashkenazi Jews demonstrates that the historical exchange between Ashkenazim and the Far East was not confined to the cultural sphere but also extended to an exchange of genes.

C onsistent with their displaced ethno-history since the ancient Northern Kingdom of Israel was invaded and occupied by the Neo-Assyrian Empire 1 , contemporary Jews, including Ashkenazi Jews, Sephardic Jews, North African Jews and Middle Eastern Jews 2 , retain a genetic imprint of their Near Eastern ancestry, but have received a substantial contribution, to a variable extent, from their neighboring populations such as Europeans, Near Easterners, and North Africans 2-8 . However, it has hitherto remained unclear whether Jews received any genetic contribution from populations outside western Eurasia. Intriguingly, frequent communication has been observed between Jews and Chinese since the early centuries of the Common Era, plausibly initiated by the Silk Road. For instance, Hebrew letters and prayers in the 8 th century from ancient Jewish merchants were found in the northwestern region of China 9 . Some unearthed pottery figurines from the Tang Dynasty (618-907AD) have Semitic characteristics 9 , and synagogues were recorded in the epigraphy from the Ming (1368-1644AD) and Qing Dynasties (1644-1912AD) 9,10 . Nonetheless, such connections, as revealed by the archaeological discoveries and historical records, have been confined to economic and cultural exchanges; so far, no direct evidence of a genetic contribution from Chinese into Jews has been reported.
To address the issue of whether Jews received any genetic contribution from the Far East, and thus shed more light on their ethno-origins, mitochondrial DNA (mtDNA) variation (mainly from the control region of the molecule, plus some coding-region variants) of 1,930 Jews and 21,191 East Asians, retrieved from previous studies as well as our unpublished data, were considered and analyzed, with especial attention to pinpointing eastern Eurasian haplogroups in Jews (Supplementary Table S1). Then, mtDNA control region variants of an additional 32,474 Eurasian individuals were analyzed to gain further insights into the phylogeographic distribution of M33c (Supplementary Table S1), so that the total number of Eurasian mtDNAs considered here was 55,595. Our results do reveal a direct genetic connection, as manifested by the sharing of some Eastern Eurasian haplogroups e.g. N9a, A, and M33c, between Jews and Chinese. Further analyses, including phylogeny reconstruction with the aid of new mtDNA genomes, confirm that this connection was established at least by a founder lineage M33c2. The differentiation time of this lineage is estimated to ,1.4 kilo-years ago (kya), which fits well with the historical records and, most importantly, indicates that the exchange between Jews and the Far East was not confined to culture but also extended to the demic. Results and Discussion Our analysis of the mtDNA variation in a total of 23,121 individuals from East Asian populations and Jews reveals that mtDNAs of four Ashkenazi Jewish individuals can be allocated into eastern Eurasian haplogroups A and N9a, suggesting that Ashkenazi Jews received a genetic contribution from East Asia (Table 1). Intriguingly, our results also disclose that 14 eastern Ashkenazi Jews belong to haplogroup M33c (Table 1), for which sister clusters, M33a, M33b and M33d, are prevalent in the Indian Subcontinent and thus most plausibly trace their origins there 11,12 .
To achieve further insight into the phylogeographic distribution of M33c, mtDNA variants (mainly from the control region) of an additional 32,474 Eurasian individuals were analyzed, so that the total number of Eurasian mtDNAs considered here was 55,595. As shown in Table 1 To shed light on the phylogeny within haplogroup M33c, 11 mtDNAs, covering the widest range of internal variation within the haplogroup, were chosen for whole-mtDNA genome sequencing. In good agreement with the previous result 13 , the resulting phylogenetic tree (Figure 2), incorporating five previously reported mtDNA genomes [13][14][15] as well as one whose information was released online (A Genetic Genealogy Community; http://eng.molgen.org), confirms that M33c is defined by mutations at positions 3316, 4079, 5894, 8227, 8848, 16111, and 16235. Of note is that five clades within M33c appear respectively characterized by diagnostic coding-region variant(s), and these are named M33c1 to M33c5 here. With the exception of M33c2, all the samples in these clades are from China. The likely origin of M33 in South Asia and the restriction to China of M33c, dating to 10 kya according to the estimation based on whole-mtDNA genome, implies some dispersal from South to East Asia in the immediate postglacial.
Intriguingly, sub-haplogroup M33c2 (defined by three additional coding-region variants at positions 4182, 4577, and 7364) consists of three different haplotypes (one seen in three Ashkenazi Jews, another in a single Chinese individual and the third in the likely European with unknown ethnicity). Although there is no control-region variant in the defining motif of M33c2, multiple lines of evidence suggest that the pinpointed 14 Ashkenazi Jewish M33c mtDNAs most likely all belong to this clade: (1) all of the 14 mtDNAs share an identical control-region motif (Table 1); (2) the three completely sequenced Ashkenazi Jewish mtDNAs with this motif (EU148486, Bel 1 and Forum 1) belong to M33c2 ( Figure 2); (3) M33c shows a virtually exclusive distribution in Ashkenazi Jews in western Eurasia, even though 55,595 mtDNAs have been checked (Table 1 and  Supplementary Table S1). Thus, it is plausible that the unknown European individual (JQ702003) was in fact from a Jewish population or had Ashkenazi Jewish ancestry.
Age estimates for M33c2 are similar whether based either on the whole genome or on the control region alone (Table 2), and the age of ,1.4 kya fits well with the medieval operation of the Silk Road. We note that this is an upper bound for the gene flow event during which the lineage was assimilated into the Ashkenazim; it is the age of the subclade overall, which most likely arose within China, and indeed there is no variation at all within the Jewish lineages, suggesting a very recent event. If we assume that the unidentified European lineage belongs within the Ashkenazi diversity, we can date the The whole-mtDNA genome information is displayed on the phylogenetic tree ( Figure 2). b The individual has coding region information confirming its haplogroup assignment from the Family Tree DNA (www.familytreedna.com). c This sequence is from A Genetic Genealogy Community (http://eng.molgen.org/viewtopic.php?f541&t5141). N.A. 5 not available.
All the samples in this study have confirmed G2361A mutation when assigning their haplogroup. The mutations (e.g. 489 and 16519) outside HVS-I and HVS-II are listed in the parentheses. The haplogroups A and N9a mtDNAs in the Far East are not shown here.

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Ashkenazi subclade itself more specifically to about 640 years agoaround 1350AD. This in turn would then provide a minimum point estimate for the age of the gene flow event (although the range taking account of errors in the estimates is of course much wider). The ancient Silk Road was an important transportation hub connecting China and the Mediterranean region from the Han Dynasty (206BC-220AD) onwards, and there are likely to have been Jewish merchants at the eastern end of the Silk Road from the early centuries AD. Moreover, Jewish merchants in Europe, referred to as Radhanites, were involved in trade between west and east as early as the ninth century 16 . It has been suggested, on the basis of contrasts between patterns of mtDNA and Y-chromosome variation 17 , that such merchants may have formed the nucleus for a number of extant Jewish communities.
Ashkenazi origins are controversial 18 . According to recent archaeological evidence, the Jewish community of Cologne, mentioned by Emperor Constantine in 321AD, existed in the city continuously until they had to leave in 1423-1424AD 19 . This suggests that Ashkenazi Jewry may date to Roman times, possibly originating in Italy, which is also suggested by analysis of mtDNA 8 and autosomal data 20 . An early eastern European Ashkenazi origin from Italy (first millennium and earlier) would also agree with the finding that an origin mainly from Germany 21 or another central or western European country 18 during the late Middle Ages, is demographically not possible. Recent work also suggests a sizable Jewish presence in eastern Germany (the Danube region, rather than the Rhineland) prior to the expansion in Poland between 1500 and 1650AD 22 . The M33c2 mtDNAs are confined to eastern European Ashkenazim in the present database (the single unknown example is of likely East European ancestry 14 ), suggesting that these groups had contacts to the east to the extent that they mediated female gene flow.
Extensive genetic admixture has been observed in populations residing around the ancient Silk Road region 23,24 . Our currently observed genetic imprint echoes the previously observed ancient communications between Jews and Chinese and, most significantly, implies that such historical exchanges were not confined to the cultural realm but involved gene flow. This unexpected ancient genetic connection between Ashkenazi Jews and the Far East, as witnessed at  least by mtDNA haplogroup M33c2, provides the first evidence for a significant genetic contribution from Chinese to eastern European Ashkenazi Jews that was most likely mediated by the Silk Road between around 640 and 1400 years ago. Although the involvement of male Jewish traders has been suggested before 17 , our results, focusing on the female line of descent, specifically point to the involvement also of women. Well-resolved evidence from the male-specific part of the Y chromosome and from the autosomes would help to further illustrate the rather complex, pan-Eurasian ethno-history of Jews.
Methods mtDNA Data collection and mining. mtDNA variation (mainly from control region) of 23,121 East Asians and Jews, retrieved from previous studies as well as our unpublished data, were considered and analyzed, with especial attention to pinpointing the eastern Eurasian haplogroups in Jews. Then, additional 32,474 individuals were analyzed to gain further insights into the phylogeographic distribution of M33c, leading the total number of Eurasian mtDNAs considered here to 55,595. The study project was approved by the Ethics Committee at Kunming Institute of Zoology, Chinese Academy of Sciences. Each participant was informed about the study and provided informed consent. All mtDNAs collected and considered in the present study were first allocated to haplogroups, based mainly on their control-region motifs, which were then further confirmed by typing specific coding-region variation according to the PhyloTree (mtDNA tree Build 16 25 ; http:// www.phylotree.org/). DNA amplification and sequencing. For haplogroups of interest, special attention was paid to the intrinsic phylogeny reconstructed on entire mitogenome information. In this way, entire mitogenomes for 11 selected representatives from haplogroup M33c were amplified, sequenced, and dealt with as described elsewhere 13,26 . The sequencing outputs were edited and aligned by Lasergene (DNAStar Inc., Madison, Wisconsin, USA) and compared with the revised Cambridge Reference Sequence (rCRS) 27 .
Data analysis. The median-joining network of M33c was constructed manually 28    htm). The most parsimonious phylogenetic tree (Figure 2) was reconstructed by hand as carried out previously 13,26 . The coalescence ages were estimated by the r 6 s method 29,30 and maximum likelihood (ML) analysis. Recently corrected calibrated mutation rates 31 were adopted in the r statistic and the ML analysis.