Disuniting Uniformity: A Pied Cladistic Canvas of mtDNA Haplogroup H in Eurasia
http://www.100md.com
分子生物学进展 2004年第11期
* Department of Evolutionary Biology, Institute of Molecular and Cell Biology, University of Tartu and Estonian Biocentre, Tartu, Estonia; Genetics Laboratory, Institute of Biological Problems of the North, Russian Academy of Sciences, Magadan, Russia; Institute of Medical Genetics, Tomsk Research Center, Russian Academy of Medical Sciences, Tomsk, Russia; Institute for Anthropological Research, Zagreb, Croatia; || Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia; ? Kharkov Clinical Genetic and Prenatal Diagnostics Center, Kharkov, Ukraine; # Institute of Biochemistry and Genetics, Ufa Research Center, Russian Academy of Sciences, Ufa, Russia; ** Laboratoire d'Etude du Polymorphisme de l'ADN, Faculté de Médecine, Nantes, France; Institute of Molecular Biology and Biotechnology and Department of Basic Sciences, University of Athens School of Medicine, Athens, Greece; Department of Forensic Sciences and Toxicology, University of Crete School of Medicine, Heraklion, Greece; Laboratoire de Génétique Moléculaire, Institut Universitaire de Recherche Clinique IURC et CHU, Montpellier, France; |||| Department of Physiology, University of Kiel, Kiel, Germany; ?? Department of Biology, Faculty of Natural Sciences, Tirana University, Tirana, Albania; and ## Department of Medical Laboratory Sciences, Kuwait University, Sulaibikhat, Kuwait
E-mail: evall@ut.ee.
Abstract
It has been often stated that the overall pattern of human maternal lineages in Europe is largely uniform. Yet this uniformity may also result from an insufficient depth and width of the phylogenetic analysis, in particular of the predominant western Eurasian haplogroup (Hg) H that comprises nearly a half of the European mitochondrial DNA (mtDNA) pool. Making use of the coding sequence information from 267 mtDNA Hg H sequences, we have analyzed 830 mtDNA genomes, from 11 European, Near and Middle Eastern, Central Asian, and Altaian populations. In addition to the seven previously specified subhaplogroups, we define fifteen novel subclades of Hg H present in the extant human populations of western Eurasia. The refinement of the phylogenetic resolution has allowed us to resolve a large number of homoplasies in phylogenetic trees of Hg H based on the first hypervariable segment (HVS-I) of mtDNA. As many as 50 out of 125 polymorphic positions in HVS-I were found to be mutated in more than one subcluster of Hg H. The phylogeographic analysis revealed that sub-Hgs H1*, H1b, H1f, H2a, H3, H6a, H6b, and H8 demonstrate distinct phylogeographic patterns. The monophyletic subhaplogroups of Hg H provide means for further progress in the understanding of the (pre)historic movements of women in Eurasia and for the understanding of the present-day genetic diversity of western Eurasians in general.
Key Words: human mitochondrial DNA ? population genetics ? phylogeography
Introduction
The mitochondrial DNA (mtDNA) sequences of Europeans are sorted into ten major phylogenetic clades, or haplogroups, alphabetically named H, J, K, N1, T, U4, U5, V, X, and W (Torroni et al. 1994, 1996; Macaulay et al. 1999; Richards et al. 2000). Haplogroup (Hg) H alone constitutes about one half of the European mtDNA pool and, along with other aforementioned lineages, is widespread also in western Asia (Macaulay et al. 1999; Richards et al. 2000; Tambets et al. 2000; Kivisild et al. 2003), Central Asia (Comas et al. 1998; Metspalu et al. 1999), Siberia (Saillard et al. 2000a; Derbeneva et al. 2002a; Derenko et al. 2003), southern Asia (Passarino et al. 1996; Kivisild et al. 1999, 2003; Bamshad et al. 2001), and northern Africa (Corte-Real et al. 1996; Rando et al. 1998; Stevanovitch et al. 2004; fig. 1A). At least 267 Hg H mtDNA genomes have been sequenced (nearly) completely (Reid, Vernham, and Jacobs 1994; Rieder et al. 1998; Levin, Cheng, and Reeder 1999; Ingman et al. 2000; Finnil?, Lehtonen, and Majamaa 2001; Maca-Meyer et al. 2001; Herrnstadt et al. 2002, correction by Herrnstadt, Preston, and Howell 2003; Mishmar et al. 2003). Out of seven Hg H sub-Hgs defined so far, Hgs H1 and H2 (Finnil?, Lehtonen, and Majamaa 2001) along with Hgs H3 and H4 (Herrnstadt et al. 2002) and Hgs H5, H6, and H7 (Quintans et al. 2004) cover 74% of Finnish, 68% of U.S./U.K. and 77% of Galician Hg H sequences, respectively.
FIG. 1.— Spatial frequency distributions of haplogroup H (A) and its subhaplogroup H1b (B). H1b frequencies are given as percentages with respect to Hg H.
Attempts to classify Hg H lineages by first hypervariable segment (HVS-I) have been hindered by a frequent occurrence of mutations at fast-evolving nucleotide sites—so-called mutational hot-spots (Richards et al. 2000; Allard et al. 2002). Furthermore, HVS-I sequence information leaves a substantial fraction of Hg H genomes phylogenetically unresolved: on average one-third of the lineages share a haplotype identical with the Cambridge reference sequence (CRS; Anderson et al. 1981). Therefore, progress in the analysis of Hg H diversity, and, indeed, of the understanding of the phylogeography of western Eurasian maternal lineages, depends critically on the use of full genome information in mtDNA samples, representative in size and geography.
Materials and Methods
The phylogeny of 267 published Hg H mtDNA coding region sequences (Reid, Vernham, and Jacobs 1994; Rieder et al. 1998; Levin, Cheng, and Reeder 1999; Ingman et al. 2000; Finnil?, Lehtonen, and Majamaa 2001; Maca-Meyer et al. 2001; Herrnstadt et al. 2002; Mishmar et al. 2003) was analyzed (fig. S1 in the Supplementary Material online). Markers for nine subclades were selected for genotyping in 563 Hg H mtDNA samples of European, Near and Middle Eastern, Central Asian and Altaian origin. To find the relative mutation rates of particular nucleotide positions, the frequencies of phylogenetically independent mutations were calculated from 987 published mtDNA coding region sequences (Marzuki et al. 1991; Reid, Vernham, and Jacobs 1994; Arnason, Xu, and Gullberg 1996; Polyak et al. 1998; Rieder et al. 1998; Levin, Cheng, and Reeder 1999; Ingman et al. 2000; Finnil?, Lehtonen, and Majamaa 2001; Maca-Meyer et al. 2001; Torroni et al. 2001b; Derbeneva et al. 2002b; Herrnstadt et al. 2002; Kivisild et al. 2002 and references therein; Kong et al. 2003; Mishmar et al. 2003).
The samples were selected at random from nine populations: 50 Finno-Ugric speakers from the Volga-Ural region (10 Udmurts, 10 Mokshas, 16 Erzyas, 7 Permyak Komis, 7 Zyrian Komis); 50 Estonians; 165 Eastern Slavs (127 Russians, 38 Ukrainians from various districts of Russia and the Ukraine); 50 Slovaks; 50 French from southern France, Lyon, Low Normandy, and Poitiers; 50 individuals from the Balkans (17 Croats, 17 Albanians, 16 Greeks); 50 Turks; 50 individuals from the Near and the Middle East (10 Jordanians, 8 Lebanese, 7 Saudis, 12 Syrians, 13 Iranians); 48 individuals from Central Asia (17 Altaians, 11 Kirghiz, 3 Kazakhs, 11 Tajiks, 6 Uzbeks). Sixteen Russian and six Ukrainian HVS-I sequences have been published by Malyarchuk and Derenko (2001a), 33 Russian HVS-I and second hypervariable segment (HVS-II) sequences by Malyarchuk et al. (2002), and all of the Volga-Ural region mtDNA HVS-I sequences by Bermisheva et al. (2002). All the samples harbored a C at nucleotide position (np) 7028, which is diagnostic for Hg H and was inferred from the absence of the AluI restriction site at np 7025 (Torroni et al. 1994). All mutations and position numbers in this study are given with respect to Anderson et al. (1981) as revised by Andrews et al. (1999).
Four hundred forty-eight samples were screened for 14 polymorphisms in the mtDNA coding region and three in HVS-II in addition to HVS-I sequence variation. A hierarchical strategy was applied to 104 Russian and 11 Ukrainian mtDNAs (Appendix S2 in the Supplementary Material online). HVS-I variation for all of the samples was scored between nps 16024–16383. Nucleotide changes at positions 73, 951, 3010, 4336, 4452, 4769, 4793, 5004, 8448, 9066, 9380, 13101, 13759, and 16482 were determined by restriction fragment length polymorphisms (RFLPs; Appendixes S1 and S2). Nucleotide states at positions 239, 456, 3915, and 6776 were detected by direct sequencing or allele-specific polymerase chain reaction (PCR; Appendixes S1 and S2). Nucleotide positions 239 and 3915 were sequenced in samples having 16362C and/or lacking a Hin6I restriction site at np 9380 and/or having a DdeI site at np 16478. We note that the transition at np 239 nearly always occurs with the 16362C allele, as it was not found in Hg H variants with 16362T in 2,350 published HVS-II sequences (Hofmann et al. 1997; Parson et al. 1998; Dimo-Simonin et al. 2000; Malyarchuk et al. 2003; Vanecek, Vorel, and Sip 2004; Pereira, Cunha, and Amorim 2004). Credible regions of the obtained haplogroup frequencies were computed with the Sampling program kindly provided by Vincent Macaulay.
The phylogeny of the samples was studied by the construction of a reduced median network (fig. 2A). In the network analysis 479 samples were included (see Appendix S1), including the 31 Finnish sequences taken from Finnil?, Lehtonen, and Majamaa (2001), while 115 Eastern Slav mtDNAs, which were analyzed hierarchically (see Appendix S2), have not been included. The reduced median network (Bandelt et al. 1995; rho set at 2) was constructed with the Network 4.0.0.0. program (Fluxus Technology Ltd., Clare, Suffolk, UK, http://www.fluxus-engineering.com) followed by a median joining algorithm (Bandelt, Forster, and R?hl 1999; epsilon set at 0), as explained at the Fluxus-Engineering Web site. Nucleotide positions were divided into three classes of transition rates—fast (16093, 16129, 16189, 16304, 16311, and 16362), intermediate (16172, 16209, 16278, 16293), and slow (the remainder of the positions between 16024 and 16383)—and assigned class weights 1, 2, and 4, respectively. Transversions and coding region mutations were weighted 8.
FIG. 2.— Backbone of the phylogenetic tree of mtDNA haplogroup H subclades studied here. Nonsynonymous (#) and synonymous ('') mutations indicated. Mutations are transitions unless an exact base change has been shown.
To obtain the frequencies of Hg H and its sub-Hg H1b in different populations, we compiled a data set of 26105 HVS-I sequences from various sources listed in table S2 in the Supplementary Material online. The frequency data in individual populations was grouped into broader geographical regions (see table S2) and summary frequencies obtained were mapped (fig. 1). Maps were obtained using Surfer version 7 (Golden Software, Inc., Golden, Colo.) with the Kriging procedure. Estimates at each grid node were obtained by consideration of the entire data set.
Altogether, 830 mitochondrial genomes were included in the coalescence analysis. A subset of the obtained coalescence estimates are presented in table 1 and all of the results in table S1. An average transitional distance from the root haplotype (rho) was calculated. Coalescence time has been calculated taking one transitional step between nucleotide positions 16090–16365 ("HVS") equal to 20,180 years (Forster et al. 1996) and one base substitution between nucleotide positions 577–16023 ("coding") equal to 5,138 years (Mishmar et al. 2003). Standard deviation of the rho estimate (sigma) was calculated as in Saillard et al. (2000b), and SD denotes the deviation in years. The 115 Eastern Slav samples analyzed hierarchically and not shown in figure 2A have been included in the coalescence analysis. Note that the coding sequence data is derived mainly from European populations.
Table 1 Coalescence Ages of Some Haplogroup H Subclusters
Results and Discussion
Figure 3 shows the backbone of the phylogenetic tree of Hg H subclades studied here. We have corrected the names of sub-Hgs H5 and H6 as defined by Quintans et al. (2004) as 4336C and 3915A, respectively, to H5a and H6a, following the hierarchical principle described by Richards et al. (1998). Note that the most parsimonious phylogenetic tree has two branching events based on shared HVS-I nucleotide transitions: one between sub-Hgs H1b and H1f and the second between sub-Hgs H6 and H8. Because the transitions, at nucleotide positions (nps) 16189 and 16362, involve mutational hot-spots, the indicated sub-Hgs, though monophyletic, are not necessarily sister clades, as depicted in figure 3.
FIG. 3.— (A) A phylogenetic network of mtDNA variants that belong to nine subhaplogroups of haplogroup H (see Appendix S1). Some related haplotypes belonging to paraphyletic H* are also shown. The legend shows the color code for studied populations. The number of individuals is shown beside the circles above the legend. Mutations are transitions unless a transversional base change has been shown. "d" denotes deletion. Letter coding for the restriction enzymes is as follows: a: AciI, b: SspI, c: Bsh1236I, d: Alw44I, e: MboI, f: Eco32I, g: DdeI, h: Eco47I, i: AluI, j: BsuRI, k: MspI. The gain of a restriction site is marked by a "+" following the site description; the mark "-" indicates the loss of a site. (B) Frequencies (%) of sub-Hgs H2a, H3, H6, and H8 relative to haplogroup H pool in eleven populations. The legend shows the patterns corresponding to different sub-Hgs. A: Finnish data of Finnil?, Lehtonen, and Majamaa (2001); B: Estonian; C: Volga-Ural region Finno-Ugric; D: Eastern Slavic; E: Slovak; F: French; G: U.K. and U.S. data of Herrnstadt et al. (2002); H: Balkan; I: Turk; J: Near and Middle Eastern: K: central Asian and Altaian. See the Materials and Methods section for details of the studied populations.
The number of internal branches in Hg H is significantly higher than in other mtDNA haplogroups widespread in Europe. In the majority of European mtDNA variants—J, T, K, X and U5—the coding region variation is described by only a few extant basal subclades (Finnil? et al. 2000; Finnil? and Majamaa 2001; Herrnstadt et al. 2002; Reidla et al. 2003). In contrast, there are 57 basic branches stemming from the founder node of Hg H in the parsimonious phylogenetic tree relating 267 Hg H coding region sequences (fig. S1).
One hundred twenty-five variable positions were detected in 594 (563 + 31 Finnish sequences of Finnil?, Lehtonen, and Majamaa 2001) Hg H HVS-I sequences. Among them, recurrent transitions were observed in 50 positions (40%) in different subclades (table 2). The sites with the highest number of recurrences match the HVS-I hot-spot sites identified previously (Hasegawa et al. 1993; Malyarchuk and Derenko 2001b; Allard et al. 2002). The most variable positions, 16093 and 16311, had received parallel hits in seven different subclusters; 16189 in six; 16092, 16304, and 16362 each in five; and 16129, 16209, 16249, and 16325 each in four subclusters. Another 12 HVS-I mutations were found in three and 28 substitutions in two different phylogenetic contexts. Because quite a few of these hot-spot mutations are present in HVS-I haplotypes that have been highlighted as having founder status in Europe (Richards et al. 2000), our results document again that additional coding region information is essential and unavoidable in defining monophyletic subclades of Hg H reliably (Torroni et al. 1993; Bandelt et al. 2001; Kivisild et al. 2002). We also found that a reversion of A to the ancestral base G at np 73 of the HVS-II, noticed in Hg H first by Torroni et al. (1996), has occurred independently at least four times in Hg H phylogeny (see also Helgason et al. 2000).
Table 2 Nucleotide Positions in the First Hypervariable Segment (HVS-I) That Have Received More Than One Transitional Hit in Haplogroup H
In the coding region, a transition at np 3010 that defines sub-Hg H1 (Finnil?, Lehtonen, and Majamaa 2001) is phylogenetically equally problematic. The derived state at np 3010 has been detected in haplogroups C, D, H, J, L2, L3, and U, making this base pair one of the fastest evolving mtDNA coding region positions (Ingman et al. 2000; Finnil?, Lehtonen, and Majamaa 2001; Maca-Meyer et al. 2001; Torroni et al. 2001b; Herrnstadt et al. 2002). Character conflicts at np 3010 and at more conserved nps 1462 (occurs also in Hgs H*, H2, T), 6272 (H*, L3), 6776 (H3), 8470 (H3), 12172 (H*, L1, U2), and 14869 (H*, K2, L3) were found (fig. S1). Given the data, the number of independent 3010A incidences in Hg H may possibly be as many as four (fig. S1).
Sub-Hg H4 was previously defined by an array of eight mutations (nps 3992, 4024, 5004, 8269, 9123, 10044, 14365, and 14582) through an analysis of haplotypes that occurred in at least two individuals (Herrnstadt et al. 2002). However, re-examination of the sequence data of Herrnstadt et al. (2002) revealed that only six mutations at nps 3992, 4024, 5004, 9123, 14365, and 14582 appear to be necessary to characterize the clade (fig. S1). Consequently, here we name the bough defined by a G-to-A mutation at np 8269, which further embraces the 10044 twig, as H4a.
While applying the RFLP method we discovered three previously unknown mutations: a transition at np 13760 abolishing the AciI site at np 13757 defining sub-Hg H11, a transition at np 5005 eliminating the H4-defining DdeI site at np 5003, and a transition at np 8449 eliminating the H11-defining np 8446 SspI site. Therefore, we confirmed the presence of H4-specific T at np 5004 by sequencing the position in all 12 samples lacking the DdeI 5003 site. The monophyly of sub-Hg H11 is well established by the combination of two RFLPs and by the characteristic HVS-I mutation pattern. These results show that classical indirect DNA polymorphism detection methods, like RFLP, should be backed-up by direct sequencing in order to avoid the ambiguous or even erroneous inference of phylogeny.
The next paragraphs address the main phylogeographic results. The largest subcluster is sub-Hg H1, which comprises about 30% of Hg H and 13% of the total European mtDNA pool. H1 is most frequent in the Iberian Peninsula, covering about 46% of local Hg H lineages (Pereira et al. 2004; Quintans et al. 2004). In the Near East the frequency of H1 does not exceed 6% (P < .025), and its relative frequency with respect to Hg H is lower than that seen in Europe (14%). In the Central Asian populations, where Hg H makes up about 11% of the local mtDNA pool, only 6% of H samples belong to sub-Hg H1 (table 3).
Table 3 Frequencies of Haplogroup H Subhaplolgroups in Studied Populations
Sub-Hg H1b is found throughout the area of the spread of Hg H, more frequently found in Eastern Europe and north central Europe (about 7% and 5% of Hg H, respectively; fig. 1B). It was also found to make up about 5% of Hg H in Siberian Mansis. A minor sub-Hg H1f constitutes a quarter of the selected subset of Finnish Hg H genomes of Finnil?, Lehtonen, and Majamaa (2001), being almost absent elsewhere in Europe. Confirmation of the high frequency of this rare variant of mtDNA among northern central Finns, characterized by HVS-I motif 16093–16189, can be found in the Finnish data of Meinil?, Finnil?, and Majamaa (2001), reflecting founder effects in the Finnish population history (Nevanlinna 1972; de la Chapelle and Wright 1998; Kittles et al. 1999; Peltonen, Palotie, and Lange 2000). In our previous study (Tambets et al. 2004), we assumed monophyly of the transition at np 16162. This mutation occurs in the motif 73–3010–16162, which defines H1a. The finding of an Eastern Slav individual bearing haplotype 73–16093–16162 and lacking H1 defining transition at np 3010 hints that motif 73–16162 may have arisen, though rarely, more than once in haplogroup H (fig. 2A). Alternatively, and bearing in mind that position 3010 is a mutational hot-spot (see above), one may consider a parallel occurrence or a reverse mutation at this position.
Like H1b, sub-Hg H2a occurs more frequently (P < .05) in Eastern than in Western European Hg H genomes, 6.5% and 1.1%, respectively, when averaged over populations (table 3 and fig. 2B). The spread of H2a extends to Central Asia, mimicking to some extent, albeit at a lower frequency, the phylogeography of Y-chromosomal Hg R1a (Rosser et al. 2000; Wells et al. 2001). In contrast, sub-Hg H3 was found to be more frequent (P < .05) in the Western (11.7%) than in the Eastern European Hg H pool (4.1%) and is virtually absent in Anatolia and the Near East (fig. 2B), resembling in its phylogeography the spread of Y-chromosomal Hg R1b associated 49a,f TaqI haplotype 15 (Semino et al. 1996; Cinnioglu et al. 2004). The high frequency of mtDNA Hg H3—in combination with Y chromosomal Ht 15—extends to the Iberian Peninsula, where H3 constitutes about 17% of Hg H and is the highest detected so far (Pereira et al. 2004; Quintans et al. 2004).
The coalescence ages of H2a1 and H3 fall to the period of postglacial recolonization in Europe (table 1), suggested first for mtDNA Hg V (Torroni et al. 1998, 2001a). We also note that mtDNA bearing "St. Luke motif," 16235–16293 (Vernesi et al. 2001), belong to sub-Hg H2 (fig. 2A), being particularly frequent in Germany and Scotland (Helgason et al. 2001; Pfeiffer et al. 2001).
The Near Eastern samples cluster together with Central Asian mtDNAs in the sub-Hgs H6b and H8, which are very rare in Europe. The finding is demonstrating a separate flow of maternal lineages south of the Caspian and the Black Sea in addition to well-known long-lasting migrations of pastoral nomads alongside the steppe belt that connects the Danube Basin, over the Pontic-Caspian, with Central Asia, Altay, and Manchuria.
In contrast to that found in Europeans, sub-Hgs H6 and H8 among Central Asian/Altaian populations are characterized by distinctly divergent haplotypes (fig. 2A). This finding may reflect a long-time separation of Asian and European H6 and H8 mtDNA pools and/or an earlier expansion of H6 in the eastern part of its present range. Indeed, the coalescence age of H6 in Central Asians is very deep—40,400 years (SD 16,400 years; table S1). Because the Asian branches of sub-Hg H6 are highly divergent and seem to be among the oldest in Hg H (table S1), they pose an interesting problem, deserving specific study with a much larger sample size at hand.
The commonly used HVS-I clock (Forster et al. 1996) places the initial expansion of Hg H in the Near East to about 23,000 to 28,000 years before the present (Richards et al. 2000). The ancestral clades of Hg H, pre-HV, and HV* have their combined present range predominantly in the Near and Middle East, and in the Caucasus (Metspalu et al. 1999; Richards et al. 2002), implying this could have been the region where the pre-HV/HV clade started to diversify and, possibly, where the earliest Hg H variants might have first appeared.
However, most subclusters of Hg H exhibit coalescence ages, corresponding to the beginning of their expansion in the Late Upper Paleolithic (tables 1 and S1). In this respect our results support an earlier proposition that Hg H was the major mtDNA haplogroup participating in the recolonization of Europe after the Last Glacial Maximum (Torroni et al. 1998; Richards et al. 2000). It is also important to note that the expansion time estimates derived from the coding region and HVS-I of Hg H are often in reasonable agreement with each other (tables 1 and S1). Sub-Hgs H1 and H3 have their highest frequencies in the Iberian Peninsula. These sub-Hgs may have been the companions of mtDNA Hg V in the postglacial repeopling of Europe from a refuge area in Iberia (Torroni et al. 1998). However, in contrast to Hg V, suggested coalescence ages of H1 and H3—13,400 ± 3,000 and 8,600 ± 2,800 years ago, respectively (Pereira et al. 2004)—do not imply deeper phylogeny of H1 and H3 in Iberia compared to the rest of Europe (tables 1 and S1).
These results demonstrate that a seemingly uniform spread of this major human mtDNA clade in western Eurasian populations hides within itself a complex structure of phylogeographically informative subclades. However, it is evident that additional knowledge at the level of complete mtDNA sequences is still needed for a truly comprehensive cataloguing of Hg H diversity, in particular more effectively covering its variation in the Mediterranean, Near and Middle Eastern, and Central Asian/Altaian populations. Nevertheless, even now it is tempting to speculate that much deeper coalescence ages, close to/overlapping with the boundary between the Middle and Upper Paleolithic, for some Hg H branches in Central Asian/Altaian populations, suggest that the time depth of this predominant haplogroup may be much deeper than its apparent general signal for expansion in Europe. It is, therefore, possible that the carriers of pre-Aurignacian industry identified in Zagros as well as in Altay (Otte and Derevianko 2001) were anatomically modern humans already possessing Hg H.
Supplementary Material
Supplementary Appendixes S1 and S2, figure S1, and tables S1 and S2 are available at the journal's Web site as well as the Web site of the University of Tartu, Department of Evolutionary Biology (http://www.evolutsioon.ut.ee/mtDNA-H/).
Acknowledgements
We thank Ille Hilpus and Jaan Lind for technical assistance and we are grateful to Vladimir Ferák for providing Slovak samples. The research of R.V. was supported by Estonian basic research grant 514 and European Community grants ICA1CT20070006 and QLG2-CT-2002–90455. T.K. was supported by Estonian Science Fund grant 5574. The research of B.A.M. was supported by the Russian Foundation for Basic Research (project number 03–04–48162). P.R. received support from the Ministry of Science and Technology of the Republic of Croatia (project number 0196005). The research of V.S. and V.P. was supported by a grant (project number 03–04–49021) from the Russian Foundation for Basic Research and by grants from the President of the Russian Federation (projects number MD-88.2003.04 and NSh-840.2003.4).
References
Allard, M. W., K. Miller, M. Wilson, K. Monson, and B. Budowle. 2002. Characterization of the Caucasian haplogroups present in the SWGDAM forensic mtDNA dataset for 1771 human control region sequences. Scientific Working Group on DNA Analysis Methods. J. Forensic Sci. 47:1215–1223.
Anderson, S., A. T. Bankier, B. G. Barrell et al. (14 co-authors). 1981. Sequence and organization of the human mitochondrial genome. Nature 290:457–465.
Andrews, R. M., I. Kubacka, P. F. Chinnery, R. N. Lightowlers, D. M. Turnbull, and N. Howell. 1999. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat. Genet. 23:147.
Arnason, U., X. Xu, and A. Gullberg. 1996. Comparison between the complete mitochondrial DNA sequences of Homo and the common chimpanzee based on nonchimeric sequences. J. Mol. Evol. 42:145–152.
Bamshad, M., T. Kivisild, W. S. Watkins et al. (18 co-authors). 2001. Genetic evidence on the origins of Indian caste populations. Genome Res. 11:994–1004.
Bandelt, H.-J., P. Forster, and A. R?hl. 1999. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16:37–48.
Bandelt, H.-J., P. Forster, B. C. Sykes, and M. B. Richards. 1995. Mitochondrial portraits of human populations using median networks. Genetics 141:743–753.
Bandelt, H. J., P. Lahermo, M. Richards, and V. Macaulay. 2001. Detecting errors in mtDNA data by phylogenetic analysis. Int. J. Legal Med. 115:64–69.
Bermisheva, M., K. Tambets, R. Villems, and E. Khusnutdinova. 2002. Diversity of mitochondrial DNA haplotypes in ethnic populations of the Volga-Ural region of Russia. Mol. Biol. (Mosk) 36:990–1001.
Cinnioglu, C., R. King, T. Kivisild et al. (15 co-authors). 2004. Excavating Y-chromosome haplotype strata in Anatolia. Hum. Genet. 114:127–148.
Comas, D., F. Calafell, E. Mateu et al. (12 co-authors). 1998. Trading genes along the silk road: mtDNA sequences and the origin of Central Asian populations. Am. J. Hum. Genet. 63:1824–1838.
Corte-Real, H. B., V. A. Macaulay, M. B. Richards, G. Hariti, M. S. Issad, A. Cambon-Thomsen, S. Papiha, J. Bertranpetit, and B. C. Sykes. 1996. Genetic diversity in the Iberian Peninsula determined from mitochondrial sequence analysis. Ann. Hum. Genet. 60:331–350.
de la Chapelle, A., and F. A. Wright. 1998. Linkage disequilibrium mapping in isolated populations: the example of Finland revisited. Proc. Natl. Acad. Sci. USA 95:12416–12423.
Derbeneva, O. A., E. B. Starikovskaya, D. C. Wallace, and R. I. Sukernik. 2002a. Traces of early Eurasians in the Mansi of northwest Siberia revealed by mitochondrial DNA analysis. Am. J. Hum. Genet. 70:1009–1014.
Derbeneva, O. A., R. I. Sukernik, N. V. Volodko, S. H. Hosseini, M. T. Lott, and D. C. Wallace. 2002b. Analysis of mitochondrial DNA diversity in the Aleuts of the Commander Islands and its implications for the genetic history of Beringia. Am. J. Hum. Genet. 71:415–421.
Derenko, M. V., T. Grzybowski, B. A. Malyarchuk et al. (11 co-authors). 2003. Diversity of mitochondrial DNA lineages in south Siberia. Ann. Hum. Genet. 67:391–411.
Dimo-Simonin, N., F. Grange, F. Taroni, C. Brandt-Casadevall, and P. Mangin. 2000. Forensic evaluation of mtDNA in a population from south west Switzerland. Int. J. Legal Med. 113:89–97.
Finnil?, S., I. E. Hassinen, L. Ala-Kokko, and K. Majamaa. 2000. Phylogenetic network of the mtDNA haplogroup U in Northern Finland based on sequence analysis of the complete coding region by conformation-sensitive gel electrophoresis. Am. J. Hum. Genet. 66:1017–1026.
Finnil?, S., M. S. Lehtonen, and K. Majamaa. 2001. Phylogenetic network for European mtDNA. Am. J. Hum. Genet. 68:1475–1484.
Finnil?, S., and K. Majamaa. 2001. Phylogenetic analysis of mtDNA haplogroup TJ in a Finnish population. J. Hum. Genet. 46:64–69.
Forster, P., R. Harding, A. Torroni, and H.-J. Bandelt. 1996. Origin and evolution of Native American mtDNA variation: a reappraisal. Am. J. Hum. Genet. 59:935–945.
Hasegawa, M., A. Di Rienzo, T. D. Kocher, and A. C. Wilson. 1993. Toward a more accurate time scale for the human mitochondrial DNA tree. J. Mol. Evol. 37:347–354.
Helgason, A., E. Hickey, S. Goodacre, V. Bosnes, K. Stefansson, R. Ward, and B. Sykes. 2001. mtDNA and the islands of the North Atlantic: estimating the proportions of Norse and Gaelic ancestry. Am. J. Hum. Genet. 68:723–737.
Helgason, A., S. Sigurdadottir, J. Gulcher, R. Ward, and K. Stefanson. 2000. mtDNA and the origins of the Icelanders: deciphering signals of recent population history. Am. J. Hum. Genet. 66:999–1016.
Herrnstadt, C., J. L. Elson, E. Fahy et al. (11 co-authors). 2002. Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups. Am. J. Hum. Genet. 70:1152–1171.
Herrnstadt, C., G. Preston, and N. Howell. 2003. Errors, phantoms and otherwise, in human mtDNA sequences. Am. J. Hum. Genet. 72:1585–1586.
Hofmann, S., M. Jaksch, R. Bezold, S. Mertens, S. Aholt, A. Paprotta, and K. D. Gerbitz. 1997. Population genetics and disease susceptibility: characterization of central European haplogroups by mtDNA gene mutations, correlation with D loop variants and association with disease. Hum. Mol. Genet. 6:1835–1846.
Ingman, M., H. Kaessmann, S. P??bo, and U. Gyllensten. 2000. Mitochondrial genome variation and the origin of modern humans. Nature 408:708–713.
Kittles, R. A., A. W. Bergen, M. Urbanek, M. Virkkunen, M. Linnoila, D. Goldman, and J. C. Long. 1999. Autosomal, mitochondrial, and Y chromosome DNA variation in Finland: evidence for a male-specific bottleneck. Am. J. Phys. Anthropol. 108:381–399.
Kivisild, T., M. J. Bamshad, K. Kaldma et al. (15 co-authors). 1999. Deep common ancestry of Indian and western-Eurasian mitochondrial DNA lineages. Curr. Biol. 9:1331–1334.
Kivisild, T., S. Rootsi, M. Metspalu et al. (18 co-authors). 2003. The genetic heritage of the earliest settlers persists both in Indian tribal and caste populations. Am. J. Hum. Genet. 72:313–332.
Kivisild, T., H.-V. Tolk, J. Parik, Y. Wang, S. S. Papiha, H.-J. Bandelt, and R. Villems. 2002. The emerging limbs and twigs of the East Asian mtDNA tree. Mol. Biol. Evol. 19:1737–1751.
Kong, Q. P., Y. G. Yao, C. Sun, H. J. Bandelt, C. L. Zhu, and Y. P. Zhang. 2003. Phylogeny of east Asian mitochondrial DNA lineages inferred from complete sequences. Am. J. Hum. Genet. 73:671–676.
Levin, B. C., H. Cheng, and D. J. Reeder. 1999. A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection. Genomics 55:135–146.
Maca-Meyer, N., A. M. Gonzalez, J. M. Larruga, C. Flores, and V. M. Cabrera. 2001. Major genomic mitochondrial lineages delineate early human expansions. BMC Genet. 2:13.
Macaulay, V. A., M. B. Richards, E. Hickey, E. Vega, F. Cruciani, V. Guida, R. Scozzari, B. Bonné-Tamir, B. Sykes, and A. Torroni. 1999. The emerging tree of West Eurasian mtDNAs: a synthesis of control-region sequences and RFLPs. Am. J. Hum. Genet. 64:232–249.
Malyarchuk, B. A., and M. V. Derenko. 2001a. Mitochondrial DNA variability in Russians and Ukrainians: implications to the origin of the Eastern Slavs. Ann. Hum. Genet. 65:63–78.
Malyarchuk, B. A., and M. V. Derenko. 2001b. Variation of human mitochondrial DNA: distribution of hot spots in hypervariable segment I of the major noncoding region. Genetika 37:991–1001.
Malyarchuk, B. A., T. Grzybowski, M. V. Derenko, J. Czarny, K. Drobnic, and D. Miscicka-Sliwka. 2003. Mitochondrial DNA variability in Bosnians and Slovenians. Ann. Hum. Genet. 67:412–425.
Malyarchuk, B. A., T. Grzybowski, M. V. Derenko, J. Czarny, M. Wozniak, and D. Miscicka-Sliwka. 2002. Mitochondrial DNA variability in Poles and Russians. Ann. Hum. Genet. 66:261–283.
Marzuki, S., A. S. Noer, P. Lertrit, D. Thyagarajan, R. Kapsa, P. Utthanaphol, and E. Byrne. 1991. Normal variants of human mitochondrial DNA and translation products: the building of a reference data base. Hum. Genet. 88:139–145.
Meinil?, M., S. Finnil?, and K. Majamaa. 2001. Evidence for mtDNA admixture between the Finns and the Saami. Hum. Hered. 52:160–170.
Metspalu, E., T. Kivisild, K. Kaldma, J. Parik, M. Reidla, K. Tambets, and R. Villems. 1999. The Trans-Caucasus and the expansion of the Caucasoid-specific human mitochondrial DNA. Pp. 121–134 in S. Papiha, R. Deka, and R. Chakraborty, eds. Genomic diversity: application in human population genetics. Kluwer Academic/Plenum Publishers, New York.
Mishmar, D., E. Ruiz-Pesini, P. Golik et al. (13 co-authors). 2003. Natural selection shaped regional mtDNA variation in humans. Proc. Natl. Acad. Sci. USA 100:171–176.
Nevanlinna, H. R. 1972. The Finnish population structure. A genetic and genealogical study. Hereditas 71:195–236.
Otte M., and A. Derevianko. 2001. The Aurignacian in Altai. Antiquity 75:44–48.
Parson, W., T. J. Parsons, R. Scheithauer, and M. M. Holland. 1998. Population data for 101 Austrian Caucasian mitochondrial DNA d-loop sequences: application of mtDNA sequence analysis to a forensic case. Int. J. Legal Med. 111:124–132.
Passarino, G., O. Semino, L. F. Bernini, and A. S. Santachiara-Benerecetti. 1996. Pre-Caucasoid and Caucasoid genetic features of the Indian population, revealed by mtDNA polymorphisms. Am. J. Hum. Genet. 59:927–934.
Peltonen, L., A. Palotie, and K. Lange. 2000. Use of population isolates for mapping complex traits. Nat. Rev. Genet. 1:182–190.
Pereira L., C. Cunha, and A. Amorim. 2004. Predicting sampling saturation of mtDNA haplotypes: an application to an enlarged Portuguese database. Int. J. Legal. Med. 118:132–136.
Pereira, L., M. Richards, A. Alonso, C. Albarran, O. Garcia, V. Macaulay, and A. Amorim. 2004. Subdividing mtDNA haplogroup H based on coding-region polymorphisms—a study in Iberia. Int. Congr. Ser. 1261:416–418.
Pfeiffer, H., P. Forster, C. Ortmann, and B. Brinkmann. 2001. The results of an mtDNA study of 1,200 inhabitants of a German village in comparison to other Caucasian databases and its relevance for forensic casework. Int. J. Legal Med. 114:169–172.
Piercy, R., K. M. Sullivan, N. Benson, and P. Gill. 1993. The application of mitochondrial DNA typing to the study of white Caucasian genetic identification. Int. J. Legal Med. 106:85–90.
Polyak, K., Y. Li, H. Zhu, C. Lengauer, J. K. Willson, S. D. Markowitz, M. A. Trush, K. W. Kinzler, and B. Vogelstein. 1998. Somatic mutations of the mitochondrial genome in human colorectal tumours. Nat. Genet. 20:291–293.
Quintans, B., V. Alvarez-Iglesias, A. Salas, C. Phillips, M. V. Lareu, and A. Carracedo. 2004. Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing. Forensic Sci. Int. 140:251–257.
Rando, J. C., F. Pinto, A. M. Gonzalez, M. Hernandez, J. M. Larruga, V. M. Cabrera, and H. J. Bandelt. 1998. Mitochondrial DNA analysis of northwest African populations reveals genetic exchanges with European, Near-Eastern, and sub-Saharan populations. Ann. Hum. Genet. 62:531–550.
Reid, F. M., G. A. Vernham, and H. T. Jacobs. 1994. Complete mtDNA sequence of a patient in a maternal pedigree with sensorineural deafness. Hum. Mol. Genet. 3:1435–1436.
Reidla, M., T. Kivisild, E. Metspalu et al. (43 co-authors). 2003. Origin and diffusion of mtDNA haplogroup X. Am. J. Hum. Genet. 73:1178–1190.
Richards, M., H. Corte-Real, P. Forster, V. Macaulay, H. Wilkinson-Herbots, A. Demaine, S. Papiha, R. Hedges, H.-J. Bandelt, and B. Sykes. 1996. Paleolithic and neolithic lineages in the European mitochondrial gene pool. Am. J. Hum. Genet. 59:185–203.
Richards, M., V. Macaulay, E. Hickey et al. (26 co-authors). 2000. Tracing European founder lineages in the Near Eastern mtDNA pool. Am. J. Hum. Genet. 67:1251–1276.
Richards, M., V. Macaulay, A. Torroni, and H. J. Bandelt. 2002. In search of geographical patterns in European mitochondrial DNA. Am. J. Hum. Genet. 71:1168–1174.
Richards, M. B., V. A. Macaulay, H.-J. Bandelt, and B. C. Sykes. 1998. Phylogeography of mitochondrial DNA in western Europe. Ann. Hum. Genet. 62:241–260.
Rieder, M. J., S. L. Taylor, V. O. Tobe, and D. A. Nickerson. 1998. Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome. Nucleic Acids Res. 26:967–973.
Rosser, Z. H., T. Zerjal, M. E. Hurles et al. (63 co-authors). 2000. Y-chromosomal diversity in Europe is clinal and influenced primarily by geography, rather than by language. Am. J. Hum. Genet. 67:1526–1543.
Saillard, J., I. Evseva, L. Tranebjaerg, and S. Norby. 2000a. Mitochondrial DNA diversity among Nenets. Pp. 255–258 in C. Renfrew and K. Boyle, eds. Archaeogenetics: DNA and and the population prehistory of Europe. McDonald Institute for Archaeological Research Monograph Series, Cambridge University, Cambridge.
Saillard, J., P. Forster, N. Lynnerup, H.-J. Bandelt, and S. N?rby. 2000b. mtDNA variation among Greenland Eskimos: the edge of the Beringian expansion. Am. J. Hum. Genet. 67:718–726.
Semino, O., G. Passarino, A. Brega, M. Fellous, and A. S. Santachiara-Benerecetti. 1996. A view of the neolithic demic diffusion in Europe through two Y chromosome-specific markers. Am. J. Hum. Genet. 59:964–968.
Stevanovitch, A., A. Gilles, E. Bouzaid, R. Kefi, F. Paris, R. P. Gayraud, J. L. Spadoni, F. El-Chenawi, and E. Beraud-Colomb. 2004. Mitochondrial DNA sequence diversity in a sedentary population from Egypt. Ann. Hum. Genet. 68:23–39.
Tambets, K., T. Kivisild, E. Metspalu et al. (13 co-authors). 2000. The topology of the maternal lineages of the Anatolian and Trans-Caucasus populations and the peopling of the Europe: some preliminary considerations. Pp. 219–235 in C. Renfrew and K. Boyle, eds. Archaeogenetics: DNA and the population prehistory of Europe. Cambridge University Press, Cambridge.
Tambets, K., S. Rootsi, T. Kivisild et al. (46 co-authors). 2004. The western and eastern Roots of the Saami—the story of genetic "outliers" told by mitochondrial DNA and Y chromosomes. Am. J. Hum. Genet. 74:661–682.
Torroni, A., H.-J. Bandelt, L. D'Urbano et al. (11 co-authors). 1998. mtDNA analysis reveals a major late Paleolithic population expansion from southwestern to northeastern Europe. Am. J. Hum. Genet. 62:1137–1152.
Torroni, A., H. J. Bandelt, V. Macaulay et al. (33 co-authors). 2001a. A signal, from human mtDNA, of postglacial recolonization in Europe. Am. J. Hum. Genet. 69:844–852.
Torroni, A., K. Huoponen, P. Francalacci, M. Petrozzi, L. Morelli, R. Scozzari, D. Obinu, M. L. Savontaus, and D. C. Wallace. 1996. Classification of European mtDNAs from an analysis of three European populations. Genetics 144:1835–1850.
Torroni, A., M. T. Lott, M. F. Cabell, Y. S. Chen, L. Lavergne, and D. C. Wallace. 1994. mtDNA and the origin of Caucasians: identification of ancient Caucasian-specific haplogroups, one of which is prone to a recurrent somatic duplication in the D-loop region. Am. J. Hum. Genet. 55:760–776.
Torroni, A., C. Rengo, V. Guida et al. (12 co-authors). 2001b. Do the four clades of the mtDNA haplogroup L2 evolve at different rates? Am. J. Hum. Genet. 69:1348–1356.
Torroni, A., R. I. Sukernik, T. G. Schurr, Y. B. Starikorskaya, M. F. Cabell, M. H. Crawford, A. G. Comuzzie, and D. C. Wallace. 1993. mtDNA variation of aboriginal Siberians reveals distinct genetic affinities with Native Americans. Am. J. Hum. Genet. 53:591–608.
Vanecek, T., F. Vorel, and M. Sip. 2004. Mitochondrial DNA D-loop hypervariable regions: Czech population data. Int. J. Legal Med. 118:14–18.
Vernesi, C., G. Di Benedetto, D. Caramelli, E. Secchieri, L. Simoni, E. Katti, P. Malaspina, A. Novelletto, V. T. Marin, and G. Barbujani. 2001. Genetic characterization of the body attributed to the evangelist Luke. Proc. Natl. Acad. Sci. USA 98:13460–13463. Epub 12001 Oct 13416.
Wells, R. S., N. Yuldasheva, R. Ruzibakiev et al. (27 co-authors). 2001. The Eurasian heartland: a continental perspective on Y-chromosome diversity. Proc. Natl. Acad. Sci. USA 98:10244–10249.(Eva-Liis Loogv?li*, Urmas)
E-mail: evall@ut.ee.
Abstract
It has been often stated that the overall pattern of human maternal lineages in Europe is largely uniform. Yet this uniformity may also result from an insufficient depth and width of the phylogenetic analysis, in particular of the predominant western Eurasian haplogroup (Hg) H that comprises nearly a half of the European mitochondrial DNA (mtDNA) pool. Making use of the coding sequence information from 267 mtDNA Hg H sequences, we have analyzed 830 mtDNA genomes, from 11 European, Near and Middle Eastern, Central Asian, and Altaian populations. In addition to the seven previously specified subhaplogroups, we define fifteen novel subclades of Hg H present in the extant human populations of western Eurasia. The refinement of the phylogenetic resolution has allowed us to resolve a large number of homoplasies in phylogenetic trees of Hg H based on the first hypervariable segment (HVS-I) of mtDNA. As many as 50 out of 125 polymorphic positions in HVS-I were found to be mutated in more than one subcluster of Hg H. The phylogeographic analysis revealed that sub-Hgs H1*, H1b, H1f, H2a, H3, H6a, H6b, and H8 demonstrate distinct phylogeographic patterns. The monophyletic subhaplogroups of Hg H provide means for further progress in the understanding of the (pre)historic movements of women in Eurasia and for the understanding of the present-day genetic diversity of western Eurasians in general.
Key Words: human mitochondrial DNA ? population genetics ? phylogeography
Introduction
The mitochondrial DNA (mtDNA) sequences of Europeans are sorted into ten major phylogenetic clades, or haplogroups, alphabetically named H, J, K, N1, T, U4, U5, V, X, and W (Torroni et al. 1994, 1996; Macaulay et al. 1999; Richards et al. 2000). Haplogroup (Hg) H alone constitutes about one half of the European mtDNA pool and, along with other aforementioned lineages, is widespread also in western Asia (Macaulay et al. 1999; Richards et al. 2000; Tambets et al. 2000; Kivisild et al. 2003), Central Asia (Comas et al. 1998; Metspalu et al. 1999), Siberia (Saillard et al. 2000a; Derbeneva et al. 2002a; Derenko et al. 2003), southern Asia (Passarino et al. 1996; Kivisild et al. 1999, 2003; Bamshad et al. 2001), and northern Africa (Corte-Real et al. 1996; Rando et al. 1998; Stevanovitch et al. 2004; fig. 1A). At least 267 Hg H mtDNA genomes have been sequenced (nearly) completely (Reid, Vernham, and Jacobs 1994; Rieder et al. 1998; Levin, Cheng, and Reeder 1999; Ingman et al. 2000; Finnil?, Lehtonen, and Majamaa 2001; Maca-Meyer et al. 2001; Herrnstadt et al. 2002, correction by Herrnstadt, Preston, and Howell 2003; Mishmar et al. 2003). Out of seven Hg H sub-Hgs defined so far, Hgs H1 and H2 (Finnil?, Lehtonen, and Majamaa 2001) along with Hgs H3 and H4 (Herrnstadt et al. 2002) and Hgs H5, H6, and H7 (Quintans et al. 2004) cover 74% of Finnish, 68% of U.S./U.K. and 77% of Galician Hg H sequences, respectively.
FIG. 1.— Spatial frequency distributions of haplogroup H (A) and its subhaplogroup H1b (B). H1b frequencies are given as percentages with respect to Hg H.
Attempts to classify Hg H lineages by first hypervariable segment (HVS-I) have been hindered by a frequent occurrence of mutations at fast-evolving nucleotide sites—so-called mutational hot-spots (Richards et al. 2000; Allard et al. 2002). Furthermore, HVS-I sequence information leaves a substantial fraction of Hg H genomes phylogenetically unresolved: on average one-third of the lineages share a haplotype identical with the Cambridge reference sequence (CRS; Anderson et al. 1981). Therefore, progress in the analysis of Hg H diversity, and, indeed, of the understanding of the phylogeography of western Eurasian maternal lineages, depends critically on the use of full genome information in mtDNA samples, representative in size and geography.
Materials and Methods
The phylogeny of 267 published Hg H mtDNA coding region sequences (Reid, Vernham, and Jacobs 1994; Rieder et al. 1998; Levin, Cheng, and Reeder 1999; Ingman et al. 2000; Finnil?, Lehtonen, and Majamaa 2001; Maca-Meyer et al. 2001; Herrnstadt et al. 2002; Mishmar et al. 2003) was analyzed (fig. S1 in the Supplementary Material online). Markers for nine subclades were selected for genotyping in 563 Hg H mtDNA samples of European, Near and Middle Eastern, Central Asian and Altaian origin. To find the relative mutation rates of particular nucleotide positions, the frequencies of phylogenetically independent mutations were calculated from 987 published mtDNA coding region sequences (Marzuki et al. 1991; Reid, Vernham, and Jacobs 1994; Arnason, Xu, and Gullberg 1996; Polyak et al. 1998; Rieder et al. 1998; Levin, Cheng, and Reeder 1999; Ingman et al. 2000; Finnil?, Lehtonen, and Majamaa 2001; Maca-Meyer et al. 2001; Torroni et al. 2001b; Derbeneva et al. 2002b; Herrnstadt et al. 2002; Kivisild et al. 2002 and references therein; Kong et al. 2003; Mishmar et al. 2003).
The samples were selected at random from nine populations: 50 Finno-Ugric speakers from the Volga-Ural region (10 Udmurts, 10 Mokshas, 16 Erzyas, 7 Permyak Komis, 7 Zyrian Komis); 50 Estonians; 165 Eastern Slavs (127 Russians, 38 Ukrainians from various districts of Russia and the Ukraine); 50 Slovaks; 50 French from southern France, Lyon, Low Normandy, and Poitiers; 50 individuals from the Balkans (17 Croats, 17 Albanians, 16 Greeks); 50 Turks; 50 individuals from the Near and the Middle East (10 Jordanians, 8 Lebanese, 7 Saudis, 12 Syrians, 13 Iranians); 48 individuals from Central Asia (17 Altaians, 11 Kirghiz, 3 Kazakhs, 11 Tajiks, 6 Uzbeks). Sixteen Russian and six Ukrainian HVS-I sequences have been published by Malyarchuk and Derenko (2001a), 33 Russian HVS-I and second hypervariable segment (HVS-II) sequences by Malyarchuk et al. (2002), and all of the Volga-Ural region mtDNA HVS-I sequences by Bermisheva et al. (2002). All the samples harbored a C at nucleotide position (np) 7028, which is diagnostic for Hg H and was inferred from the absence of the AluI restriction site at np 7025 (Torroni et al. 1994). All mutations and position numbers in this study are given with respect to Anderson et al. (1981) as revised by Andrews et al. (1999).
Four hundred forty-eight samples were screened for 14 polymorphisms in the mtDNA coding region and three in HVS-II in addition to HVS-I sequence variation. A hierarchical strategy was applied to 104 Russian and 11 Ukrainian mtDNAs (Appendix S2 in the Supplementary Material online). HVS-I variation for all of the samples was scored between nps 16024–16383. Nucleotide changes at positions 73, 951, 3010, 4336, 4452, 4769, 4793, 5004, 8448, 9066, 9380, 13101, 13759, and 16482 were determined by restriction fragment length polymorphisms (RFLPs; Appendixes S1 and S2). Nucleotide states at positions 239, 456, 3915, and 6776 were detected by direct sequencing or allele-specific polymerase chain reaction (PCR; Appendixes S1 and S2). Nucleotide positions 239 and 3915 were sequenced in samples having 16362C and/or lacking a Hin6I restriction site at np 9380 and/or having a DdeI site at np 16478. We note that the transition at np 239 nearly always occurs with the 16362C allele, as it was not found in Hg H variants with 16362T in 2,350 published HVS-II sequences (Hofmann et al. 1997; Parson et al. 1998; Dimo-Simonin et al. 2000; Malyarchuk et al. 2003; Vanecek, Vorel, and Sip 2004; Pereira, Cunha, and Amorim 2004). Credible regions of the obtained haplogroup frequencies were computed with the Sampling program kindly provided by Vincent Macaulay.
The phylogeny of the samples was studied by the construction of a reduced median network (fig. 2A). In the network analysis 479 samples were included (see Appendix S1), including the 31 Finnish sequences taken from Finnil?, Lehtonen, and Majamaa (2001), while 115 Eastern Slav mtDNAs, which were analyzed hierarchically (see Appendix S2), have not been included. The reduced median network (Bandelt et al. 1995; rho set at 2) was constructed with the Network 4.0.0.0. program (Fluxus Technology Ltd., Clare, Suffolk, UK, http://www.fluxus-engineering.com) followed by a median joining algorithm (Bandelt, Forster, and R?hl 1999; epsilon set at 0), as explained at the Fluxus-Engineering Web site. Nucleotide positions were divided into three classes of transition rates—fast (16093, 16129, 16189, 16304, 16311, and 16362), intermediate (16172, 16209, 16278, 16293), and slow (the remainder of the positions between 16024 and 16383)—and assigned class weights 1, 2, and 4, respectively. Transversions and coding region mutations were weighted 8.
FIG. 2.— Backbone of the phylogenetic tree of mtDNA haplogroup H subclades studied here. Nonsynonymous (#) and synonymous ('') mutations indicated. Mutations are transitions unless an exact base change has been shown.
To obtain the frequencies of Hg H and its sub-Hg H1b in different populations, we compiled a data set of 26105 HVS-I sequences from various sources listed in table S2 in the Supplementary Material online. The frequency data in individual populations was grouped into broader geographical regions (see table S2) and summary frequencies obtained were mapped (fig. 1). Maps were obtained using Surfer version 7 (Golden Software, Inc., Golden, Colo.) with the Kriging procedure. Estimates at each grid node were obtained by consideration of the entire data set.
Altogether, 830 mitochondrial genomes were included in the coalescence analysis. A subset of the obtained coalescence estimates are presented in table 1 and all of the results in table S1. An average transitional distance from the root haplotype (rho) was calculated. Coalescence time has been calculated taking one transitional step between nucleotide positions 16090–16365 ("HVS") equal to 20,180 years (Forster et al. 1996) and one base substitution between nucleotide positions 577–16023 ("coding") equal to 5,138 years (Mishmar et al. 2003). Standard deviation of the rho estimate (sigma) was calculated as in Saillard et al. (2000b), and SD denotes the deviation in years. The 115 Eastern Slav samples analyzed hierarchically and not shown in figure 2A have been included in the coalescence analysis. Note that the coding sequence data is derived mainly from European populations.
Table 1 Coalescence Ages of Some Haplogroup H Subclusters
Results and Discussion
Figure 3 shows the backbone of the phylogenetic tree of Hg H subclades studied here. We have corrected the names of sub-Hgs H5 and H6 as defined by Quintans et al. (2004) as 4336C and 3915A, respectively, to H5a and H6a, following the hierarchical principle described by Richards et al. (1998). Note that the most parsimonious phylogenetic tree has two branching events based on shared HVS-I nucleotide transitions: one between sub-Hgs H1b and H1f and the second between sub-Hgs H6 and H8. Because the transitions, at nucleotide positions (nps) 16189 and 16362, involve mutational hot-spots, the indicated sub-Hgs, though monophyletic, are not necessarily sister clades, as depicted in figure 3.
FIG. 3.— (A) A phylogenetic network of mtDNA variants that belong to nine subhaplogroups of haplogroup H (see Appendix S1). Some related haplotypes belonging to paraphyletic H* are also shown. The legend shows the color code for studied populations. The number of individuals is shown beside the circles above the legend. Mutations are transitions unless a transversional base change has been shown. "d" denotes deletion. Letter coding for the restriction enzymes is as follows: a: AciI, b: SspI, c: Bsh1236I, d: Alw44I, e: MboI, f: Eco32I, g: DdeI, h: Eco47I, i: AluI, j: BsuRI, k: MspI. The gain of a restriction site is marked by a "+" following the site description; the mark "-" indicates the loss of a site. (B) Frequencies (%) of sub-Hgs H2a, H3, H6, and H8 relative to haplogroup H pool in eleven populations. The legend shows the patterns corresponding to different sub-Hgs. A: Finnish data of Finnil?, Lehtonen, and Majamaa (2001); B: Estonian; C: Volga-Ural region Finno-Ugric; D: Eastern Slavic; E: Slovak; F: French; G: U.K. and U.S. data of Herrnstadt et al. (2002); H: Balkan; I: Turk; J: Near and Middle Eastern: K: central Asian and Altaian. See the Materials and Methods section for details of the studied populations.
The number of internal branches in Hg H is significantly higher than in other mtDNA haplogroups widespread in Europe. In the majority of European mtDNA variants—J, T, K, X and U5—the coding region variation is described by only a few extant basal subclades (Finnil? et al. 2000; Finnil? and Majamaa 2001; Herrnstadt et al. 2002; Reidla et al. 2003). In contrast, there are 57 basic branches stemming from the founder node of Hg H in the parsimonious phylogenetic tree relating 267 Hg H coding region sequences (fig. S1).
One hundred twenty-five variable positions were detected in 594 (563 + 31 Finnish sequences of Finnil?, Lehtonen, and Majamaa 2001) Hg H HVS-I sequences. Among them, recurrent transitions were observed in 50 positions (40%) in different subclades (table 2). The sites with the highest number of recurrences match the HVS-I hot-spot sites identified previously (Hasegawa et al. 1993; Malyarchuk and Derenko 2001b; Allard et al. 2002). The most variable positions, 16093 and 16311, had received parallel hits in seven different subclusters; 16189 in six; 16092, 16304, and 16362 each in five; and 16129, 16209, 16249, and 16325 each in four subclusters. Another 12 HVS-I mutations were found in three and 28 substitutions in two different phylogenetic contexts. Because quite a few of these hot-spot mutations are present in HVS-I haplotypes that have been highlighted as having founder status in Europe (Richards et al. 2000), our results document again that additional coding region information is essential and unavoidable in defining monophyletic subclades of Hg H reliably (Torroni et al. 1993; Bandelt et al. 2001; Kivisild et al. 2002). We also found that a reversion of A to the ancestral base G at np 73 of the HVS-II, noticed in Hg H first by Torroni et al. (1996), has occurred independently at least four times in Hg H phylogeny (see also Helgason et al. 2000).
Table 2 Nucleotide Positions in the First Hypervariable Segment (HVS-I) That Have Received More Than One Transitional Hit in Haplogroup H
In the coding region, a transition at np 3010 that defines sub-Hg H1 (Finnil?, Lehtonen, and Majamaa 2001) is phylogenetically equally problematic. The derived state at np 3010 has been detected in haplogroups C, D, H, J, L2, L3, and U, making this base pair one of the fastest evolving mtDNA coding region positions (Ingman et al. 2000; Finnil?, Lehtonen, and Majamaa 2001; Maca-Meyer et al. 2001; Torroni et al. 2001b; Herrnstadt et al. 2002). Character conflicts at np 3010 and at more conserved nps 1462 (occurs also in Hgs H*, H2, T), 6272 (H*, L3), 6776 (H3), 8470 (H3), 12172 (H*, L1, U2), and 14869 (H*, K2, L3) were found (fig. S1). Given the data, the number of independent 3010A incidences in Hg H may possibly be as many as four (fig. S1).
Sub-Hg H4 was previously defined by an array of eight mutations (nps 3992, 4024, 5004, 8269, 9123, 10044, 14365, and 14582) through an analysis of haplotypes that occurred in at least two individuals (Herrnstadt et al. 2002). However, re-examination of the sequence data of Herrnstadt et al. (2002) revealed that only six mutations at nps 3992, 4024, 5004, 9123, 14365, and 14582 appear to be necessary to characterize the clade (fig. S1). Consequently, here we name the bough defined by a G-to-A mutation at np 8269, which further embraces the 10044 twig, as H4a.
While applying the RFLP method we discovered three previously unknown mutations: a transition at np 13760 abolishing the AciI site at np 13757 defining sub-Hg H11, a transition at np 5005 eliminating the H4-defining DdeI site at np 5003, and a transition at np 8449 eliminating the H11-defining np 8446 SspI site. Therefore, we confirmed the presence of H4-specific T at np 5004 by sequencing the position in all 12 samples lacking the DdeI 5003 site. The monophyly of sub-Hg H11 is well established by the combination of two RFLPs and by the characteristic HVS-I mutation pattern. These results show that classical indirect DNA polymorphism detection methods, like RFLP, should be backed-up by direct sequencing in order to avoid the ambiguous or even erroneous inference of phylogeny.
The next paragraphs address the main phylogeographic results. The largest subcluster is sub-Hg H1, which comprises about 30% of Hg H and 13% of the total European mtDNA pool. H1 is most frequent in the Iberian Peninsula, covering about 46% of local Hg H lineages (Pereira et al. 2004; Quintans et al. 2004). In the Near East the frequency of H1 does not exceed 6% (P < .025), and its relative frequency with respect to Hg H is lower than that seen in Europe (14%). In the Central Asian populations, where Hg H makes up about 11% of the local mtDNA pool, only 6% of H samples belong to sub-Hg H1 (table 3).
Table 3 Frequencies of Haplogroup H Subhaplolgroups in Studied Populations
Sub-Hg H1b is found throughout the area of the spread of Hg H, more frequently found in Eastern Europe and north central Europe (about 7% and 5% of Hg H, respectively; fig. 1B). It was also found to make up about 5% of Hg H in Siberian Mansis. A minor sub-Hg H1f constitutes a quarter of the selected subset of Finnish Hg H genomes of Finnil?, Lehtonen, and Majamaa (2001), being almost absent elsewhere in Europe. Confirmation of the high frequency of this rare variant of mtDNA among northern central Finns, characterized by HVS-I motif 16093–16189, can be found in the Finnish data of Meinil?, Finnil?, and Majamaa (2001), reflecting founder effects in the Finnish population history (Nevanlinna 1972; de la Chapelle and Wright 1998; Kittles et al. 1999; Peltonen, Palotie, and Lange 2000). In our previous study (Tambets et al. 2004), we assumed monophyly of the transition at np 16162. This mutation occurs in the motif 73–3010–16162, which defines H1a. The finding of an Eastern Slav individual bearing haplotype 73–16093–16162 and lacking H1 defining transition at np 3010 hints that motif 73–16162 may have arisen, though rarely, more than once in haplogroup H (fig. 2A). Alternatively, and bearing in mind that position 3010 is a mutational hot-spot (see above), one may consider a parallel occurrence or a reverse mutation at this position.
Like H1b, sub-Hg H2a occurs more frequently (P < .05) in Eastern than in Western European Hg H genomes, 6.5% and 1.1%, respectively, when averaged over populations (table 3 and fig. 2B). The spread of H2a extends to Central Asia, mimicking to some extent, albeit at a lower frequency, the phylogeography of Y-chromosomal Hg R1a (Rosser et al. 2000; Wells et al. 2001). In contrast, sub-Hg H3 was found to be more frequent (P < .05) in the Western (11.7%) than in the Eastern European Hg H pool (4.1%) and is virtually absent in Anatolia and the Near East (fig. 2B), resembling in its phylogeography the spread of Y-chromosomal Hg R1b associated 49a,f TaqI haplotype 15 (Semino et al. 1996; Cinnioglu et al. 2004). The high frequency of mtDNA Hg H3—in combination with Y chromosomal Ht 15—extends to the Iberian Peninsula, where H3 constitutes about 17% of Hg H and is the highest detected so far (Pereira et al. 2004; Quintans et al. 2004).
The coalescence ages of H2a1 and H3 fall to the period of postglacial recolonization in Europe (table 1), suggested first for mtDNA Hg V (Torroni et al. 1998, 2001a). We also note that mtDNA bearing "St. Luke motif," 16235–16293 (Vernesi et al. 2001), belong to sub-Hg H2 (fig. 2A), being particularly frequent in Germany and Scotland (Helgason et al. 2001; Pfeiffer et al. 2001).
The Near Eastern samples cluster together with Central Asian mtDNAs in the sub-Hgs H6b and H8, which are very rare in Europe. The finding is demonstrating a separate flow of maternal lineages south of the Caspian and the Black Sea in addition to well-known long-lasting migrations of pastoral nomads alongside the steppe belt that connects the Danube Basin, over the Pontic-Caspian, with Central Asia, Altay, and Manchuria.
In contrast to that found in Europeans, sub-Hgs H6 and H8 among Central Asian/Altaian populations are characterized by distinctly divergent haplotypes (fig. 2A). This finding may reflect a long-time separation of Asian and European H6 and H8 mtDNA pools and/or an earlier expansion of H6 in the eastern part of its present range. Indeed, the coalescence age of H6 in Central Asians is very deep—40,400 years (SD 16,400 years; table S1). Because the Asian branches of sub-Hg H6 are highly divergent and seem to be among the oldest in Hg H (table S1), they pose an interesting problem, deserving specific study with a much larger sample size at hand.
The commonly used HVS-I clock (Forster et al. 1996) places the initial expansion of Hg H in the Near East to about 23,000 to 28,000 years before the present (Richards et al. 2000). The ancestral clades of Hg H, pre-HV, and HV* have their combined present range predominantly in the Near and Middle East, and in the Caucasus (Metspalu et al. 1999; Richards et al. 2002), implying this could have been the region where the pre-HV/HV clade started to diversify and, possibly, where the earliest Hg H variants might have first appeared.
However, most subclusters of Hg H exhibit coalescence ages, corresponding to the beginning of their expansion in the Late Upper Paleolithic (tables 1 and S1). In this respect our results support an earlier proposition that Hg H was the major mtDNA haplogroup participating in the recolonization of Europe after the Last Glacial Maximum (Torroni et al. 1998; Richards et al. 2000). It is also important to note that the expansion time estimates derived from the coding region and HVS-I of Hg H are often in reasonable agreement with each other (tables 1 and S1). Sub-Hgs H1 and H3 have their highest frequencies in the Iberian Peninsula. These sub-Hgs may have been the companions of mtDNA Hg V in the postglacial repeopling of Europe from a refuge area in Iberia (Torroni et al. 1998). However, in contrast to Hg V, suggested coalescence ages of H1 and H3—13,400 ± 3,000 and 8,600 ± 2,800 years ago, respectively (Pereira et al. 2004)—do not imply deeper phylogeny of H1 and H3 in Iberia compared to the rest of Europe (tables 1 and S1).
These results demonstrate that a seemingly uniform spread of this major human mtDNA clade in western Eurasian populations hides within itself a complex structure of phylogeographically informative subclades. However, it is evident that additional knowledge at the level of complete mtDNA sequences is still needed for a truly comprehensive cataloguing of Hg H diversity, in particular more effectively covering its variation in the Mediterranean, Near and Middle Eastern, and Central Asian/Altaian populations. Nevertheless, even now it is tempting to speculate that much deeper coalescence ages, close to/overlapping with the boundary between the Middle and Upper Paleolithic, for some Hg H branches in Central Asian/Altaian populations, suggest that the time depth of this predominant haplogroup may be much deeper than its apparent general signal for expansion in Europe. It is, therefore, possible that the carriers of pre-Aurignacian industry identified in Zagros as well as in Altay (Otte and Derevianko 2001) were anatomically modern humans already possessing Hg H.
Supplementary Material
Supplementary Appendixes S1 and S2, figure S1, and tables S1 and S2 are available at the journal's Web site as well as the Web site of the University of Tartu, Department of Evolutionary Biology (http://www.evolutsioon.ut.ee/mtDNA-H/).
Acknowledgements
We thank Ille Hilpus and Jaan Lind for technical assistance and we are grateful to Vladimir Ferák for providing Slovak samples. The research of R.V. was supported by Estonian basic research grant 514 and European Community grants ICA1CT20070006 and QLG2-CT-2002–90455. T.K. was supported by Estonian Science Fund grant 5574. The research of B.A.M. was supported by the Russian Foundation for Basic Research (project number 03–04–48162). P.R. received support from the Ministry of Science and Technology of the Republic of Croatia (project number 0196005). The research of V.S. and V.P. was supported by a grant (project number 03–04–49021) from the Russian Foundation for Basic Research and by grants from the President of the Russian Federation (projects number MD-88.2003.04 and NSh-840.2003.4).
References
Allard, M. W., K. Miller, M. Wilson, K. Monson, and B. Budowle. 2002. Characterization of the Caucasian haplogroups present in the SWGDAM forensic mtDNA dataset for 1771 human control region sequences. Scientific Working Group on DNA Analysis Methods. J. Forensic Sci. 47:1215–1223.
Anderson, S., A. T. Bankier, B. G. Barrell et al. (14 co-authors). 1981. Sequence and organization of the human mitochondrial genome. Nature 290:457–465.
Andrews, R. M., I. Kubacka, P. F. Chinnery, R. N. Lightowlers, D. M. Turnbull, and N. Howell. 1999. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat. Genet. 23:147.
Arnason, U., X. Xu, and A. Gullberg. 1996. Comparison between the complete mitochondrial DNA sequences of Homo and the common chimpanzee based on nonchimeric sequences. J. Mol. Evol. 42:145–152.
Bamshad, M., T. Kivisild, W. S. Watkins et al. (18 co-authors). 2001. Genetic evidence on the origins of Indian caste populations. Genome Res. 11:994–1004.
Bandelt, H.-J., P. Forster, and A. R?hl. 1999. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol. 16:37–48.
Bandelt, H.-J., P. Forster, B. C. Sykes, and M. B. Richards. 1995. Mitochondrial portraits of human populations using median networks. Genetics 141:743–753.
Bandelt, H. J., P. Lahermo, M. Richards, and V. Macaulay. 2001. Detecting errors in mtDNA data by phylogenetic analysis. Int. J. Legal Med. 115:64–69.
Bermisheva, M., K. Tambets, R. Villems, and E. Khusnutdinova. 2002. Diversity of mitochondrial DNA haplotypes in ethnic populations of the Volga-Ural region of Russia. Mol. Biol. (Mosk) 36:990–1001.
Cinnioglu, C., R. King, T. Kivisild et al. (15 co-authors). 2004. Excavating Y-chromosome haplotype strata in Anatolia. Hum. Genet. 114:127–148.
Comas, D., F. Calafell, E. Mateu et al. (12 co-authors). 1998. Trading genes along the silk road: mtDNA sequences and the origin of Central Asian populations. Am. J. Hum. Genet. 63:1824–1838.
Corte-Real, H. B., V. A. Macaulay, M. B. Richards, G. Hariti, M. S. Issad, A. Cambon-Thomsen, S. Papiha, J. Bertranpetit, and B. C. Sykes. 1996. Genetic diversity in the Iberian Peninsula determined from mitochondrial sequence analysis. Ann. Hum. Genet. 60:331–350.
de la Chapelle, A., and F. A. Wright. 1998. Linkage disequilibrium mapping in isolated populations: the example of Finland revisited. Proc. Natl. Acad. Sci. USA 95:12416–12423.
Derbeneva, O. A., E. B. Starikovskaya, D. C. Wallace, and R. I. Sukernik. 2002a. Traces of early Eurasians in the Mansi of northwest Siberia revealed by mitochondrial DNA analysis. Am. J. Hum. Genet. 70:1009–1014.
Derbeneva, O. A., R. I. Sukernik, N. V. Volodko, S. H. Hosseini, M. T. Lott, and D. C. Wallace. 2002b. Analysis of mitochondrial DNA diversity in the Aleuts of the Commander Islands and its implications for the genetic history of Beringia. Am. J. Hum. Genet. 71:415–421.
Derenko, M. V., T. Grzybowski, B. A. Malyarchuk et al. (11 co-authors). 2003. Diversity of mitochondrial DNA lineages in south Siberia. Ann. Hum. Genet. 67:391–411.
Dimo-Simonin, N., F. Grange, F. Taroni, C. Brandt-Casadevall, and P. Mangin. 2000. Forensic evaluation of mtDNA in a population from south west Switzerland. Int. J. Legal Med. 113:89–97.
Finnil?, S., I. E. Hassinen, L. Ala-Kokko, and K. Majamaa. 2000. Phylogenetic network of the mtDNA haplogroup U in Northern Finland based on sequence analysis of the complete coding region by conformation-sensitive gel electrophoresis. Am. J. Hum. Genet. 66:1017–1026.
Finnil?, S., M. S. Lehtonen, and K. Majamaa. 2001. Phylogenetic network for European mtDNA. Am. J. Hum. Genet. 68:1475–1484.
Finnil?, S., and K. Majamaa. 2001. Phylogenetic analysis of mtDNA haplogroup TJ in a Finnish population. J. Hum. Genet. 46:64–69.
Forster, P., R. Harding, A. Torroni, and H.-J. Bandelt. 1996. Origin and evolution of Native American mtDNA variation: a reappraisal. Am. J. Hum. Genet. 59:935–945.
Hasegawa, M., A. Di Rienzo, T. D. Kocher, and A. C. Wilson. 1993. Toward a more accurate time scale for the human mitochondrial DNA tree. J. Mol. Evol. 37:347–354.
Helgason, A., E. Hickey, S. Goodacre, V. Bosnes, K. Stefansson, R. Ward, and B. Sykes. 2001. mtDNA and the islands of the North Atlantic: estimating the proportions of Norse and Gaelic ancestry. Am. J. Hum. Genet. 68:723–737.
Helgason, A., S. Sigurdadottir, J. Gulcher, R. Ward, and K. Stefanson. 2000. mtDNA and the origins of the Icelanders: deciphering signals of recent population history. Am. J. Hum. Genet. 66:999–1016.
Herrnstadt, C., J. L. Elson, E. Fahy et al. (11 co-authors). 2002. Reduced-median-network analysis of complete mitochondrial DNA coding-region sequences for the major African, Asian, and European haplogroups. Am. J. Hum. Genet. 70:1152–1171.
Herrnstadt, C., G. Preston, and N. Howell. 2003. Errors, phantoms and otherwise, in human mtDNA sequences. Am. J. Hum. Genet. 72:1585–1586.
Hofmann, S., M. Jaksch, R. Bezold, S. Mertens, S. Aholt, A. Paprotta, and K. D. Gerbitz. 1997. Population genetics and disease susceptibility: characterization of central European haplogroups by mtDNA gene mutations, correlation with D loop variants and association with disease. Hum. Mol. Genet. 6:1835–1846.
Ingman, M., H. Kaessmann, S. P??bo, and U. Gyllensten. 2000. Mitochondrial genome variation and the origin of modern humans. Nature 408:708–713.
Kittles, R. A., A. W. Bergen, M. Urbanek, M. Virkkunen, M. Linnoila, D. Goldman, and J. C. Long. 1999. Autosomal, mitochondrial, and Y chromosome DNA variation in Finland: evidence for a male-specific bottleneck. Am. J. Phys. Anthropol. 108:381–399.
Kivisild, T., M. J. Bamshad, K. Kaldma et al. (15 co-authors). 1999. Deep common ancestry of Indian and western-Eurasian mitochondrial DNA lineages. Curr. Biol. 9:1331–1334.
Kivisild, T., S. Rootsi, M. Metspalu et al. (18 co-authors). 2003. The genetic heritage of the earliest settlers persists both in Indian tribal and caste populations. Am. J. Hum. Genet. 72:313–332.
Kivisild, T., H.-V. Tolk, J. Parik, Y. Wang, S. S. Papiha, H.-J. Bandelt, and R. Villems. 2002. The emerging limbs and twigs of the East Asian mtDNA tree. Mol. Biol. Evol. 19:1737–1751.
Kong, Q. P., Y. G. Yao, C. Sun, H. J. Bandelt, C. L. Zhu, and Y. P. Zhang. 2003. Phylogeny of east Asian mitochondrial DNA lineages inferred from complete sequences. Am. J. Hum. Genet. 73:671–676.
Levin, B. C., H. Cheng, and D. J. Reeder. 1999. A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection. Genomics 55:135–146.
Maca-Meyer, N., A. M. Gonzalez, J. M. Larruga, C. Flores, and V. M. Cabrera. 2001. Major genomic mitochondrial lineages delineate early human expansions. BMC Genet. 2:13.
Macaulay, V. A., M. B. Richards, E. Hickey, E. Vega, F. Cruciani, V. Guida, R. Scozzari, B. Bonné-Tamir, B. Sykes, and A. Torroni. 1999. The emerging tree of West Eurasian mtDNAs: a synthesis of control-region sequences and RFLPs. Am. J. Hum. Genet. 64:232–249.
Malyarchuk, B. A., and M. V. Derenko. 2001a. Mitochondrial DNA variability in Russians and Ukrainians: implications to the origin of the Eastern Slavs. Ann. Hum. Genet. 65:63–78.
Malyarchuk, B. A., and M. V. Derenko. 2001b. Variation of human mitochondrial DNA: distribution of hot spots in hypervariable segment I of the major noncoding region. Genetika 37:991–1001.
Malyarchuk, B. A., T. Grzybowski, M. V. Derenko, J. Czarny, K. Drobnic, and D. Miscicka-Sliwka. 2003. Mitochondrial DNA variability in Bosnians and Slovenians. Ann. Hum. Genet. 67:412–425.
Malyarchuk, B. A., T. Grzybowski, M. V. Derenko, J. Czarny, M. Wozniak, and D. Miscicka-Sliwka. 2002. Mitochondrial DNA variability in Poles and Russians. Ann. Hum. Genet. 66:261–283.
Marzuki, S., A. S. Noer, P. Lertrit, D. Thyagarajan, R. Kapsa, P. Utthanaphol, and E. Byrne. 1991. Normal variants of human mitochondrial DNA and translation products: the building of a reference data base. Hum. Genet. 88:139–145.
Meinil?, M., S. Finnil?, and K. Majamaa. 2001. Evidence for mtDNA admixture between the Finns and the Saami. Hum. Hered. 52:160–170.
Metspalu, E., T. Kivisild, K. Kaldma, J. Parik, M. Reidla, K. Tambets, and R. Villems. 1999. The Trans-Caucasus and the expansion of the Caucasoid-specific human mitochondrial DNA. Pp. 121–134 in S. Papiha, R. Deka, and R. Chakraborty, eds. Genomic diversity: application in human population genetics. Kluwer Academic/Plenum Publishers, New York.
Mishmar, D., E. Ruiz-Pesini, P. Golik et al. (13 co-authors). 2003. Natural selection shaped regional mtDNA variation in humans. Proc. Natl. Acad. Sci. USA 100:171–176.
Nevanlinna, H. R. 1972. The Finnish population structure. A genetic and genealogical study. Hereditas 71:195–236.
Otte M., and A. Derevianko. 2001. The Aurignacian in Altai. Antiquity 75:44–48.
Parson, W., T. J. Parsons, R. Scheithauer, and M. M. Holland. 1998. Population data for 101 Austrian Caucasian mitochondrial DNA d-loop sequences: application of mtDNA sequence analysis to a forensic case. Int. J. Legal Med. 111:124–132.
Passarino, G., O. Semino, L. F. Bernini, and A. S. Santachiara-Benerecetti. 1996. Pre-Caucasoid and Caucasoid genetic features of the Indian population, revealed by mtDNA polymorphisms. Am. J. Hum. Genet. 59:927–934.
Peltonen, L., A. Palotie, and K. Lange. 2000. Use of population isolates for mapping complex traits. Nat. Rev. Genet. 1:182–190.
Pereira L., C. Cunha, and A. Amorim. 2004. Predicting sampling saturation of mtDNA haplotypes: an application to an enlarged Portuguese database. Int. J. Legal. Med. 118:132–136.
Pereira, L., M. Richards, A. Alonso, C. Albarran, O. Garcia, V. Macaulay, and A. Amorim. 2004. Subdividing mtDNA haplogroup H based on coding-region polymorphisms—a study in Iberia. Int. Congr. Ser. 1261:416–418.
Pfeiffer, H., P. Forster, C. Ortmann, and B. Brinkmann. 2001. The results of an mtDNA study of 1,200 inhabitants of a German village in comparison to other Caucasian databases and its relevance for forensic casework. Int. J. Legal Med. 114:169–172.
Piercy, R., K. M. Sullivan, N. Benson, and P. Gill. 1993. The application of mitochondrial DNA typing to the study of white Caucasian genetic identification. Int. J. Legal Med. 106:85–90.
Polyak, K., Y. Li, H. Zhu, C. Lengauer, J. K. Willson, S. D. Markowitz, M. A. Trush, K. W. Kinzler, and B. Vogelstein. 1998. Somatic mutations of the mitochondrial genome in human colorectal tumours. Nat. Genet. 20:291–293.
Quintans, B., V. Alvarez-Iglesias, A. Salas, C. Phillips, M. V. Lareu, and A. Carracedo. 2004. Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing. Forensic Sci. Int. 140:251–257.
Rando, J. C., F. Pinto, A. M. Gonzalez, M. Hernandez, J. M. Larruga, V. M. Cabrera, and H. J. Bandelt. 1998. Mitochondrial DNA analysis of northwest African populations reveals genetic exchanges with European, Near-Eastern, and sub-Saharan populations. Ann. Hum. Genet. 62:531–550.
Reid, F. M., G. A. Vernham, and H. T. Jacobs. 1994. Complete mtDNA sequence of a patient in a maternal pedigree with sensorineural deafness. Hum. Mol. Genet. 3:1435–1436.
Reidla, M., T. Kivisild, E. Metspalu et al. (43 co-authors). 2003. Origin and diffusion of mtDNA haplogroup X. Am. J. Hum. Genet. 73:1178–1190.
Richards, M., H. Corte-Real, P. Forster, V. Macaulay, H. Wilkinson-Herbots, A. Demaine, S. Papiha, R. Hedges, H.-J. Bandelt, and B. Sykes. 1996. Paleolithic and neolithic lineages in the European mitochondrial gene pool. Am. J. Hum. Genet. 59:185–203.
Richards, M., V. Macaulay, E. Hickey et al. (26 co-authors). 2000. Tracing European founder lineages in the Near Eastern mtDNA pool. Am. J. Hum. Genet. 67:1251–1276.
Richards, M., V. Macaulay, A. Torroni, and H. J. Bandelt. 2002. In search of geographical patterns in European mitochondrial DNA. Am. J. Hum. Genet. 71:1168–1174.
Richards, M. B., V. A. Macaulay, H.-J. Bandelt, and B. C. Sykes. 1998. Phylogeography of mitochondrial DNA in western Europe. Ann. Hum. Genet. 62:241–260.
Rieder, M. J., S. L. Taylor, V. O. Tobe, and D. A. Nickerson. 1998. Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome. Nucleic Acids Res. 26:967–973.
Rosser, Z. H., T. Zerjal, M. E. Hurles et al. (63 co-authors). 2000. Y-chromosomal diversity in Europe is clinal and influenced primarily by geography, rather than by language. Am. J. Hum. Genet. 67:1526–1543.
Saillard, J., I. Evseva, L. Tranebjaerg, and S. Norby. 2000a. Mitochondrial DNA diversity among Nenets. Pp. 255–258 in C. Renfrew and K. Boyle, eds. Archaeogenetics: DNA and and the population prehistory of Europe. McDonald Institute for Archaeological Research Monograph Series, Cambridge University, Cambridge.
Saillard, J., P. Forster, N. Lynnerup, H.-J. Bandelt, and S. N?rby. 2000b. mtDNA variation among Greenland Eskimos: the edge of the Beringian expansion. Am. J. Hum. Genet. 67:718–726.
Semino, O., G. Passarino, A. Brega, M. Fellous, and A. S. Santachiara-Benerecetti. 1996. A view of the neolithic demic diffusion in Europe through two Y chromosome-specific markers. Am. J. Hum. Genet. 59:964–968.
Stevanovitch, A., A. Gilles, E. Bouzaid, R. Kefi, F. Paris, R. P. Gayraud, J. L. Spadoni, F. El-Chenawi, and E. Beraud-Colomb. 2004. Mitochondrial DNA sequence diversity in a sedentary population from Egypt. Ann. Hum. Genet. 68:23–39.
Tambets, K., T. Kivisild, E. Metspalu et al. (13 co-authors). 2000. The topology of the maternal lineages of the Anatolian and Trans-Caucasus populations and the peopling of the Europe: some preliminary considerations. Pp. 219–235 in C. Renfrew and K. Boyle, eds. Archaeogenetics: DNA and the population prehistory of Europe. Cambridge University Press, Cambridge.
Tambets, K., S. Rootsi, T. Kivisild et al. (46 co-authors). 2004. The western and eastern Roots of the Saami—the story of genetic "outliers" told by mitochondrial DNA and Y chromosomes. Am. J. Hum. Genet. 74:661–682.
Torroni, A., H.-J. Bandelt, L. D'Urbano et al. (11 co-authors). 1998. mtDNA analysis reveals a major late Paleolithic population expansion from southwestern to northeastern Europe. Am. J. Hum. Genet. 62:1137–1152.
Torroni, A., H. J. Bandelt, V. Macaulay et al. (33 co-authors). 2001a. A signal, from human mtDNA, of postglacial recolonization in Europe. Am. J. Hum. Genet. 69:844–852.
Torroni, A., K. Huoponen, P. Francalacci, M. Petrozzi, L. Morelli, R. Scozzari, D. Obinu, M. L. Savontaus, and D. C. Wallace. 1996. Classification of European mtDNAs from an analysis of three European populations. Genetics 144:1835–1850.
Torroni, A., M. T. Lott, M. F. Cabell, Y. S. Chen, L. Lavergne, and D. C. Wallace. 1994. mtDNA and the origin of Caucasians: identification of ancient Caucasian-specific haplogroups, one of which is prone to a recurrent somatic duplication in the D-loop region. Am. J. Hum. Genet. 55:760–776.
Torroni, A., C. Rengo, V. Guida et al. (12 co-authors). 2001b. Do the four clades of the mtDNA haplogroup L2 evolve at different rates? Am. J. Hum. Genet. 69:1348–1356.
Torroni, A., R. I. Sukernik, T. G. Schurr, Y. B. Starikorskaya, M. F. Cabell, M. H. Crawford, A. G. Comuzzie, and D. C. Wallace. 1993. mtDNA variation of aboriginal Siberians reveals distinct genetic affinities with Native Americans. Am. J. Hum. Genet. 53:591–608.
Vanecek, T., F. Vorel, and M. Sip. 2004. Mitochondrial DNA D-loop hypervariable regions: Czech population data. Int. J. Legal Med. 118:14–18.
Vernesi, C., G. Di Benedetto, D. Caramelli, E. Secchieri, L. Simoni, E. Katti, P. Malaspina, A. Novelletto, V. T. Marin, and G. Barbujani. 2001. Genetic characterization of the body attributed to the evangelist Luke. Proc. Natl. Acad. Sci. USA 98:13460–13463. Epub 12001 Oct 13416.
Wells, R. S., N. Yuldasheva, R. Ruzibakiev et al. (27 co-authors). 2001. The Eurasian heartland: a continental perspective on Y-chromosome diversity. Proc. Natl. Acad. Sci. USA 98:10244–10249.(Eva-Liis Loogv?li*, Urmas)