The Druze population of the Galilee represents a contemporary refugium of this past genetic landscape. Therefore it seems highly unlikely that the contemporary Galilee Druze haplogroup X subpopulation reflects either random migration as in the island migration model[33] or a colonization process originating from nearby Near-Eastern populations. The quest for genes associated with diseases is widely recognized as an essential task in the effort to investigate the genetic basis of complex human disorders and traits. The p-value is the fraction of samples in which the gene diversity was lower than that in the previous study. 5 (2008), e2105. In 311 samples, comprising 208 surnames. Low migration rates were also evident between the Galilee Druze and Druze from other subregions. We consider an ideal situation where surname households are completely correlated with mitochondrial lineages. Shlush, L.I., Behar, D.M., Yudkovsky, G., Templeton, A., Hadid, Y., Basis, F., Hammer, M., Itzkovitz, S. and Skorecki, K. (2008). The historical events and time frame for the loss or dilution of haplogroup X individuals is consistent with the population upheavals and patterns of migration that have characterized the Near East during the past two millennia at least [41], [42]. To assess the probability that the fraction of X haplogroups in the Druze population may arise from a sampling of a larger source population, we assumed a source population of size N, fs of which are X haplogroup, and a target population (the original Druze) of size n, ft of which are X haplogroup (for example fs = 6/218 for the Turks and ft = 41/311 for the Druze). The mean number of pairwise differences[21] in the sample set was 5.42 +/−2.56 (n = 311), and the distribution of the pairwise difference was unimodal as expected in populations that have undergone population expansion[18]. The divergence time is in thousand of years. We employed a paternal household-based sampling method (Figure S1) from 20 Druze villages in northern Israel, (Table S1). Since the application of such estimates to a non-random sample has not been ascertained, we have restricted ourselves to analysis of the mean mismatch distribution for the HVS-I segment (16090–16365)[18], [20]. The probability of obtaining k haplotypes using random sampling is:Using this formula, the p value – probability to obtain N haplotypes (maximum diversity) under a random sampling scheme can be calculated. To assign a p-value to the null hypothesis that the low diversity of previous studies [4], [7] is a result of smaller sample size, we randomly chose 1000 sample sets of identical sizes (n = 45) from the current data set and calculated the resulting gene diversity. http://www.journals.plos.org/plosone/article?id=10.1371/journal.pone.0002105. This was attributed to a founder effect, genetic drift, and population expansion[4]. The finding of a surprisingly high frequency and high diversity of X haplogroup lineages, and the rejection of a recent bottleneck or founder effect, encouraged us to conduct a more detailed analysis of the distribution of the X haplogroup lineages according to geographic sub-regions. This finding remained significant after Bonferroni correction for multiple hypothesis testing. 280 males were typed for 12 short tandem repeats (STR) and haplogroup defining SNPs (see Methods). The geographical distribution of Haplogroup K ranges from Northwest Europe to regions of the Eastern Mediterranean and the Middle East. [7] found 25 haplotypes, based on part of the HVS-I region (nucleotides 16090–16365), yielding a calculated genetic diversity of Ĥ = 0.94±0.02. Haplogroup X is one of rarest matrilinear haplogroups in Europe, being found only is about 1% of the overall population. . These findings have been uncovered due to the unique demographic features of the Druze population, and the adjusted sampling method employed in the current study. Furthermore, the formal rejection of the alternate hypothesis relating to immigration to the region of individuals sharing the same mtDNA haplogroup but with lineages that diverged in antiquity, further strengthens the Druze refugium model. Analysis of population substructure by NRY haplogroups among different geographic regions indicated a significant difference in the frequency of haplogroup K among the different localities. DNA samples were extracted from buccal swabs, with written informed consent according to procedures approved by the Institutional Human Subjects Review Committee. We gratefully acknowledge the assistance of the Druze community contact persons: Mr. Atila Tayseer, and the helpful advice and input of Dr. Sara Selig. Source Link: http://www.journals.plos.org/plosone/article?id=10.1371/journal.pone.0002105. Analysis of Nei's genetic diversity (Ĥ) among the different Druze subregions for both NRY lineages (for definition see Methods) and mtDNA lineages (defined by nucleotides 16024–16569 and 1–310), revealed similar diversities for both the NRY and the mtDNA for all Druze geographic subregions (Table S5), excluding matrilocality or patrilocality among the Druze[12]. Complete mtDNA sequencing for each of the D-loop defined haplotypes was carried out for haplogroups H and K samples, which together with haplogroup X were the 3 most prevalent haplogroups among the Druze population. 1992; 1993a).Together, these haplogroups account for ∼97% of modern Native American mtDNAs surveyed to date (Torroni and Wallace 1994; Merriwether et al. Affiliation Lineage analysis within the mtDNA X-haplogroup was particularly enlightening. Discover a faster, simpler path to publishing in a high-quality journal. The Druze: a population genetic refugium of the Near East. The MJ[51] algorithm was implemented within the Network 4112 program to draw the MJ tree of each NRY haplogroup by using the 12 STR data. Taken together these findings support the hypothesis that the Galilee Druze are a further more isolated subpopulation of the Druze, who in turn represents a refugium of the population genetic architecture of the Near East in antiquity. Defining Mutations. We also used a numerical model to test and exclude the possibility of non-random migration and colonization [30]–[32]such as might result from fission of kin structured and hence correlated samples from nearby populations. Conceived and designed the experiments: AT SI DB YH KS LS FB. Introduction. Although, we cannot exclude the possibility that some ratio between non-random migration and colonization did occur during and following the “Dawa” period and generated the differences in haplogroup frequencies among the current Druze subregions, and between the Druze and other populations, this explanation is highly unlikely in face of the demographic modeling results. Using this model, we demonstrate that the Druze have low migration rates with all nearby populations. Analysis of the same HVS-I region in our Druze database revealed 82 haplotypes with an estimated genetic diversity Ĥ = 0.968±0.004 (P<0.005 for comparison of genetic diversity in the two sample sets). The origin of haplogroup G is controversial. We also used a more detailed model in which we included the relative weights of each one of the observed haplotypes, so the number of haplotypes in each lineage is not constant. Retrieved 2011-01-28. Click through the PLOS taxonomy to find articles in your field. CRS = the Cambridge Reference Sequence. Enrichment analysis[22] revealed that both X1 and X2 were highly enriched in this region (P = 9.3*10̂−5 and  = 3*10̂−4, respectively). In studies designed to uncover population allele frequencies, or population based association studies with complex clinical phenotypes, the first step will have to be elucidation of the population substructure by directed sampling, followed by random sampling from a regional subpopulation. We observed a striking overall pattern of heterogeneous parental origins, consistent with Druze oral tradition, together with both a high frequency and a high diversity of the mitochondrial DNA (mtDNA) X haplogroup within a confined regional subpopulation. Asterisks designate samples whose genotype does not match any of the currently designated subhaplogroups. The distribution of X haplogroup individuals in such a sampling scheme follows a hypergeomtric distribution, from which a p-value can be obtained. al, submitted). No population or geographic region has been identified to date, in which haplogroup X and its major subhaplogroups are found at both high frequency and high diversity, which could provide a potential clue as to their geographic origin. It should be noted though, that directed sampling might miss localized founder effects and bottlenecks, since it does not provide the real haplotype frequencies, but rather is designed to uncover as many diverse lineages as possible for a given sample size. We adopted the consensus haplogroup nomenclature schemes for both the NRY and mtDNA [50]. HGDP00600 Druze, mtDNA R0a2, Y-DNA J2(xJ2f2) HGDP00628 Bedouin, mtDNA R0a2, Y-DNA E3b3 HGDP00616 Bedouin, mtDNA R0a2c, Y-DNA J(xJ2) HGDP00230 Pathan, mtDNA R0a2d, Y-DNA R1a1 HGDP00678 Palestinian, mtDNA R0a2, Y-DNA J(xJ2) HGDP00281 Kalash, mtDNA R0a, Y-DNA L HGDP00285 Kalash, mtDNA R0a, Y-DNA G HGDP00319 Kalash, mtDNA R0a, Y-DNA H1 Various studies have shown that when a new colony is formed from several potential source populations, either lower[25] or higher[26] genetic differentiation can occur depending upon how colonizing groups of individuals are formed, and depending upon the quantitative relationship between colonization and migration. In contrast to the overall high NRY haplogroup diversity for the entire sample, village based pockets of low diversity were observed, consistent with local paternal founder effects. Shutterstock Our findings explain a 1,000-year saga of two people living side by side in these lands. (2008) The Druze: A Population Genetic Refugium of the Near East. First consider the hypothesis that the current population genetic structure is the result of a random or non-random migration process contributing to original members of the Druze religion. The source population of Haplogroup X in the Druze is still unknown but remains an intriguing detail. Node names and colors are identical to those in figure 1A. Therefore the Druze would seem to have an even greater degree of genetic isolation, than indicated by these results of the demographic modeling. For 32 D-loop defined haplotypes within haplogroup H, we observed 26 different coding region variant defined lineages, of which 12 represent novel lineages (Table 2). The data were analyzed with Genescan (v. 3.7, Applied Biosystems) and Genotyper (v. 1.1, Applied Biosystems). https://doi.org/10.1371/journal.pone.0002105.g002. Popular tradition, however, … Yes Druze women in Isfiya, one of the largest Druze villages in Israel. It is found throughout Europe, North Africa, the Near/Middle East, in most of Central Asia, parts of North Asia, and among Native North Americans (X2a). The mode of each marginal posterior distribution was considered as a point estimate of the corresponding parameter value. Laboratory of Molecular Medicine, Rambam Health Care Campus, Haifa, Israel, Affiliation Each subject reported the birthplace of his/her maternal and paternal ancestry. The 41 mtDNA X haplogroup samples were available for complete mtDNA sequencing, of which 11 could be assigned to different HVS-I and HVS-II D-loop sequence based haplotypes and were therefore subject to complete mtDNA sequence. We have used the isolation with migration model [29], which is a Markov chain Monte Carlo calculation using DNA sequence data to estimate the relative effects of migration and isolation on genetic diversity in a pair of populations. • The DNA studies done on the Druze so far prove their genetic diversity; with Y-DNA haplogroups: (J, E, R, G, L, Q, C, K/T), & mtDNA Haplogroups: (H, K, X, U, T, HV, I, J, L2, M1, N1, W, R0). Copyright: © 2008 Shlush et al. Haplogroup H is a human mitochondrial DNA (mtDNA) haplogroup. To assess the probability that the fraction of X haplogroups in the Druze population may arise from a sampling of a larger source population, we assumed a source population of size N, f s of which are X haplogroup, and a target population (the original Druze) of size n, f t of which are X haplogroup (for example f s = 6/218 for the Turks and f t = 41/311 for the Druze). For the microsatellite analysis, twelve short tandem repeats (Y-STRs: DYS19, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS426, DYS439, DYS438 and DYS457) were genotyped in two multiplex reactions following the protocol of Redd et al. Furthermore demographic modeling indicated low migration rates with nearby populations. This village, is believed to be one of the oldest Druze villages in Israel, and is mentioned in historical records dating from the 13th century. Demographic modeling can also provide estimates of divergence times for populations with shared ancestries. Demographic modeling using the IM application[40] applied to mtDNA HVS-I sequences (nucleotides 16067–16384) from various Near East populations. Previous studies described a high frequency with a low diversity of both the X1 and X2 subhaplogroups in the Druze population[4], [7]. None of them belonged to R1b-M269 or R1b-L23 clades, which dominated during the Yamna period. ... Haifa, Israel. Furthermore, unlike other monotheistic religions, the Druze tenets strictly close their religion to new adherents, thus forbidding admixture with other populations. https://doi.org/10.1371/journal.pone.0002105.s001, https://doi.org/10.1371/journal.pone.0002105.s002, https://doi.org/10.1371/journal.pone.0002105.s003, https://doi.org/10.1371/journal.pone.0002105.s004, https://doi.org/10.1371/journal.pone.0002105.s005, https://doi.org/10.1371/journal.pone.0002105.s006, https://doi.org/10.1371/journal.pone.0002105.s007, https://doi.org/10.1371/journal.pone.0002105.s008. The next step in the lineage assignment included calculating coalescence time for each of the sub-branches using Network 4112 program. here. No, Is the Subject Area "Regional geography" applicable to this article? Considering a population of N haplotypes (or mitochondrial lineages), each containing x individuals. Mitochondrial haplogroup H is today predominantly found in Europe, and is believed to have evolved before the Last Glacial Maximum (LGM). Performed the experiments: MH DB GY LS. The Druze: A Population Genetic Refugium of the Near East, PLoS ONE, vol. Thus in the absence of a readily available approach to ascertain distinct maternal households, a paternal household based sampling strategy also facilitates identification of the maximum number of diverse maternal lineages in a given sample set. Sequences reported by Hammer et.al [53]: Bedouins (Bedouin) 58 samples. https://doi.org/10.1371/journal.pone.0002105, Editor: Neil John Gemmell, University of Canterbury, New Zealand, Received: August 31, 2007; Accepted: March 20, 2008; Published: May 7, 2008. Background For millennia, the southern part of the Mesopotamia has been a wetland region generated by the Tigris and Euphrates rivers before flowing into the Gulf. Phylogenetic mitochondrial DNA haplogroups are highly partitioned across global geographic regions. We sample m individuals (without loss of generality m≥N). It should be noted that the estimated coalescence times for the major mtDNA X subhaplogroups X1 and X2 are 42,900±18,100 and 17,900±2,900 respectively[4]. Mitochondrial DNA analysis (nucleotides 16024–16569 and 1–310) carried out on all samples, revealed 106 haplotypes (Ĥ = 0.979±0.003) (Table S2 and S3). The highest frequencies of Haplogroup K was found to be in Belgium, The Netherlands, Cyprus and Lebanon. Run-time parameters included a maximum migration rate of 10, a maximum divergence time of 10 Ne generations, and a burn in of 100,000 steps followed by a run of at least 10,000,000 steps with a linear heating scheme of increment 0.1. In the period immediately preceding the fall of the Second Temple in … A very high global genetic diversity has been reported for haplogroup X[4]. The project is open to males with Druze patrilineal ancestry as well as other individuals with close Y-DNA genetic matches to our project members. The secret is out, says Druze MK Ayoub Kara: "The Druze are actually descended from the Jewish people, and genetic evidence proves it." The cutoff of 1000 years was used in order to estimate the number of lineages that were included in the Druze genetic pool before the “Dawa” period. Altogether we sampled 311 different paternal households from 20 Druze villages in Northern Israel, (Table S1,[5], [8]), and 208 surnames were identified, consistent with the fact that certain kindreds have adopted the same surname, despite family records clearly indicating distinct paternal origins. A study of 329 Druze men found 12.5% were haplogroup G. This study was not confined to Israel, but was probably mostly Israeli. Members will be divided into subgroups based on their actual (or predicted) Y-DNA Haplogroups. The demographic modeling in the current study indicates most recent divergence of the Druze from an ancestral population shared with Egyptians, Ashkenazi Jews, Adygeis and Greeks (Figure 2). Sequences reported in Macaulay et al [7]: Egyptians (Egypt) 67 samples; Iraqis (Iraq) 116 samples; Syrians (Syria) 69 samples; Palestinians (Pales) 110 samples; Turks (Turk) 218 samples; Armenians (Armenian) 191 samples; Adygei (Adyg) 50 samples; Greeks (Greece) 65 samples; Swedes (Swe) 32 samples; Nowegians (Nor) 231 samples; Basques (Basq) 156 samples. Yes These findings were enabled through the use of a paternal kindred based sampling approach, and suggest that the Galilee Druze represent a population isolate, and that the combination of a high frequency and diversity of the mtDNA X haplogroup signifies a phylogenetic refugium, providing a sample snapshot of the genetic landscape of the Near East prior to the modern age. One possible model for the colonization of a new or vacant habitat occurs when some subpopulations in nearby geographic regions contribute migrants to a common pool, the “migrant pool,” from which colonists are drawn at random to fill vacant sites, with mixing of individuals from different populations[27]. The Druze harbor a remarkable diversity of mitochondrial DNA types or lineages that appear to have separated from each other many thousands of … Jordan. Yes The issue of haplogroups only affects the SAAP analysis. The fourth character defines the Druze Galilee village in which the haplotype was the most prevalent, according to maternal ancestry. The following populations were used: 311 Druze from the current study. No, Is the Subject Area "Biogeography" applicable to this article? In order to evaluate the differences in mtDNA genetic diversity between the current dataset and that reported previously[28], we have used t-test combined with bootstrap standard error analysis according to Nei [11]. It is also less likely that haplogroup X came from the Turks because although Druze DNA is most like current Turkish DNA, Turks don't exhibit haplogroup X to the same degree as the Druze. Jabal al-Druze (جبل الدروز, jabal ad-durūz, Mountain of the Druze), officially Jabal al-Arab (جبل العرب, jabal al-ʿarab, Mountain of the Arabs), is an elevated volcanic region in the As-Suwayda Governorate of southern Syria. No, Is the Subject Area "Haplotypes" applicable to this article? In several cases, individuals from the same village having the same surname were sampled if they could show that their households were unrelated, despite sharing an identical surname, thus yielding a total of 311 samples for analysis. For Native Americans, extensive RFLP and control region (CR; also known as the “D-loop”) sequence analysis has unambiguously identified four major founding mtDNA haplogroups, designated “A”–“D” (Torroni et al. This project is for people who have tested and been assigned the paternal haplogroup G2a2b (L91) and also for people who are believed to have belonged to this paternal haplogroup based on tests done on descendants.. Subhaplogroup X1 was found to be largely restricted to the Afro-Asiatic-speaking populations of northern Africa and the neighboring areas, suggesting a possible geographic diffusion of X1 along the coast of the Mediterranean and the Red Sea. A small sample set which might include individuals from the same kindred or from a confined geographic region in which there is a high rate of consanguinity and endogamy is expected to yield a lower calculated genetic diversity than a larger sample set gathered from individuals belonging to different parental households from different subpopulations. Therefore, we tested the possibility of obtaining an X haplogroup frequency as high as observed in our study (41/311), from random sampling of Turkish individuals as a metapopulation contributing to the migrant pool, (Turks have a current X haplogroup frequency of 6/218[28]). This site uses Akismet to reduce spam. The refugium hypothesis based on mtDNA haplogroup X analysis was corroborated by the finding of high diversity for the Druze mtDNA haplogroups H and K, with the added finding of novel lineages not shared with nearby populations. The overall low migration rate between the Druze and all other nearby populations (Figure 2) cannot explain the high diversity and high frequency of X haplogroup lineages in the Galilee region. Therefore, in order to obtain a comprehensive phylogenetic picture of lineage diversity in a sample set from the Druze population or populations with similar socio-demographic patterns, it is necessary to either collect a very large random sample, or to use a directed sampling strategy to ensure coverage of all of the regionally endogamous cryptic subpopulations. Mutations were scored relative to the revised Cambridge Reference Sequence (rCRS)[46]. Moreover, a high degree of consanguinity (47% first cousin marriages[9]) and local endogamy, generate a situation in which each paternal household in a specific geographic region is a subpopulation isolate in which there is a strong correlation between paternal and maternal household identity[10]. With regard to population substructure and genetic distances directed sampling is more appropriate to uncover cryptic diversity and regional substructure in populations such as the Druze, characterized by high levels of endogamy and local founder effects. The highest incidence of haplogroup X is observed in Greece (4%), Macedonia (3%), Romania (2.5%) and around the Caucasus, notably among the Avars (5%), Adyghe-Kabardin (5%), Karachay-Balkars (4.5%), Nogays (4%), Dargins (3.5%), Armenians (3.5%), Azeri (3.5%), North Ossetians (3%) and Georgians … Sequences reported by Metspalu et.al [54]: Iranian (Iran) 436 samples. It is striking that those different lineages (from the same parental haplogroup) whose genetic divergence date back more than ten thousand years would remain so concentrated within such a small geographic region. Wrote the paper: AT SI DB KS LS. Ruth and Bruce Rappaport Faculty of Medicine and Research Institute, Technion – Israel Institute of Technology, Haifa, Israel, Therefore we extended the mtDNA analysis to other Druze haplogroups. We have examined mitochondrial DNA sequence variation together with Y-chromosome-based haplogroup structure among the Druze, a religious minority with a unique socio-demographic history residing in the Near East. The effect of sampling strategy was particularly evident for the mtDNA X-haplogroup, in which we found both a high frequency (13.1%, 41/311 samples) together with a high diversity of haplotypes, as defined in Macaulay et al(7) on the basis of sequence variation at the HVS-I region (nucleotides 16090–16365) (Table 1). Sampling was carried out according to paternal rather than maternal households, since the Druze kindred structure is based on paternal family identity, though knowledge of maternal ancestry is often also retained. The combination of a high frequency and diversity of the Druze mtDNA haplogroup X lineages, in a confined geographic region, and the low migration rate with nearby populations make it unlikely that this diversity was imported. Analyzed the data: AT SI LS. Phylogenetic mitochondrial DNA haplogroups are highly partitioned across global geographic regions. The probability values for non-random migration and colonization were all <0.05 for a wide range of population parameters (Table S4). Yes For example, the village of Beit -Jaan included 5 samples from haplogroup K, all of which belonged to the same lineage (Ĥ = 0). Copyright © 2013 - 2021 Rebekah A. Canada | All Rights Reserved | Powered by WordPress & The PODs Framework. We observed a striking overall pattern of heterogeneous parental origins, consistent with Druze oral tradition, together with both a high frequency and a high diversity of the mitochondrial DNA (mtDNA) X haplogroup within a confined regional subpopulation. Although the Druze represent a small percentage of the total population of the countries of the Near East in which they reside, their concentration in mountainous districts has produced a compact social structure, resulting in a nearly exclusive majority in some geographical regions, and therefore a low frequency of admixture with other populations. In the case of the NRY the term lineage is used to denote a cluster of related evolving STR-based haplotypes within a haplogroup. Purple bars = Near East populations among themselves. Furthermore, comparison of the Druze X haplogroup sequences to Turkish and Egyptian X haplogroup sequences[4] revealed different lineages whose coalescence antedates the “Dawa” period. The MJ[51] algorithm was implemented within the Network 4112 program. Dnalc.org. We used a simplified analytical model to assess the probability of obtaining a diversity level with random sampling, comparable to that obtained by surname directed sampling. Yes Our findings of high diversity and high frequency of X haplogroup concentrated among the Galilee Druze provides new insights regarding both sampling methodologies in population genetics, and the understanding of the effects of sociological patterns on the population genetic landscape of the Near East. Subclades X2a and X2g are found in North American Ojibwe. Your email address will not be published. In ancestry dna i saw many romanians score around 2 % middle east. Same in myorigins 1.0 i saw many romanians score some percentage of west middle east. These findings were enabled through the use of a paternal kindred based sampling approach, and suggest that the Galilee Druze represent a population isolate, and that the combination of a high frequency and diversity of the mtDNA X haplogroup signifies a phylogenetic refugium, providing a sample snapshot of the genetic landscape of the Near East prior to the modern age. Sequences reported by Thomas et.al [55]:Ashkenazi Jews (Ashkenazi) 78 samples Ethiopians (Ethiop) 74 samples. In order to determine the total number of distinct lineages, we followed the approach previously described for several other populations[13]–[15], of first sequencing the complete HVS-I and HVS-II D-loop sequences for all samples in the haplogroup, followed by complete sequencing of the entire mtDNA for each of the D-loop defined haplotypes. Therefore we extended the mtDNA analysis to other Druze haplogroups. These findings were enabled through the use of a paternal kindred based sampling approach, and suggest that the Galilee Druze represent a population isolate, and that the combination of a high frequency and diversity of the mtDNA X haplogroup signifies a phylogenetic refugium, providing a sample snapshot of the genetic landscape of the Near East prior to the modern age. In human mitochondrial genetics, Haplogroup X is a human mitochondrial DNA (mtDNA) haplogroup which can be used to define genetic populations.The genetic sequences of haplogroup X diverged originally from haplogroup N, and subsequently further diverged about 20,000 to 30,000 years ago to give two sub-groups, X1 and X2.Overall haplogroup X accounts for about 2% of the population of Europe, … In order to exclude the effect of sample size alone in generating this difference, we conducted a bootstrap analysis designed to account for the different sample sizes in the two studies (see Methods), and found that, the difference in diversity remained significant (P<0.04). There were 10 STR haplotypes and 7 lineages among the 24 haplogroup K was found to be in Belgium the. Total number of migrants per generation sampling effects during the “ Dawa ” period 74. Settings were used ( -q1 10 -m1 10 -m2 10 -t 10 -b 100000 -L 0.5 -s123 ). 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Genetic matches to our project members are found in North American Ojibwe Shlush al... L can also provide estimates of migration rates and population divergence times for with... To migration events every time, Druze tradition considers the population to have before... Defined as a point estimate of the sub-branches using Network 4112 program of..

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