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Am J Hum Genet. 2004 May; 74(5): 827–845. Published online 2004 April 7. | PMCID: PMC1181978 |
Copyright © 2004 by The American Society of Human Genetics. All rights reserved. Where West Meets East: The Complex mtDNA Landscape of the Southwest and Central Asian Corridor Lluís Quintana-Murci,1,2,3 Raphaëlle Chaix,4 R. Spencer Wells,5 Doron M. Behar,6 Hamid Sayar,12 Rosaria Scozzari,7 Chiara Rengo,9 Nadia Al-Zahery,9 Ornella Semino,9 A. Silvana Santachiara-Benerecetti,9 Alfredo Coppa,8 Qasim Ayub,10 Aisha Mohyuddin,10 Chris Tyler-Smith,11 S. Qasim Mehdi,10 Antonio Torroni,9 and Ken McElreavey3 1Centre National de la Recherche Scientifique (CNRS) URA 1961, 2Unit of Molecular Prevention and Therapy of Human Diseases, and 3Unit of Reproduction, Fertility and Populations, Institut Pasteur, and 4Musée de l’Homme, Paris; 5Wellcome Trust Center for Human Genetics, Headington, United Kingdom; 6Bruce Rappaport Faculty of Medicine and Research Institute, Technion and Rambam Medical Center, Haifa, Israel; 7Dipartimento di Genetica e Biologia Molecolare and 8Dipartimento di Biologia Animale e dell’Uomo, Università “La Sapienza,” Rome; 9Dipartimento di Genetica e Microbiologia, Università di Pavia, Pavia, Italy; 10Biomedical & Genetic Engineering Division, Dr. A. Q. Khan Research Laboratories, Islamabad, Pakistan; 11The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, United Kingdom; and 12Department of Medicine, University of Arizona, Tucson Received December 3, 2003; Accepted January 20, 2004. The southwestern and Central Asian corridor has played a pivotal role in the history of humankind, witnessing numerous waves of migration of different peoples at different times. To evaluate the effects of these population movements on the current genetic landscape of the Iranian plateau, the Indus Valley, and Central Asia, we have analyzed 910 mitochondrial DNAs (mtDNAs) from 23 populations of the region. This study has allowed a refinement of the phylogenetic relationships of some lineages and the identification of new haplogroups in the southwestern and Central Asian mtDNA tree. Both lineage geographical distribution and spatial analysis of molecular variance showed that populations located west of the Indus Valley mainly harbor mtDNAs of western Eurasian origin, whereas those inhabiting the Indo-Gangetic region and Central Asia present substantial proportions of lineages that can be allocated to three different genetic components of western Eurasian, eastern Eurasian, and south Asian origin. In addition to the overall composite picture of lineage clusters of different origin, we observed a number of deep-rooting lineages, whose relative clustering and coalescent ages suggest an autochthonous origin in the southwestern Asian corridor during the Pleistocene. The comparison with Y-chromosome data revealed a highly complex genetic and demographic history of the region, which includes sexually asymmetrical mating patterns, founder effects, and female-specific traces of the East African slave trade. The southwestern Asian corridor is a wide geographical area that extends from Anatolia and the trans-Caucasus area through the Iranian plateau to the Indo-Gangetic plains of Pakistan and northwestern India. This region is characterized by a patchwork of different physical-anthropology types with complex boundaries and gradients and by the coexistence of several language families (e.g., Indo-European, Turkic, and Sino-Tibetan) as well as relict linguistic outliers. The southwestern Asian corridor, located at the crossroads of major population expansions, was the first portion of Eurasia to be inhabited by the Homo sapiens sapiens population(s) that left Africa ~60,000 years before the present (YBP) (Tishkoff et al. 1996; Watson et al. 1997; Quintana-Murci et al. 1999), and from this region modern humans migrated to the rest of the world. Although Paleolithic and Mesolithic people left their mark in the area, major prehistorical and historical events with possible genetic consequences occurred during the Neolithic period and later. Important agricultural developments occurred in the eastern horn of the Fertile Crescent ~8,000 YBP, notably in Elam (southwestern Iran). The highly urban Elamite civilization had close contacts with Mesopotamians but exhibited an extensive differentiation from the rest of the Fertile Crescent populations, including a language that is thought to belong to the Dravidian family. It is hypothesized that the proto-Elamo-Dravidian language (McAlpin 1974, 1981), spoken by the Elamites in southwestern Iran, spread eastwards with the movement of farmers from this region to the Indus Valley and the Indian subcontinent (Cavalli-Sforza et al. 1994; Cavalli-Sforza 1996; Renfrew 1996). Starting ~5,000 YBP, animal domestication, particularly the horse, gave the inhabitants of the Central Asian steppes the opportunity to expand geographically in different directions (Zvelebil 1980). These Central Asian nomads, probably from the Andronovo and Srubnaya cultures, migrated through Iran and Afghanistan, reaching Pakistan and India, and their arrival is contemporaneous with the decline of the strong agricultural South Asian civilizations, such as the Harappans. Most likely, their arrival on the Iranian plateau ~4,000 YBP brought the Indo-Iranian branch of the Indo-European language family and, eventually, caused the replacement of Dravidian languages in Iran, Pakistan, and most of northern and central India (Renfrew 1987, 1996; Cavalli-Sforza 1996). Starting in the 3rd century b.c., the eastern part of the Eurasian steppes witnessed similar pastoral movements. By the time of the 3rd century a.d., Turkic-speaking peoples from the Altai region began to migrate westwards, replacing Indo-European languages in parts of Central Asia and, eventually, in what is now modern Turkey. Later, the Mongols also moved westward and, by the 13th century a.d., established their rule over a vast region, including parts of India, Pakistan, and Iran and reaching as far west as the Caucasus and Turkey (Cavalli-Sforza et al. 1994). In the past decade, studies of mtDNA variation have provided a substantial contribution to the understanding of human origins and diffusion patterns. mtDNA surveys in worldwide populations have shown a continent-specific distribution of mtDNA lineages (Wallace et al. 1999; Ingman et al. 2000; Maca-Meyer et al. 2001; Herrnstadt et al. 2002; Mishmar et al. 2003). African populations are characterized by the oldest superhaplogroups, L1, L2, and L3 (Bandelt et al. 1995, 2001; Chen et al. 1995, 2000; Graven et al. 1995; Soodyall et al. 1996; Bandelt and Forster 1997; Watson et al. 1997; Alves-Silva et al. 2000; Torroni et al. 2001b; Salas et al. 2002), but it seems that only L3 radiated out of Africa, mainly in the form of haplogroups M and N, ~60,000 YBP, giving rise to the extant Eurasian variation (Watson et al. 1997; Quintana-Murci et al. 1999; Wallace et al. 1999). Most western Eurasians are characterized by clades within haplogroup N (Torroni et al. 1996; Macaulay et al. 1999; Richards et al. 2000), whereas N and M contributed almost equally to the current eastern Eurasian mtDNA pool (Stoneking et al. 1990; Ballinger et al. 1992; Torroni et al. 1993; Horai et al. 1996; Kolman et al. 1996; Comas et al. 1998; Starikovskaya et al. 1998; Redd and Stoneking 1999; Schurr et al. 1999; Derbeneva et al. 2002; Kivisild et al. 2002; Yao et al. 2002). Despite the major role played by the transect between the Near East and India in human origin and population dispersals, the extent and nature of mtDNA variation in the populations of the area are still not well resolved. In this context, mtDNA studies have focused on the western and eastern extremities of the southwestern Asian corridor, including the Near East/Caucasus region (Macaulay et al. 1999; Comas et al. 2000; Richards et al. 2000; Tambets et al. 2000; Nasidze and Stoneking 2001) and India (Mountain et al. 1995; Kivisild et al. 1999 a, 1999 b; Bamshad et al. 2001; Roychoudhury et al. 2001; Kivisild et al. 2003). In addition, Central Asian mtDNA variation is poorly characterized and is based only on HVS-I sequence data (Comas et al. 1998). Some populations of the region have been also analyzed for Y-chromosome variation, including Iranian (Quintana-Murci et al. 2001), Pakistani (Qamar et al. 2002), and, especially, Central Asian populations (Pérez-Lezaun et al. 1999; Karafet et al. 2001; Wells et al. 2001; Zerjal et al. 2002). To obtain a global mtDNA perspective of the entire region, we have now analyzed 910 mtDNAs from 23 different populations, located mainly in the southwestern Asian corridor but also, for comparison, in Central Asia. As a first step in the study, we performed high-resolution RFLP analysis and control-region sequencing of 208 mtDNAs, 108 from the western part of the corridor (Anatolia and the Caucasus), and 100 mtDNAs from its southeastern counterpart (southeast Pakistan). This allowed a clear-cut definition of the haplogroups (and their diagnostic markers) existing in the area. The phylogenetic information retrieved from this initial data set, together with previously published RFLP and HVS-I data, was then used to classify an extended collection of 702 newly obtained HVS-I sequences from the Iranian plateau, the Indus Valley, and Central Asia. The observed patterns of variation revealed different genetic contributions from western and eastern Eurasians and South Asians and evince complex demographic processes in some specific populations, including sexually asymmetrical mating patterns, founder effects, and differential migration patterns. Population Samples The approximate location of the 23 populations from which the 910 mtDNAs were sampled is shown in . Each sample comprises unrelated healthy donors from whom appropriate informed consent was obtained. For the preliminary part of the study, 208 individuals from three different geographic regions were analyzed: 58 individuals from the Caucasus, 50 from Turkey and 100 from southeastern Pakistan. The three samples were heterogeneous; the sample from the Caucasus region was made up of three different ethnic groups: Georgians, Balkarians, and Chechens. The sample from Turkey was collected mainly in Konya (Anatolia). The Pakistani sample was collected in Karachi and comprised mainly Sindhis, who are a mix of tribes of different religions and ethnicities from the southeastern province of Sindh. The extended population sample included 702 individuals from 20 different populations living in the Iranian plateau, the Indus Valley, the Karakorum and Hindu Kush mountains, and Central Asia. Further details of the whole sample collection are reported in table 1 and in the work of Wells et al. ( 2001) and Qamar et al. ( 2002). The term “Makrani” refers to the so-called “Negroid Makrani” population living in the Makran coast of Baluchistan, distinct from the Makrani Baluch population, which is not considered in this study. | Table 1Description of the Populations Included in the Study |
mtDNA Analysis High-resolution RFLP haplotypes were determined for the samples from the Caucasus region, Anatolia and Karachi. The entire mtDNA of each subject was PCR amplified using primer pairs and procedures previously described (Torroni et al. 1997). Each of the PCR segments was then digested with 14 restriction endonucleases ( AluI, AvaII, BamHI, DdeI, HaeII, HaeIII, HhaI, HincII, HinfI, HpaI, MspI, MboI, RsaI, and TaqI). In addition, all mtDNAs were screened for the NlaIII sites at nucleotide positions (nps) 4216 and 4577. The presence/absence of the BstOI/ BstNI site at np 13704, the AccI sites at nps 14465 and 15254, the BfaI site at np 4914, the XbaI site at np 7440, the MseI sites at nps 14766 and 16297, the MnlI site at np 10871, the MboII site at np 12703, and the HphI site at np 10237 were also analyzed in all the Pakistani-Karachi mtDNAs but only hierarchically in the mtDNAs from the Caucasus and Anatolia. Polymorphisms at nps 12308 and 11719 were also tested, the first by use of a mismatched primer that generates a HinfI site when the transition at 12308 is present (Torroni et al. 1996) and the second by use of a mismatched primer that generates a HaeIII site when the transition at 11719 is present (Saillard et al. 2000). The sequencing of the mtDNA control-region in the 208 individuals from the Caucasus region, Anatolia, and Karachi was performed as described elsewhere (Torroni et al. 2001a) and, in most cases, encompassed a large region (generally from np 16000 to nps 100–200). For the remaining 702 individuals, sequence data encompassed a shorter region (from np 16000 to np 16401), which includes the entire HVS-I, and variable positions were determined between nps 16024–16383, relative to the reference sequence (Anderson et al. 1981; Andrews et al. 1999). The published RFLP data (Macaulay et al. 1999; Quintana-Murci et al. 1999; Richards et al. 2000) and the new data obtained from the high resolution RFLP analyses of the 208 mtDNAs (see appendix A [online only]) were used to identify the RFLP and HVS-I sites (), which are diagnostic of the main haplogroups and subhaplogroups within the mtDNA phylogeny. These markers were then selectively assayed, on the basis of the HVS-I information, in the remaining 702 mtDNAs by PCR amplification of the appropriate fragment and digestion with the informative restriction enzyme. Data Analysis Descriptive statistical indexes, the Tajima’s D (Tajima 1989) and Fu’s FS (Fu 1997) neutrality tests, and the analysis of molecular variance (AMOVA) (Excoffier et al. 1992) were calculated using the Arlequin software, version 2.001 (Schneider et al. 2000). For the AMOVA analysis, we used the number of pairwise differences for the HVS-I sequence data and haplogroup frequencies for haplogroup data. We performed the AMOVA analyses either with all populations in a single group or divided into several groups, according to their geographic location or linguistic affiliation. For the geographic grouping, we divided populations into four regions: the Anatolian/Caucasus region (Anatolians and Caucasus populations), the Iranian plateau (Persians, Iranian Turks, Lurs, Iranian Kurds, Mazandarans, and Gilaks), the Indus Valley (Baluchi, Brahui, Parsi, Sindhi, Pakistani-Karachi, Pathans, Makrani, Hazara, and Gujarat) and Central Asia (Uzbeks, Turkmen, Kurds from Turkmenistan, Shugnan, Hunza Burusho, and Kalash). For the linguistic division, we grouped populations according to their linguistic affiliation: Indo-Europeans (Persians, Lurs, Iranian Kurds, Mazandarans, Gilaks, Baluchi, Parsi, Sindhi, Pakistani-Karachi, Pathans, Makrani, Hazara, Shugnan, Kalash, and Gujarat), Altaic (Anatolian, Iranian Turks, Turkmen, and Uzbek), Dravidian (Brahui), Caucasian (Caucasus), and language isolates (Burusho). The population genetic structure was also explored through the spatial analysis of molecular variance ( SAMOVA) approach (Dupanloup et al. 2002), which defines groups of populations that are geographically homogeneous and maximally differentiated from each other. This method is based on a simulated annealing procedure that aims at maximizing the proportion of total genetic variance due to differences between groups of populations without any a priori definition of groups of populations that is based on geographic or linguistic features. The SAMOVA analyses were based on HVS-I sequence data and were done using the SAMOVA 1.0 software. Median-joining networks (Bandelt et al. 1995, 1999) were constructed by hand and confirmed by the Network program (A. Röhl; Shareware Phylogenetic Network Software Web site). For network construction of some specific lineages, sequence data from other populations were taken from the literature. From the Anatolia/Caucasus region, we included Armenians (AM), Azerbaijanis (AZ), Turks (TR), and Kurds (KR) from Richards et al. ( 2000); Turks (TC) from Calafell et al. ( 1996); Turks (TT) from Tambets et al. ( 2000); and Kurds (KC) from Comas et al. ( 2000). From the Middle East/Arabian Peninsula, we included Iraqis (IQ), Syrians (SY), Yemenites (YM), Palestinians (PL), and Druze (DZ) from Richards et al. ( 2000); individuals from Dubai (DB) from A.T. (unpublished data); and Egyptians (EG) from Krings et al. ( 1999). From Pakistan/India, we included Pakistanis (PK) and Indians from Andhra Pradesh (AP), Gujarat (GK), Haryana (HY), Kashmir (KS), Maharashtra (MH), Punjab (PN), Rajasthan (RJ), Uttar Pradesh (UP), and Tamil Nadu (TN) from Kivisild et al. ( 1999a); and Indians (IN) from Mountain et al. ( 1995). From Central Asia, we included Kirghiz (KG), Uighur (UG), and Kazakh (KZ) samples from Comas et al. ( 1998). From western Eurasia, we included Basques (BS), Sicilians (SC), Bulgarians (BL), and Italians from Tuscany (TS) from Richards et al. ( 2000); Russians (RS) from Malyarchuk et al. ( 2002); Mansi (MN) from Derbeneva et al. ( 2002); and Sardinians (SD) from Di Rienzo and Wilson ( 1991). We also included Chinese (CH) from Yao et al. ( 2002). The time to the most recent common ancestor of some clades and their SEs were calculated by means of the estimator ρ, the averaged distance to a specified founder haplotype, and were determined as described by Forster et al. ( 1996) and Saillard et al. ( 2000). Time estimates were also calculated, using the Network program. Principal-components (PC) analyses were performed using SPSS version 10.0.7 software, with basal mtDNA haplogroup frequencies as input vectors. Admixture proportions (mY) and their SEs were calculated, using information from all haplogroups, by means of the program Admix 2.0 (Dupanloup and Bertorelle 2001), on the basis of 1,000 bootstraps. The parental populations used for the analysis were Iranian populations and Gujarati for the Parsi population, and Pakistani populations (excluding the Makrani) and a geographically dispersed set of sub-Saharan African samples (Krings et al. 1999; Brakez et al. 2001; Brehm et al. 2002, Salas et al. 2002) for the Makrani population. The Topology of the Southwest and Central Asian mtDNA Tree The complete high-resolution RFLP haplotypes and HVS-I sequence data of the 208 individuals from the Caucasus region, Anatolia, and southeastern Pakistan and the detailed haplogroup classification and HVS-I sequence data of the extended database of 702 individuals are reported in the online-only material. The phylogenetic relationships of the 51 different named haplogroups observed in the 910 samples, along with the diagnostic sites used for the mtDNA haplogroup classification, are shown in . The vast majority of the mtDNAs clustered into macrohaplogroups M, N, and R, but a limited number were found to belong to the sub-Saharan haplogroups L1a, L2a, L3b, and L3d. Five haplogroups, N1d, HV2, U9, R5, and R6, are defined here for the first time, whereas others (U2a, U2b, and U2c) represent newly identified subclades. Moreover, for some previously known haplogroups (R2 and U8b), we detected diagnostic coding-region markers that allow a better definition of the haplogroup topology within the tree. Macrohaplogroup N in southwestern and Central Asia is partitioned into several branches: N1 (which also encompasses haplogroup I), N9a, A, W, X, and R. Within the N trunk, the new haplogroup N1d stems from the node of N1 and is defined by three characteristic RFLP sites (−951 MboI, −5003 DdeI, −8616 MboI) and two HVS-I transitions (nps 16301 and 16356). The internal topology of superhaplogroup R has also been improved. The novel lineage R5 is defined by −8592 MboI and transitions at nps 16266 and 16304, whereas the new other haplogroup, R6, is characterized by −12282 AluI and transitions at nps 16129 and 16362. Moreover, the R2 mtDNAs, previously recognizable only by the HVS-I transition at np 16071, are now identifiable through the diagnostic coding-region motif +4216 NlaIII, +4769 AluI, −14304 AluI. It is worth noting that +4216 NlaIII is also one defining mutation of the lineage-cluster J-T (). However, the comparison of entire mtDNA sequences belonging to both R2 and J-T (A.T., unpublished data) indicates that +4216 NlaIII has indeed occurred independently on the two branches of the phylogeny. An improvement of the classification within HV was also obtained. A haplogroup, named HV2, was found to bear the HVS-I transition at np 16217 and most likely is also characterized by +9336 RsaI, since 16 of the 20 mtDNAs with the HVS-I 16217 mutation harbor this coding region site. This lineage corresponds to an internal node of HV that Tambets et al. ( 2000) tentatively identified as P*. The improvement of the haplogroup U subclassification was even more extensive. This major western Eurasian haplogroup is also found in the Middle East and India and, at lower frequencies, in northern and eastern Africa, but the frequency distributions of its subclades appear to differ considerably among geographical regions (Kivisild et al. 1999 a, 2003; Macaulay et al. 1999; Richards et al. 2000). Subclade U2 (characterized by the rather variable HVS-I transition at np 16051) was previously subdivided into two branches, the “European” U2e characterized by a further HVS-I transversion at np 16129, and the “Indian” U2i lacking such a transversion (Kivisild et al. 1999a). We show that U2e also harbors the distinguishing RFLP motif +13730 HinfI, +15907 RsaI, and U2i is indeed made up of three clusters, here termed “U2a,” “U2b,” and “U2c.” U2a is characterized by the rare and stable HVS-I transversion 16206C, U2b is defined by the diagnostic site −15047 HaeIII, and U2c harbors the RFLP motif +5789 TaqI, +8020 MboI/+8022 TaqI, −15060 MboI (). A subset of U that was already known but is now better defined is U8b. Finnilä et al. ( 2001) observed that, on the basis of the shared transition at np 9698, haplogroup U8 formed a sister clade with haplogroup K. Our data reveal that at least a subset of U8, here termed “U8b,” is also characterized by −9052 HaeII, which is indeed also the diagnostic marker of haplogroup K. This observation strengthens the sister haplogroup status of U8b and K. Finally, our data reveal the presence of a new—and rare—previously undefined subgroup of U, termed “U9,” that is characterized by −6383 HaeIII. This haplogroup does not correspond to the U9 of Herrnstadt et al. ( 2002), which, in reality, corresponds to a subset of the previously defined U3. The comparison of the RFLP and HVS-I data obtained from our data set identified some pitfalls when classifying the internal lineages within some haplogroups (e.g., J and M) on the basis of the HVS-I sequence data alone. Thus, we classified all our J mtDNAs according only to their differential RFLP status (), and, since an accurate RFLP classification of the South Asian branches of haplogroup M remains to be defined, we adopted a conservative classification and merged all South Asian M mtDNAs into M*. Haplogroup Profile Distribution The haplogroup repertoire present in the study populations is shaped mainly by the presence of lineages that can be attributed to eastern Eurasia, South Asia, and western Eurasia (; table 2). Sub-Saharan African lineages, represented by haplogroups L1, L2, and L3A and their internal derivatives, are virtually absent from all populations analyzed except the Makrani from southern Pakistan, among whom they reach high frequencies (39%). | Table 2mtDNA Haplogroup and Subcluster Frequencies for the 23 Study Populations[Note] |
The eastern Eurasian component is represented by haplogroups A, B, F, and N9a, all of which belong to the major N trunk, and the East Asian branches of macrohaplogroup M, such as the C, D, G, and Z haplogroups. The latter lineages are particularly widespread among northern and East Asians and, to a lesser extent, Central Asians (Torroni et al. 1993, 1994a, 1994b; Kivisild et al. 2002; Yao et al. 2002; Kong et al. 2003). The eastern Eurasian lineage cluster shows, with some exceptions, a decreasing gradient of frequencies towards the west (; table 2). The highest frequencies of these branches were found among the Central Asian populations, reaching their maximum in the Turkmen and Uzbeks (37% and 31%, respectively). Interestingly, Kurds from Turkmenistan showed the lowest frequencies of eastern Eurasian lineages (9%) in Central Asia, in sharp contrast to the local Turkmen population. These eastern Eurasian–specific lineages were absent—or at very low frequencies—in populations from the Anatolian/Caucasus region, the Iranian plateau, and the Indus Valley, with one exception: the Hazaras from northern Pakistan, among whom they reach 35%. The South Asian influence is mainly represented by the nodal type of macrohaplogroup M (M*) and the three sister clades U2a, U2b, and U2c. The M* haplogroup is absent or infrequent in all the populations west of the Indus Valley and is present at low frequencies in our Central Asian populations (<12%). Conversely, it is present at high frequencies (30%–55%) in populations living in the southern coasts of Pakistan and northwestern India. The three sister clades U2a, U2b, and U2c show a similar geographic pattern to that of haplogroup M*, although their distribution is somewhat more restricted to the Indo-Pakistani region. Also, N1d and HV2 and some lineages within paragroup R* are at higher frequencies in populations located east of the Iranian plateau, and this will be discussed in more detail below. The proportion of western Eurasian lineages (HV, pre-HV, N1, J-T, U-K, I, W, and X) showed the opposite pattern of that exhibited by eastern Eurasian lineages (; table 2). They exhibit their highest frequencies in the Anatolian/Caucasus and Iranian regions and their prevalence decreases eastwards. Despite this decreasing frequency cline towards the East, they are still present at relatively high frequencies in the Indus Valley and Central Asia. Indeed, the western Eurasian presence in the Kalash population reaches a frequency of 100%, the most prevalent haplogroups being U4, (pre-HV)1, U2e, and J2. Phylogeography of Specific Haplogroups The phylogeography of several haplogroups suggests that they are either autochthonous to the southwestern Asian corridor or that at least they underwent a major expansion in this region. Among these lineages, haplogroup U7 presents the most widespread distribution. U7 is virtually absent in western and eastern European populations and is present at low frequencies (2%–4%) in the Near East, the Caucasus region, Central Asia, and the Indian subcontinent (Kivisild et al. 1999a, 2003; Macaulay et al. 1999; Richards et al. 2000; Tambets et al. 2000; Malyarchuk and Derenko 2001; Malyarchuk et al. 2002). Our data show that this haplogroup is present in most of the populations linking the Near East with Central and South Asia, reaching its highest frequencies in some Iranian and Indus Valley populations ( table 2), in agreement with recent data reporting a frequency of 9% in a composite Iranian sample (Kivisild et al. 2003). shows the median-joining network for this haplogroup. The topology of the network shows that this haplogroup is divided into two major well-defined star-like subclades separated by a transition at np 16309. The time-depth calculated for paragroup U7* (without 16309) is 35,100 ± 8,500 years, whereas that for U7a (with 16309) is 22,500 ± 5,400 years. These coalescence times support the idea that the 16318T mutation is indeed the ancestral feature of U7. The overall coalescence time calculated for U7 is 38,200 ± 13,900 years. The phylogeography of haplogroups HV2 and R2 resembles that of U7 but has a more restricted geographic distribution. Both haplogroups are concentrated in southern Pakistan and India, with some overflow into adjacent areas, including the Near East/Caucasus region, the Iranian plateau, the Arabian Peninsula, and Central Asia, where most of the derived types are observed (a and ). The coalescence times were estimated at 27,700 ± 9600 years for HV2 and 31,200 ± 8200 years for R2. The distribution of the three sister clades within haplogroup U2 (U2a, U2b, and U2c) is essentially restricted to the Indo-Pakistani regions (). They have not been observed in Europe and the Near East and, according to our data, they are absent in the Iranian plateau and Central Asian populations. They are, however, common in populations from Pakistan and India. The estimated coalescence times for these haplogroups are: 45,700 ± 14,400 years for U2a, 35,900 ± 9000 years for U2b, and 45,200 ± 10,400 years for U2c. The R5 lineage showed a similar distribution to the U2 subclades (d), but its root types are more concentrated in the Indus Valley region, with the derivatives in central and southern India. The estimated time depth of this lineage is 51,800 ± 13,800 years. Finally, three small haplogroups (R6, N1d, and U9) have been observed so far only in south Pakistan. R6 was found in three individuals from the mixed sample from Karachi; N1d in one Baluchi, one Brahui, one Makrani, and three individuals from Karachi; and U9 in three Makrani, one Pathan, and one individual from Karachi. Population Diversity and Demographic Regimes HVS-I sequences have also been used to gain information on the internal population diversity ( table 3). Most populations showed similar sequence diversity values, with the Kalash showing the lowest (0.830) and the Indian Gujarati the highest (0.998). The low diversity exhibited by the Kalash population is also evident in the low mean number of pairwise differences (3.857). This is the lowest value of all the populations studied, which otherwise ranged from 4.399 in the Baluchi and the Caucasus populations to 6.633 in the Makrani. As shown in table 3, most populations yielded significantly negative values for both Tajima’s D and Fu’s FS neutrality tests. The only exceptions were the Mazandarians, the Kurds from Turkmenistan, and the Kalash. The former two groups exhibited significantly negative Fu’s FS values and unimodal mismatch distributions (not shown) but the Tajima’s D statistic was not significantly different from 0. This contrasting pattern may be the result of mutation rate heterogeneity along the HVS-I region; this effect has been shown to confound the signature of population expansion in Tajima’s test, leading to higher D values (Aris-Brosou and Excoffier 1996). For the Kalash population, both neutrality tests gave nonsignificantly negative values ( table 3), and the mismatch distribution was unequivocally multimodal (data not shown). | Table 3Diversity Indices and Neutrality Tests for the Study Populations |
Population Relationships The basal mtDNA haplogroup frequencies of the 23 populations were used as input vectors to perform a PC analysis. shows the PC plot for the first two principal components, which account for 43% and 12% of the total variation respectively. Leaving aside the two outliers, the Kalash and the Makrani, geographic grouping of populations are apparent in the diagram. The first PC (PC1) mainly reveals a west-to-east cline by separating a group of closely related populations from the Iranian plateau from those inhabiting the Indus Valley and northwest India. The Central Asian Uzbeks, Turkmen, and Shugnan tend to be closer to populations from the Anatolian/Caucasus/Iranian regions, rather than to Indus Valley populations, as a consequence of the high prevalence of western Eurasian lineages observed in these populations. The Hazara from Pakistan shows an intermediate position between populations from the Indus Valley and those from Central Asia. PC2 essentially displays the outlier genetic position of the Makrani and the Kalash populations, who are separated from the rest of populations of the Iranian plateau and the Indus Valley. Population Genetic Structure: AMOVA and SAMOVA Analyses We investigated how the proportion of variance, based on haplogroup (main lineages) and haplotype (HVS-I sequences) frequencies, was distributed in a hierarchical mode by an AMOVA analysis (Excoffier et al. 1992). When the 23 populations were treated as a single group, populations turned out to show overall differentiation: the FST value for the haplogroup data was 0.067 ( P<.001) and the ![[var phi]](/corehtml/pmc/pmcents/x03C6.gif) ST for the sequence data was 0.032 ( P<.001). The fraction of genetic variance due to differences among linguistic groups (see the “ Materials and Methods” section) was not statistically different from 0, independently of the genetic system used (i.e., haplogroup or sequence data), indicating that genetic variance within any population or among populations within groups was larger than that between groups and, therefore, that the division by linguistic affiliation is not reflected in mtDNA variation. Finally, when populations were regrouped into four geographic groups (see the “ Materials and Methods” section), a small but significant differentiation among groups was detected ( FCT=0.043 and P<.001 for haplogroup data and CT=0.016 and P<.001 for HVS-I data). To investigate in greater detail the genetic structure of the populations and the amount of genetic variation due to differences among population groups, we applied the SAMOVA algorithm (Dupanloup et al. 2002), on the basis of HVS-I data, searching for two, three, and four groups. The inclusion of the Kalash population, which is among the most differentiated ( table 3; ), gave inconsistent results (data not shown), so this population was excluded from further analyses. A search for two significantly differentiated population clusters revealed one group consisting of all populations from the Anatolian/Caucasus region and the Iranian plateau (including the Kurds from Turkmenistan), and a second group made up of populations from the Indus Valley and Central Asia ( FCT=0.021; P<.001). In the three-group search, the previous two remained unchanged, and a third group, represented by the Hazara, emerged from the analysis ( FCT=0.021; P<.001). Finally, the search for four groups revealed the Makrani of southern Pakistan as the fourth most differentiated group ( FCT=0.022; P<.001). This study provides the first comprehensive survey of mtDNA variation in a part of the world that was among the first regions to be inhabited after the “out of Africa” exit, and has subsequently experienced numerous waves of migration during the last 50,000 years. We now discuss the events, both ancient and modern, that are likely to have led to the current mtDNA distribution, and compare the mtDNA data with that from other loci, particularly the Y chromosome. The mtDNA Landscape of the Southwestern Asian Corridor A simple pattern underlies the mtDNA variation in this region: a west-to-east divide with a sharp boundary. Populations located west of the Indus basin, including those from Iran, Anatolia and the Caucasus, exhibit a common mtDNA lineage composition, consisting mainly of western Eurasian lineages, with a very limited contribution from South Asia and eastern Eurasia (). Indeed, the different Iranian populations show a striking degree of homogeneity. This is revealed not only by the nonsignificant FST values and the PC plot () but also by the SAMOVA results, in which a significant genetic barrier separates populations west of Pakistan from those east and north of the Indus Valley (results not shown). These observations suggest either a common origin of modern Iranian populations and/or extensive levels of gene flow amongst them. There is a virtual absence of both common South Asian lineages (M*, U2a, U2b, and U2c) and the more autochthonous U9, R*, R2, R5, R6, N1d, and HV2 lineages in the Anatolian/Caucasus region and Iranian plateau, whereas these lineages make up >50% of the haplogroup profile in the adjacent Indus Valley. Most of these lineages appear to be restricted to the eastern part of the corridor (). Whereas geographical clustering and the coalescent age of U7 (~38,000 YBP; see table 2 and ) suggest that it is the most widespread local lineage of Pleistocene origin connecting the western and eastern extremes of the corridor, the Indus Valley and India show signals of an in situ differentiation of deep-rooting lineages (HV2, R2, R5, U2a, U2b, and U2c), the distribution of which appears to be limited to this region (figs. and ). All these lineages have high time depths (28,000–52,000 YBP), similar to haplogroup M* (32,000–53,000) in the region (Kivisild et al. 1999b; Quintana-Murci et al. 1999; Roychoudhury et al. 2001). Notably, haplogroup M* has also not penetrated west of the Indus Valley, although it is present at high frequencies in south Pakistani and Indian populations. Thus, the distribution and ages of these lineages suggest that they are the legacy of the first inhabitants of the southwestern Asian region who underwent important expansions during the Paleolithic period. It is interesting that the newly identified haplogroup U9, found in the Indus Valley, has also been observed in Ethiopia (A.T., unpublished data), supporting the link between East Africa and the southwestern and southern coasts of Asia (Kivisild et al. 1999a; Quintana-Murci et al. 1999). The absence of these lineages west of Pakistan may be due either to limited gene flow from the Indus basin or to important demographic expansions in the Fertile Crescent (including its eastern lobe, represented by present-day Iran), associated with a substantial increase in frequency and diversity of western Eurasian lineages. Geographical features such as the Dash-e Kavir and Dasht-e Lut deserts in Iran could have acted as significant barriers to gene flow, and this is consistent with Y-chromosomal data (e.g., the distribution of lineage R-M17) from these regions (Quintana-Murci et al. 2001; Wells et al. 2001; Qamar et al. 2002). Gene flow from the Fertile Crescent to India has, however, been more common than that from east to west (). Eastern populations within the corridor mostly exhibit a rich variety of west Eurasian lineages at high frequencies (26%–57%), with a gradient towards the Indian subcontinent, with lower frequencies in caste (<10%) and tribal groups (<1%) (Kivisild et al. 1999a; 2003; Bamshad et al. 2001). The substantial western Eurasian presence in the Indus Valley and northwestern India may have been the result of repeated gene flow received from further west at different periods, including the first Paleolithic arrivals to the corridor region from the Middle East and subsequent dispersals associated with Neolithic urban civilizations, such as Mesopotamians and Elamites, who may have carried farming towards the eastern part of the corridor. The exact mode and tempo in which the different western Eurasian lineages reached the Indo-Gangetic plains remains to be elucidated. However, it appears that J, T1, and U3, which have been proposed as the main marker haplogroups for the Neolithic diffusion of agriculture from the Middle East to the West (Richards et al. 2000, 2002), did not play an equivalent role in the diffusion of farming toward the East. The eastern Eurasian contribution to the west, in contrast, is negligible (), in agreement with HVS-I sequence data in Turkish populations (Calafell et al. 1996; Comas et al. 1996, 1998). This pattern may seem surprising in view of the historically documented repeated waves of Altaic-speaking nomads (e.g., Turks, Huns, and Mongols) starting in the 3rd century a.d., who traveled from east to west, imposing Altaic languages in some western regions (e.g., Anatolia and Azerbaijan), probably through an elite-dominance process. In this context, it is interesting that five of the seven individuals belonging to eastern Eurasian lineages west of the Indus Valley are Turkic-speaking. The east-to-west differences in the genetic contribution of the eastern invaders along the corridor may be due to the existing population densities in these regions at the time of arrival. The genetic contribution of the newcomers was strong in the sparsely populated arid lands of eastern Central Asia. Conversely, the eastern influence in western territories was much lower, since the invading eastern nomads probably found higher population densities. Our results reinforce recent Y-chromosome data from Central Asia, which show that the paternal genetic contribution of the eastern invaders is barely detectable west of Uzbekistan (Zerjal et al. 2002). The Effects of Admixture and Drift: Demographic Events in Central Asia Central Asians exhibit high frequencies of East Asian lineages, which are otherwise virtually absent in populations from the Indo-Gangetic region and westwards, concomitantly with a high prevalence of lineages of western Eurasian origin (). Two explanations have been put forward: Central Asians could represent an early incubator of Eurasian variation, or their current genetic diversity could result from later admixture between western and eastern Eurasian populations. Y-chromosome data have been interpreted as indicating that Central Asian populations are amongst the oldest on the continent and were the source of at least three major migration events (Wells et al. 2001) but were also a receiver of migrations (Zerjal et al. 2002). mtDNA studies (Comas et al. 1998) based on HVS-I variation in four populations of Central Asia found that they contained both European and East Asian motifs. This was interpreted as evidence for admixture between Europeans and East Asians, a conclusion that is substantiated by our more thorough analysis. Indeed, if Central Asia had been the source of modern Eurasian diversity, one would expect to observe (i) substantial overlap between present-day western and eastern Eurasian haplogroups and (ii) extensive divergence between the HVS-I types found in Central Asia and those observed in western and eastern Eurasia. This is not the case. Our data, which take into consideration coding-region information and provide a more clear-cut phylogeography, show a major demarcation in the Eurasian landscape between European and East Asian mtDNA lineages within both the R and N branches, and with M playing virtually no role in western Eurasia. Moreover, most Central Asian HVS-I types match sequences that are observed today in either western or eastern Eurasians, suggesting recent arrival in Central Asia. The complexity of the peopling of the region is well illustrated by the Kalash population from the Hindu Kush valleys, where western Eurasian mtDNAs reach fixation with no detectable East or South Asian lineages ( and table 2). Their outlying genetic position is seen in all analyses ( table 3 and ). Moreover, although this population is composed of western Eurasian lineages, the most prevalent (i.e., U4, (pre-HV)1, U2e, and J2) are rare or absent in the surrounding populations and usually characterize populations from Eastern Europe, the Middle East, and the Caucasus (Macaulay et al. 1999; Richards et al. 2000; Tambets et al. 2000). Also, the internal HVS-I sequence diversity within each of these haplogroups was surprisingly low: 12 of 15 samples belonging to U4 were associated with the motif 16134–16356, all (pre-HV)1 samples harbored 16362, all U2e samples were characterized by the motif 16051-16129C-16154-16248-16362, and all J2 mtDNAs showed the motif 16069-16126-16193-16274-16278. These sequence motifs are almost entirely restricted to the Kalash community, except for those associated with U4. All these observations bear witness to the strong effects of genetic drift on the Kalash population. This distinctive demographic scenario is supported by the nonsignificantly negative values of Tajima’s D and Fu’s FS neutrality tests ( table 3), which reject the hypothesis of population growth, the unambiguous multimodal mismatch distribution (not shown), and the small census size of the population, 3,000–6,000. It has been suggested that this population descends from Greeks or from Slavic peoples, and they claim descent from a place called Tsyam, possibly in Syria (Robertson 1896; Decker 1992). The strong effects of drift and the small population size make genetic inference about the geographic origin of the Kalash difficult. However, a western Eurasian origin for this population is likely, in view of their maternal lineages, which can ultimately be traced back to the Middle East. Correlation of Genes and Languages in the Southwestern Asian Corridor The study of the mtDNA pool of present-day populations living in the southwest and Central Asian corridor shows that the linguistic differences in these regions (i.e., mainly Indo-European vs. Altaic) are not reflected in the patterns of mtDNA diversity. However, there are two linguistic outliers that merit further consideration: the Hunza Burusho and the Brahui. The Hunzas live mainly in the remote Hunza Valley of northern Pakistan and speak Burushaski, a language isolate of uncertain origin. Our analysis shows that the Hunza mtDNAs, like the Y haplotypes (Qamar et al. 2002), are shared with neighboring populations, particularly with southern Pakistanis (see PC plot in ). This genetic pattern could have been established before the linguistic differentiation took place, or there could have been substantial gene flow with neighboring populations. In any case, no distinctive genetic signature accompanies the linguistic and geographic isolation of the Hunza Burusho population, in agreement with recent data based on 182 autosomal microsatellites (Ayub et al. 2003). The second linguistic outlier is the Brahui population, located in central Baluchistan, which represents a Dravidian-speaking enclave outside India. Historical records indicate that the Brahui are descendants of Turko-Iranian tribes from west Asia (Hughes-Buller 1991). Today, Dravidian languages are essentially restricted to south India and Sri Lanka, but the proto-Elamo-Dravidian hypothesis (McAlpin 1974, 1981) proposes that they originated in the Iranian province of Elam and were once spoken over a much larger area, including Iran, Pakistan, Afghanistan, and all India. The Brahui population is characterized by high prevalences (55%) of western Eurasian mtDNAs and the lowest frequency in the region (21%) of haplogroup M*, which otherwise is common (~60%) among Dravidian-speaking Indian populations. As shown in the PC1 (), the Brahui lie in an intermediate position between Iranian and Indus Valley populations, far from the Gujaratis and even farther from Dravidian-speaking Indian groups (results not shown). These observations exclude the possibility that the Dravidian presence in Baluchistan has resulted from recent incursions of Dravidian speakers from India and show that the maternal gene pool of the Brahui is similar to that of Indo-Iranian speakers from the southwestern Asian corridor. Although the present Brahui population could represent an ancient Indian Dravidian-speaking population relocated to Pakistan, where they admixed with local populations, no historical record supports this hypothesis. Thus, this suggests that they are the last northern survivors of a larger Dravidian-speaking region predating the arrival of Indo-Iranian speakers, thus reinforcing the proto-Elamo-Dravidian hypothesis (McAlpin 1974, 1981). Traces of Recent and Sexually Asymmetrical Events The phylogeographical cross-comparison of mtDNA and Y-chromosomal data is very useful for tracing differential male and female histories. Some populations studied here (Iranian, Pakistani, and Central Asian) have been analyzed previously for Y-chromosomal variation (Quintana-Murci et al. 2001; Qamar et al. 2002; Zerjal et al. 2002). In most cases, mtDNA variation is in good agreement with the Y-chromosomal data, suggesting that the patterns reflect general population processes. A good, although surprising, example of concordance between the two systems is the Hazara, who claim to be the direct male-line descendants of Genghis Khan’s army. The presence and time depth of the Y-chromosomal haplogroup C* (xC3c) in the Hazara, along with its absence from neighboring populations, has been interpreted as the genetic legacy of Genghis Khan and his male relatives (Qamar et al. 2002; Zerjal et al. 2003). Our results indicate that the Hazara are also characterized by very high frequencies of eastern Eurasian mtDNAs (35%, table 2, ), which are virtually absent from bordering populations, suggesting that the male descendants of Genghis Khan, or other Mongols, were accompanied by women of East Asian ancestry. In contrast to the parallelism between mtDNA and Y-chromosomal data in most populations, the Parsis and the Makrani both show a sharp contrast between these loci. The Parsis live in southeastern Pakistan, and historical records indicate an Iranian origin (Nanavutty 1997). These followers of the prophet Zoroaster started their migration from Iran in the 7th century a.d., settling in the northwestern Indian province of Gujarat around 900 a.d. and eventually moving to Mumbai in India and Karachi in Pakistan. Y-chromosome data show that they resemble Iranian populations rather than their neighbors in Pakistan: an admixture estimate of 100% from Iran was obtained (Qamar et al. 2002), supporting the historical records. However, when the Parsi mtDNA pool was compared with those of the Iranians and Gujaratis (their putative parental populations), a strong contrast with the Y-chromosomal data emerged. About 60% of their maternal gene pool belongs to South Asian haplogroups, which make up only 7% of the combined Iranian sample ( table 2). The very high frequency of haplogroup M among the Parsis (55%), similar to those of Indian populations and much higher than that of the combined Iranian sample (1.7%), highlights their close affinities with India (). Our results lead to an admixture estimate of 100% from Gujarat and provide a strong contrast between the maternal and paternal components of this population. Although the small population size of the Parsis (a few thousand) may have distorted haplogroup frequencies in this population, diversity of both Y-chromosome and mtDNA lineages remains high, making a strong drift effect unlikely. Our results therefore support a male-mediated migration of the ancestors of the present-day Parsi population from Iran to India, where they admixed with local females, or directional mating in Gujarat between Iranian males and local women, leading ultimately to the loss of mtDNAs of Iranian origin. Another example of an unequal sex-specific contribution is seen in the so-called “Negroid” Makrani of Baluchistan. This population lives in the Makran coastal region and shows distinct African physical traits (Sultana 1995). We observed a high presence (39%) of lineages L3d, L3b, L2a, and L1a, generally restricted to sub-Saharan African populations (Chen et al. 1995, 2000; Salas et al. 2002) and otherwise present in only 4 of the remaining 877 individuals examined. The presence of African mtDNAs among the Makrani seems to be of recent origin, since the Makrani haplotypes are identical to those observed in modern sub-Saharan African populations (Salas et al. 2002), particularly in Bantu-speaking populations from Mozambique. Indeed, all but one of the Makrani L1, L2, and L3A types matched Mozambique sequences, and these were the most frequent haplotypes in the Mozambique samples (L1a2, L2a1a, and L2a1b) (Pereira et al. 2001; Salas et al. 2002). Our results contrast with the Makrani Y-chromosome profile, which is similar to that of other Pakistani populations and is dominated by western Eurasian lineages (Qamar et al. 2002). The sub-Saharan African male-specific contribution, represented primarily by Hg E-M2, occurred at only 9% in the Makrani and is also present in neighboring populations, although at a lower prevalence (2%–4%). We estimated the maternal and paternal contributions of sub-Saharan Africans to the current Makrani gene pool, using information from all haplogroups, at 12% (±7%) for the Y chromosome and 40% (±9%) for the mtDNA. These findings must be interpreted in the light of known historical data. Forced migration from Africa began in the 7th century and increased considerably during the Omani Empire. The latter formed a strong slave-trade connection between the Makran port of Gwadar, the principal ports of Oman, and ports located in East Africa, including Mozambique (Clarence-Smith 1989; Sultana 1995). In the 16th and 17th centuries, the Portuguese also traded between Mozambique and southwestern Asia. The African component in the Makrani community may therefore represent the genetic legacy of this slave trade. Whereas the Atlantic slave trade dealt mainly with male labor, the East African slave trade seemingly favored females over males (Lovejoy 2000). Slave women were mainly domestics and/or concubines, and children fathered by the master were freed. In addition, strong cultural barriers hindered male slaves from fathering children, a situation exacerbated by the proportion of slaves imported as eunuchs (Lovejoy 2000). As a consequence of these practices, the contribution of paternal African genes to the population is expected to be low. Indeed, the contrast between male and female African contributions observed among the Makrani strongly supports historical records of a female sex bias during the East African slave trade. Other factors, such as asymmetrical mating patterns between African women and autochthonous males during the process of genetic admixture, and/or unequal reproductive success among Makrani males, might have accelerated the loss of African Y chromosomes from the population. In this context, a similar pattern has been reported recently in the Yemeni Hadramawt population (Richards et al. 2003), geographically adjacent to East Africa, where the African maternal contribution has also been interpreted as the result of the East African slave trade. Our data not only confirm a female-biased slave trade towards the East but also show that this pattern, which includes differential mating patterns between the sexes, extended to the eastern limits of the East African slave trade. Our analysis of mtDNAs from the southwestern and Central Asian corridor shows that the highest variation is observed in populations located in the Indus Valley and Central Asia, highlighting this region as the place where western Eurasian lineages meet both the South Asian and eastern Eurasian genetic strata, respectively. The amalgamation of different genetic components in this area may have resulted from the successive and continuous waves of migration from diverse geographical sources at different time periods, from the early human settlements in the region after the “out of Africa” dispersal to migrations associated with the diffusion of new technologies, such as farming and/or pastoral nomadism, and accompanied by new languages, like the incursions of Indo-Iranian speakers from the northwest. In addition, the Indo-Gangetic region is characterized by the presence of autochthonous genetic footprints of Pleistocene origin and traces of recent historical events, such as the East African slave trade. This extraordinarily rich and heterogeneous genetic portrait testifies to the numerous and complex movements in the region and evinces more subtle demographic episodes in some populations, including founder effects and sexually asymmetrical events associated with differential migration patterns between males and females. We warmly acknowledge Hans-Jürgen Bandelt, for stimulating remarks and quality check of the data; Francesca Luca, for help in data analysis; and two anonymous reviewers, for helpful and constructive criticisms. This work was supported by CNRS and a North Atlantic Treaty Organization collaborative linkage grant (LST.CLG.977507) (to L.Q.-M.). Financial support was also provided by The Wellcome Trust (to C.T.-S. and S.Q.M.), the Italian Ministry of the University (Progetti Ricerca Interesse Nazionale 2001, 2002, 2003) (to A.T., R.S., and A.C.), Progetto CNR-MIUR Genomica Funzionale-Legge 449/97 (to A.T.), Fondo Investimenti Ricerca di Base 2001 (to A.T.), Fondo d’Ateneo per la Ricerca 2002 dell’Università di Pavia (to A.T.), Progetto Finalizzato C.N.R. “Beni Culturali”(to A.S.S.-B.), Grandi Progetti di Ateneo (to R.S.), and the Istituto Pasteur Fondazione Cenci Bolognetti (to R.S.). N.A.-Z. was supported by The International Center for Genetic Engineering and Biology (Trieste) and University of Pavia fellowships. Electronic-Database Information The URLs for data presented herein are as follows: Alves-Silva J, da Silva Santos M, Guimaraes PE, Ferreira AC, Bandelt HJ, Pena SD, Prado VF (2000) The ancestry of Brazilian mtDNA lineages. 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