ral ssBioMed CentBMC Evolutionary Biology Open AcceResearch article Phylogeography of mtDNA haplogroup R7 in the Indian peninsula Gyaneshwer Chaubey1,2, Monika Karmin1, Ene Metspalu1, Mait Metspalu1, Deepa Selvi-Rani2, Vijay Kumar Singh2, Jüri Parik1, Anu Solnik1, B Prathap Naidu2, Ajay Kumar2,5, Niharika Adarsh2,5, Chandana Basu Mallick2,5, Bhargav Trivedi2,5, Swami Prakash2,5, Ramesh Reddy2,5, Parul Shukla2,5, Sanjana Bhagat2,5, Swati Verma2,5, Samiksha Vasnik2,5, Imran Khan2,5, Anshu Barwa2,5, Dipti Sahoo2,5, Archana Sharma2,5, Mamoon Rashid2,5, Vishal Chandra2,5, Alla G Reddy2, Antonio Torroni3, Robert A Foley4, Kumarasamy Thangaraj2, Lalji Singh2, Toomas Kivisild*1,4 and Richard Villems1 Address: 1Department of Evolutionary Biology, Institute of Molecular and Cell Biology, University of Tartu and Estonian Biocentre, Tartu, Estonia, 2Centre for Cellular and Molecular Biology, Hyderabad, India, 3Dipartimento di Genetica e Microbiologia, Università di Pavia, Via Ferrata 1, 27100 Pavia, Italy, 4Leverhulme Centre of Human Evolutionary Studies, The Henry Wellcome Building, University of Cambridge, Fitzwilliam Street, Cambridge, CB2 1QH, UK and 5Students of different Universities and Colleges of India studied (as a part of their curriculum) in CCMB Hyderabad, India Email: Gyaneshwer Chaubey - gyanc@ebc.ee; Monika Karmin - monikaka@ut.ee; Ene Metspalu - emetspal@ebc.ee; Mait Metspalu - mait@ebc.ee; Deepa Selvi-Rani - deepa@ccmb.res.in; Vijay Kumar Singh - vijay81200@yahoo.com; Jüri Parik - jparik@ebc.ee; Anu Solnik - ann4@ebc.ee; B Prathap Naidu - bpn_naidu@ccmb.res.in; Ajay Kumar - ajay_biotech1@rediffmail.com; Niharika Adarsh - niharika.adarsh@gmail.com; Chandana Basu Mallick - cbm25@yahoo.com; Bhargav Trivedi - Bhargavtrivedi@gmail.com; Swami Prakash - swami.prakash@gmail.com; Ramesh Reddy - ramesh.reddy@gmail.com; Parul Shukla - parul.shukla@gmail.com; Sanjana Bhagat - sanjana_bhagat@rediffmail.com; Swati Verma - swatigole@gmail.com; Samiksha Vasnik - samiksha.vasnik@gmail.com; Imran Khan - btimran@gmail.com; Anshu Barwa - anshu_barwa@yahoo.com; Dipti Sahoo - dipti@nomail.com; Archana Sharma - arc_1231@rediffmail.com; Mamoon Rashid - amara4u1980@yahoo.com; Vishal Chandra - vishal.chandra@gmail.com; Alla G Reddy - agreddy@ccmb.res.in; Antonio Torroni - torroni@ipvgen.unipv.it; Robert A Foley - r.foley@human-evol.cam.ac.uk; Kumarasamy Thangaraj - thangs@ccmb.res.in; Lalji Singh - lalji@ccmb.res.in; Toomas Kivisild* - tk331@cam.ac.uk; Richard Villems - rvillems@ebc.ee * Corresponding author Abstract Background: Human genetic diversity observed in Indian subcontinent is second only to that of Africa. This implies an early settlement and demographic growth soon after the first 'Out-of-Africa' dispersal of anatomically modern humans in Late Pleistocene. In contrast to this perspective, linguistic diversity in India has been thought to derive from more recent population movements and episodes of contact. With the exception of Dravidian, which origin and relatedness to other language phyla is obscure, all the language families in India can be linked to language families spoken in different regions of Eurasia. Mitochondrial DNA and Y chromosome evidence has supported largely local evolution of the genetic lineages of the majority of Dravidian and Indo-European Published: 4 August 2008 BMC Evolutionary Biology 2008, 8:227 doi:10.1186/1471-2148-8-227 Received: 11 April 2008 Accepted: 4 August 2008 This article is available from: http://www.biomedcentral.com/1471-2148/8/227 © 2008 Chaubey et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 12 (page number not for citation purposes) speaking populations, but there is no consensus yet on the question of whether the Munda (Austro- BMC Evolutionary Biology 2008, 8:227 http://www.biomedcentral.com/1471-2148/8/227 Asiatic) speaking populations originated in India or derive from a relatively recent migration from further East. Results: Here, we report the analysis of 35 novel complete mtDNA sequences from India which refine the structure of Indian-specific varieties of haplogroup R. Detailed analysis of haplogroup R7, coupled with a survey of ~12,000 mtDNAs from caste and tribal groups over the entire Indian subcontinent, reveals that one of its more recently derived branches (R7a1), is particularly frequent among Munda-speaking tribal groups. This branch is nested within diverse R7 lineages found among Dravidian and Indo-European speakers of India. We have inferred from this that a subset of Munda- speaking groups have acquired R7 relatively recently. Furthermore, we find that the distribution of R7a1 within the Munda-speakers is largely restricted to one of the sub-branches (Kherwari) of northern Munda languages. This evidence does not support the hypothesis that the Austro-Asiatic speakers are the primary source of the R7 variation. Statistical analyses suggest a significant correlation between genetic variation and geography, rather than between genes and languages. Conclusion: Our high-resolution phylogeographic study, involving diverse linguistic groups in India, suggests that the high frequency of mtDNA haplogroup R7 among Munda speaking populations of India can be explained best by gene flow from linguistically different populations of Indian subcontinent. The conclusion is based on the observation that among Indo-Europeans, and particularly in Dravidians, the haplogroup is, despite its lower frequency, phylogenetically more divergent, while among the Munda speakers only one sub-clade of R7, i.e. R7a1, can be observed. It is noteworthy that though R7 is autochthonous to India, and arises from the root of hg R, its distribution and phylogeography in India is not uniform. This suggests the more ancient establishment of an autochthonous matrilineal genetic structure, and that isolation in the Pleistocene, lineage loss through drift, and endogamy of prehistoric and historic groups have greatly inhibited genetic homogenization and geographical uniformity. Background More than one sixth of humanity currently lives on the Indian subcontinent. This population is spread across up to 40,000 endogamous and semi-endogamous culturally, linguistically, and socially differentiated groups [1]. The majority of these groups or populations are castes, but they also include nearly 500 'scheduled tribes' [2] and ca. 500 'scheduled castes' [3]. Thus, the Indian subcontinent is an ideal region for studying the relationships between culture, geography and genes, and for developing interdis- ciplinary models concerning the demographic history of Homo sapiens or anatomically modern humans (AMH). Moreover, the large number of deep-rooting mtDNA line- ages emerging from the basal nodes of both superhaplo- group M and N (including R) [4-11] indicate that the Indian subcontinent was probably the first major out- come of the dispersals of AMH from Africa. Furthermore, these deep-rooted mtDNA haplogroups generally cross cultural and social boundaries; this suggests a common origin to the highly diverse peoples of the Indian sub-con- tinent, with indigenous or autochthonous diversification of the maternal gene pool [12-16]. These results have been generally corroborated by data from the Y chromosome [17,18] and autosomal DNA share about half of their maternal genetic heritage with populations living further east of India [14,21]. It has been argued, that following the initial colonization of Indian subcontinent, maternal gene flow from the west has been rather limited and largely restricted to the west- ern states of contemporary India and Pakistan [14,15,22]. Consequently, the haplogroup richness of the Indian sub- continent appears to have formed in situ, and date back to some point in the later Pleistocene, most probably between 40 Ka and 60 Ka ago. Furthermore, this high level of genetic diversity may also be linked to the possibility that the South Asian population in the Pleistocene was demographically large in global terms. Comparisons of relative regional population sizes through time, deduced by Bayesian coalescent inference methods applied to glo- bal mtDNA complete sequence data, indicate that between approximately 45 Ka and 20 Ka ago most of humanity lived in Southern Asia [23]. Two language families, Indo-European and Dravidian, account for the majority of linguistic diversity in India. However, apart from a number of linguistic isolates, there are two other major families – Tibeto-Burman and Austro- Asiatic (AA). The origin of the Austro-Asiatic language family is a highly debated issue. Building on archaeologi-Page 2 of 12 (page number not for citation purposes) [13,19,20]. The only exception, for mtDNA, are the Tibeto-Burman speakers of north-eastern India, who cal and linguistic evidence, and the assumption that rice domestication was a single event, the currently preferred BMC Evolutionary Biology 2008, 8:227 http://www.biomedcentral.com/1471-2148/8/227 hypothesis places the origin of this language family in Southeast Asia [24-26]. The alternative model, based on genetic evidence (that shows multiple domestications of rice varieties [27]), and comparative phonology, advo- cates an East Indian cradle for the AA language group [28]. The AA language family tree has two basic branches – Munda and Mon-Khmer. The former is distributed exclu- sively in the Indian subcontinent; the latter is predomi- nantly Southeast Asian, although there are a few Indian representatives (Khasian and Nicobarese) [26]. The genetic origin(s) of extant AA speakers, however, may or may not coincide with the origin of the language group. Studies of mtDNA diversity have shown that the AA speakers from Southeast Asia and the Indian subcontinent carry mtDNAs of different sources [7,9,14,16,29,30]. Although the data on Southeast Asian populations, which speak languages of the Mon-Khmer branch of the AA tree, are still somewhat limited, it seems safe to conclude, that their mtDNA characteristics are similar to those of the sur- rounding Southeast Asian populations, and distinct from AA tribes of India (Munda-speakers) [30]. Similarly, the Indian tribes speaking different Munda languages show generally the same mtDNA haplogroup composition as the Indo European and Dravidic groups of India [9,14,16,29]. In contrast, the Y chromosomes of Indian and Southeast Asian AA speaking populations share a common marker, M95, which defines a single branch (O2a) in the overwhelmingly East Asian specific tree of haplogroup O. This evidence provides a strong basis for proposing a Southeast Asian origin of the paternal line- ages of the Munda speaking populations of India [13,17,18]. The AA speaking populations of Myanmar, which is a likely dispersal route, or original location, for the ances- tral populations of Munda speakers of India, have not yet been sampled for their mtDNA. It is still possible that some of the mtDNA clades present among the AA speak- ers of India (and in their neighbours) could, in fact, be due to gene flow to India from further east. In an attempt to identify mtDNA lineages that would reveal a phylogeo- graphic distribution similar to that of the Y chromosome marker M95, we analyzed mtDNA samples representing all the major linguistic groups of India, with a particular focus to haplogroup R derived lineages. The first thorough study of complete mtDNA sequences from India [4] identified numerous indigenous clades emerging directly from the roots of superhaplogroups N, R and U, such as N5, R5-R8, R30, R31, U2a-d and U7. West Eurasian specific haplogroups HV, JT, N1, and U (xU2a-d, U7) occur at lower frequencies, suggesting lim- and northwest Eurasia [14]. Here we have now extended the complete mtDNA sequencing by determining 35 new complete sequences, in order to further refine the phylog- eny of the Indian subcontinent-specific segment of haplo- group R. Furthermore, to explore the correlations between genes, languages and geography in Indian subcontinent, we have carried out high resolution genotyping and phyl- ogeographic detailed analyses on R7, which occurs at high frequency among the Austro-Asiatic (Munda) speaking groups of India. Results and Discussion The inclusion of our 35 novel sequences (Table 1) into the phylogeny of haplogroup R allows the recognition of eight new subclades within six haplogroup R branches unique to the Indian subcontinent (Fig. 1, [see Additional file 1]). We refine here the internal topology of haplo- groups R5, R6 and R8, and describe two novel sub-clades of hg R7, to be discussed below in detail. Subclade R5a is defined by a deletion at nucleotide positions (np) 522– 523 and one control region mutation at np16266. R6a is defined by two control region substitutions (at sites 16129 and 16266). In haplogroup R7, two new subclades R7a and R7b can be identified (for details see further down). A new subclade of R8, called R8a, is defined by a single coding region substitution at np 5510. Haplogroup R30 splits into two subclades R30a and R30b, the former supported by ten coding region substitutions and the lat- ter by 24 coding and control region mutations. Similarly, in haplogroup R31 a new subclade R31a can be distin- guished by 17 control and coding region mutations. Coa- lescent estimates suggest an ancient branching pattern in hgs R30 and R31, dating back almost to the earliest diver- sification of the superhaplogroup R itself. This most prob- ably occurred soon after the out of Africa dispersals into the Indian subcontinent [see Additional file 1]. Comparison of patterns of haplogroup distribution in relation to linguistic groups reveals that the frequency of the R7 clade is several times greater among AA (Munda) speakers than among Dravidian and Indo-European speaking populations (Table 2, [see Additional file 2]). Geographically, the distribution of R7 in India is centered on the AA "heartland" (Bihar, Jharkhand, and Chhattis- garh) [see Additional file 3]. Similar to R7, haplogroup R6 is significantly more frequent among the AA speakers than among other linguistic groups (Table 2, [see Additional file 2]). PC analysis based on frequency data of the hg R subclades confirms that the majority of Munda speaking populations cluster separately from others mainly because of higher hg R7 frequency (Fig. 2). However, only 50.6% of the variation can be explained by the first two principal components. Interestingly, hg R6 is placed within thePage 3 of 12 (page number not for citation purposes) ited but phylogeographically well detectable gene flow into the Indian subcontinent, most probably from west main cluster, which is comprised of populations from all BMC Evolutionary Biology 2008, 8:227 http://www.biomedcentral.com/1471-2148/8/227 language groups. Based on these preliminary results we focused on R7 as a potential AA-associated marker. In general, the elevated frequency of hg R7 among the AA speakers of India can be explained by two alternative sce- narios. Firstly, one may consider a possible origin of R7 among AA (Munda) speakers, possibly already outside India. Under this scenario the presence of R7 in some Dra- vidian and Indo-European speaking communities would be explained by its later introgression from the Munda communities, or by language shift of some Munda speak- ing groups into Dravidian/Indo-European languages. Sec- ondly, an origin of R7 may lie among non-AA populations of India, with the presently observable higher frequency of R7 among AA resulting from founder effect(s) due to random genetic drift. To test these two scenarios, we car- ried out a detailed analysis of R7 mtDNAs in populations among IE and Dravidian-speaking populations of Indian subcontinent. Complete mtDNA sequence-based topology of hg R7 divulges two deep-rooted subclades (Fig. 1). R7a is defined by four and R7b by six coding region mutations and, in addition, by two control-region substitutions (146 and 16311). We calculated the time to the most recent common ancestor (MRCA) for all R7 major sub-clades (Fig. 1 and 3, Table 3), applying different calibration methods [30,31]. All the AA individuals coalesce to the founder R7a1 that dates back to between approximately 3 Ka and 7 Ka ago, depending on the mutation rate used. The coalescent times of R7 variation among Dravidians and Indo-Europeans are older. In other words, the only R7 lineage found by us in AA speakers of India – R7a1 – is nested within the R7 lineages found among Dravidian Table 1: Geographical, Linguistic and Haplogroup Affiliations of Completely Sequenced mtDNAs. Si No. Sample code Haplogroup Population Location Lingustic affiliation 1 Kol77 R5a1 Koli Gujarat Indo-European 2 Ben46 R5a1a Bengal West Bengal Indo-European 3 Up41 R5a1a Middle caste Uttar Pradesh Indo-European 4 Kall43 R5a2b Kallar Tamil Nadu Dravidian 5 K35 R5a2b Kota Tamil Nadu Dravidian 6 Ori74 R5a2b2 Oraon Orissa Dravidian 7 Mo38 R5a2b3 Moor Sri Lanka Dravidian 8 Gu35 R5a2b3 Gujarat Gujarat Indo-European 9 Pn32 R5a2b4 Paniya Kerala Dravidian 10 Mal33 R5a2b4 Malayan Kerala Dravidian 11 Ko 5 R6a1a Koya Andhra Pradesh Dravidian 12 Ko31 R6a1a Koya Andhra Pradesh Dravidian 13 Lam43 R7a1 Lambadi Andhra-Pradesh Dravidian 14 As426 R7a1 Asur Jharkhand Austro-Asiatic 15 Mw1 R7a1a Mawasi Chhattisgarh Austro-Asiatic 16 Tor45 R7a1a Sindhi Pakistan Indo-European 17 Ho433 R7a1b1 Ho Jharkhand Austro-Asiatic 18 Ori7 R7a1b1 Oraon Jharkhand Dravidian 19 Ori37 R7b1a Oraon Orissa Dravidian 20 A474 R7a1b2 Oraon Jharkhand Dravidian 21 G39 R7a1b2 Santhal Bihar Austro-Asiatic 22 G19 R7a1b2 Kanwar Madhya-Pradesh Indo-European 23 KO18 R7b Koya Andhra-Pradesh Dravidian 24 KO55 R7b1a Koya Andhra-Pradesh Dravidian 25 G66 R7b1a Gond Madhya-Pradesh Dravidian 26 Ko74 R8a Koya Andhra Pradesh Dravidian 27 Lam10 R8a1a1 Lambadi Andhra Pradesh Dravidian 28 Ko30 R8a1a2 Koya Andhra Pradesh Dravidian 29 Ko37 R8a1a2 Koya Andhra Pradesh Dravidian 30 CoB41 R8a1b Konkanastha Brahmin Maharashtra Indo-European 31 CoB23 R30 Konkanastha Brahmin Maharashtra Indo-European 32 Sin49 R30a Sinhalese Sri Lanka Indo-European 33 Pun47 R30b Punjab Punjab Indo-European 34 Raj25 R31a1 Rajput Rajasthan Indo-European 35 Raj48 R31a1 Rajput Rajasthan Indo-EuropeanPage 4 of 12 (page number not for citation purposes) speaking different subgroups of AA languages, as well as and Indo-European speakers of India (Table 3). BMC Evolutionary Biology 2008, 8:227 http://www.biomedcentral.com/1471-2148/8/227 Page 5 of 12 (page number not for citation purposes) The most parsimonious tree of haplogroup R7 complete mtDNA sequences observed in the Indian subcontinentFigure 1 The most parsimonious tree of haplogroup R7 complete mtDNA sequences observed in the Indian subconti- nent. This tree was redrawn manually from the output of median joining/reduced network obtained using NETWORK pro- gram (version 4.1) [34]http://www.fluxus-engineering.com. The samples were selected through a preliminary sequence analysis of the control region in order to include the widest possible range of R7 variation, language and geographical groups. Coales- cent times were calculated by a calibration method described elsewhere [32]. 16182C, 16183C and 16519 polymorphisms were omitted. Suffixes A, C, G, and T indicate transversions, recurrent mutations are underlined. Synonymous (s) and non-syn- onymous (ns) mutations are distinguished. DRA-Dravidian, AA-Austro-Asiatic, IE-Indo-European. The ethnic affiliation of the samples is as follows: Lam, Lambadi; As, Asur; Mw, Mawasi; Tor45, Pakistan; Ho, Ho; Ori&A, Oraon; G19, Kanwar; G39, San- thal; G66, Gond; KO, Koya. Two sequences, T35 (Thogataveera) and C35 (Brahmin), were taken from the literature [4]. BMC Evolutionary Biology 2008, 8:227 http://www.biomedcentral.com/1471-2148/8/227 Geographically, the distribution of R7a frequency is con- centrated towards Bihar, Jharkhand and Chhattisgarh States, while R7b has its frequency peak in Andhra- Pradesh (Fig. 4a and 4b). The frequency of R7a is higher among AA (Munda) speakers, while R7b is most common among Dravidian speakers from Andhra-Pradesh, although the overall frequency of R7b is much lower than that of R7a (Fig. 4c and 4d). A Mantel test showed a sig- nificant correlation between genes and geography for the Indian R sub-clades, but no such correlation for the rela- tionship between genes and languages (Table 4). The spa- tial autocorrelation analysis favoured a clinal pattern for the distribution of hg R7 [see Additional file 4]. At the local (i.e. district) level, R7 is present in Bihar, Jharkhand, Chhattisgarh, Madhya-Pradesh and the northern districts of Andhra-Pradesh (Adilabad, Warangal and Khammam), whereas elsewhere in India it is virtually absent, including among other AA groups inhabiting Orissa and Maharash- tra states [see Additional file 5]. The overall higher than average frequency of R7 among the AA speakers of India may superficially be seen as sup- porting the model that places the origin of this haplo- group among AA speakers, possibly even outside India, assuming the language phylum would have arisen else- where. Indirectly, such a scenario would be also sup- Table 2: Frequency of Autochthonous R Subgroups Among Different Language Groups of India. R5 R6 R7 R8 R30 R31 Total Samples Austro-Asiatic 1.12% 4.27% 5.90% 2.64% 0.61% 0.00% 983 Indo-European 3.62% 1.70% 0.58% 1.61% 2.63% 0.85% 2240 Dravidian 3.65% 1.69% 1.37% 1.64% 2.15% 0.32% 2190 Tibeto-Burman 1.74% 0.00% 0.00% 0.58% 0.58% 0.00% 172 Principal component (PC) analysis of R5-8, R30 and R31 lineages in Indian populationsFigure 2 Principal component (PC) analysis of R5-8, R30 and R31 lineages in Indian populations. Munda group and a few Indo-European/Dravidian populations collected from Bihar, Jharkhand and Chhattisgarh states, predominantly cluster with hap-Page 6 of 12 (page number not for citation purposes) logroup R7. Haplogroup frequencies were obtained from published sources [14] and our unpublished data. BMC Evolutionary Biology 2008, 8:227 http://www.biomedcentral.com/1471-2148/8/227 ported by the Y chromosome evidence (haplogroup O2a, for details, see Introduction). However, the much higher diversity of R7a and R7b sub-clades among non Austro- Asiatic populations of India suggests that the source of haplogroup R7 is not among the maternal ancestors of all Austro-Asiatic tribal groups, but that they acquired this The reduced-median network of 152 mtDNAs belonging to haplogroup R7Figu e 3 The reduced-median network of 152 mtDNAs belonging to haplogroup R7. Each sample represented on the dia- gram has been sequenced for the HVS-I region and genotyped for the coding region mutations that are indicated. Circle sizes are proportional to the number of mtDNAs with that haplotype. Recurrent mutations are underlined. Table 3: Coalescent times of hg R7 subclades estimated from HVS-I data. Clade Number of Samples Motif (Coding region) rho (ρ) sigma (σ) Time (SD) R7 152 1442-6248-7870-9051-9110-10289-13105-13830 0.796 0.31 16.064 (6.260) R7(Austro-Asiatic) 47 1442-6248-7870-9051-9110-10289-13105-13830 0.234 0.102 4.723 (2.059) R7(Indo-European) 29 1442-6248-7870-9051-9110-10289-13105-13830 0.793 0.306 16.005 (6.185) R7(Dravidian) 76 1442-6248-7870-9051-9110-10289-13105-13830 1.145 0.536 23.101 (10.822) R7a(Overall) 107 10143-10915-13404-15346 0.389 0.102 7.848 (2.064) R7a(Dravidian) 37 10143-10915-13404-15346 0.514 0.151 10.363 (3.037) R7a(Indo-European) 24 10143-10915-13404-15346 0.5 0.24 10.090 (4.757) R7b(Overall) 45 1804-2282-8557-12432-14064-15942 0.797 0.292 16.052 (5.891) R7b(Dravidian) 39 1804-2282-8557-12432-14064-15942 0.744 0.268 15.006 (5.402) R7a1(Overall) 86 12406-13674 0.337 0.115 6.805 (2.311) R7a1(Austro-Asiatic) 47 12406-13674 0.234 0.102 4.723 (2.059) R7a1(Indo-European) 23 12406-13674 0.522 0.246 10.529 (4.663)Page 7 of 12 (page number not for citation purposes) R7a1(Dravidian) 16 12406-13674 0.375 0.153 7.568 (3.090) BMC Evolutionary Biology 2008, 8:227 http://www.biomedcentral.com/1471-2148/8/227 Page 8 of 12 (page number not for citation purposes) The frequency distribution of R7a and R7b clades in Indian subcontinentFigu 4 The frequency distribution of R7a and R7b clades in Indian subcontinent. The upper panel (a, b) shows the spatial distribution (%) of these clades in Indian populations. Isofrequency maps were generated by using Surfer7 of Golden Software (Golden Software Inc., Golden, Colorado), following the Kriging procedure. These isofrequency maps illustrate the geographic spread of the respective mtDNA haplogroups. It should be cautioned, however, that these illustrative maps should not be used to predict the frequency of the clade in geographical areas with missing data. The lower panel (c, d) depicts the frequencies of R7a and R7b in different social and language groups. DRA-Dravidian, AA-Austro-Asiatic, IE-Indo-European. Table 4: Mantel correlation test of Autochthonous R Subgroups to assess the significance of correlations between gene and geography, or language. Haplogroup Gene vs Geography p Gene vs Language p R5 0.1276 0.0475 0.1748 0.2 R6 0.2654 0.037 0.13248 0.19 R7 0.299 0.023 0.219 0.225 R8 0.211496 0.01753 0.23248 0.31 R30 0.189917 0.127 0.1348 0.28 R31 0.172 0.1873 0.141 0.25 BMC Evolutionary Biology 2008, 8:227 http://www.biomedcentral.com/1471-2148/8/227 haplogroup via local admixture, together with the rest of the South Asian mtDNA lineages that make up their extant maternal lineage pool. Furthermore, the presence of only a single recent founder branch of R7, i.e. R7a1, among widely dispersed AA populations of India supports the founder event scenario by introgression of this lineage from the local non-AA populations before the range expansion of Munda speaking populations within India. If indeed R7 did have its origin among some so far unsam- pled populations of the present-day Myanmar or Cambo- dia, we would then expect to see different sub-divided AA populations losing by drift different sub-branches of R7a and R7b (to explain their reduced diversity), and the admixed Dravidian and Indo-European speaking popula- tions would be expected to have obtained a subset of the R7 variation observed in AA speakers, which is not the case. While the occurrence of R7a1 among Dravidian and Indo-European-speaking populations living close to the AA populations (Fig. 3) could be explained by language shift or secondary admixture with AA speakers, sub-haplo- group R7b appears to be restricted to Dravidian-speakers of the southern part of India (Fig. 4b and 4d). Neverthe- less, this haplogroup is also reported in two Indo-Euro- pean populations (Kolcha and Rathwa) whose local tradition speaks about their ancient split from the Gond (Gondi subfamily of Dravidian language group) popula- tion of Central India and further migration to Gujarat. Thus, from the data and analyses shown here, it is most parsimonious to conjecture that R7 originated in India among non-AA, possibly in Dravidian speaking popula- tions. To test further the two hypotheses, a Dravidian origin for R7 with admixture and founder effects, versus an external AA origin of R7, we examined whether the spread of R7 among the different Munda sub-groups in India, as defined by the language trees [26,27], is uniform. This would be expected if R7 was present among the ancestral AA speakers prior to the diversification of the language family into numerous branches. Consistent with the non- AA origin of R7, we found the distribution of R7a1 among AA populations to be profoundly skewed towards the Kherwari sub-branch of the North Munda languages which accounts for ~90% of the AA R7 samples (Fig. 5). Conversely, R7 is very rare in the South Munda group. It is completely absent in Koraput Munda speakers and mar- ginally present only in the Kharia tribe of Madhya Pradesh (in total 3 out of 431 South Munda samples) (Fig. 5). This finding yet again strengthens the argument that only a subset of Indian AA groups has acquired one sublineage of R7a1 in situ after their arrival to Indian subcontinent from local non-AA groups through admixture. Thus, we fail to find from the evidence of the extant maternal lineage pool the enigma of the origins and demographic past of the AA speakers in India remains, for while the East Asian contri- bution to their paternal gene pool seems evident, the maternal side of their genetic heritage appears to be auto- chthonous to Indian subcontinent. This suggests that introduction and spread of AA speakers into India involved a complex and sex-differentiated demography, involving both exogenous males and local females. In brief, our high-resolution study of haplogroup R7 sug- gests that this haplogroup originated in India among non- AA population most probably Dravidian, and that the Munda (mainly Kherwari group) speaking populations have acquired a subset of it only relatively recently. The highest frequency of haplogroup R7 among Austro-Asiatic tribal groups can be explained, thus, by their regional admixture with other local Indian subcontinental popula- tions followed by random genetic drift, rather than being a genetic marker of their own. The spread of R7 as well as other ancient sub-clades of haplogroup R in India follows predominantly the geographic rather than linguistic land- scape of the subcontinent. The geographic correlations are further manifested in the distribution patterns of the sub- clades: R7a being more common in northern India while R7b is more frequent in the southern parts of the subcon- tinent. Because Dravidian speakers harbour all the twigs of R7 identified so far, the haplogroup may have arisen among the matrilineal ancestry of the present day Dravid- ian speakers. However, it is important to caution that autochthonous basal mtDNA lineages in South as well as Southeast and East Asia appear to be significantly more ancient than any linguistic reconstruction offers to present day language families. This would imply that linguisti- cally significant relationships among Indian populations may be superimposed on, and masking, demographic events of much greater antiquity. Our results also remind us, once again, that phylogenetically established within- haplogroup diversity is more informative than mere fre- quency in establishing the direction of gene flow between populations, language groups and geographically defined regions. Methods To refine the phylogeny of superhaplogroup R we sequenced complete mitochondrial genomes of 35 sam- ples selected from different regions and language groups of India (Table 1). The results were incorporated into a phylogenetic tree [see Additional file 1]; for detailed tree for hg R7 see Fig. 1) together with previously published complete mtDNA sequence data from India [4]. For hap- logroup R7 we performed a high-resolution survey of phy- logenetically diagnostic markers, using information from complete mtDNA sequences. We studied ~12,000 sam-Page 9 of 12 (page number not for citation purposes) of the Austro Asiatic speakers of India any major lineages that show signs of potential origin outside India. Overall, ples collected from all over Indian subcontinent [see Additional file 6]. These samples cover all the language BMC Evolutionary Biology 2008, 8:227 http://www.biomedcentral.com/1471-2148/8/227 groups and most of the Indian states and union territories. The samples were screened for the presence of R7 mtDNAs based on HVS-I information (motif: 16260-16261- 16319-16362). Previously this motif has, together with the restriction enzyme AluI cutting site polymorphism at np. 10143, been used to define haplogroup R20 [14]. However, with the support of new complete mtDNA sequences information the lineage with this HVSI motif was subsequently named R7 [4] and we follow this update of the nomenclature. Further, the identified R7 samples were analyzed for coding region markers by sequencing. Sequencing was carried out in ABI 3730 and 3730XL DNA Analyzers (Applied Biosystems, USA) and mutations were scored against the rCRS [33]. To minimize errors, both strands were double-sequenced. Principal component analysis (PCA) of R subgroups was performed using POP- STR, kindly provided by H. Harpending. Median-joining and reduced median networks were reconstructed with NETWORK program (version 4.1) [34]http://www.fluxus- engineering.com. Reduced median and median-joining procedures were applied sequentially. Coalescence time has been calculated between nucleotide positions 16090– 16365 (HVS-I) considering one transition equals to sidering substitution rate estimate for protein-coding syn- onymous changes of 3.5 × 10-8, which gives 6,764 years per synonymous transition. Standard deviation of the rho estimate (σ) was calculated as in Saillard et al. [35]. Hap- logroup isofrequency maps were generated by using Surfer 7 of Golden Software (Golden Software Inc., Golden, Colorado), following the Kriging procedure. To determine whether language or geography has the strong- est impact on genetic differentiation, spatial autocorrela- tion, SAAP [36] and Mantel [37] tests were performed using ARLEQUIN version 2.0 [38]. For Mantel test genetic distance matrixes were generated from ARLEQUIN, and geographic distance calculated from latitude and longi- tude information. For language groups linguistic distances (ranging from 10–100) assigned manually to each branch, based on published linguistic information and vocabulary match [26-28,39,40]. Electronic database information Accession numbers for data presented herein are as fol- lows (for the complete mtDNA sequence accession num- bers FJ004804-FJ004838 and for the HVS-I region sequence accession numbers FJ010662- FJ010785). The frequency distribution of haplogroup R7 in different branches of the Austro-Asiatic language family of India [26]Figu 5 The frequency distribution of haplogroup R7 in different branches of the Austro-Asiatic language family of India[26].Page 10 of 12 (page number not for citation purposes) 20,180 years [31], while for the coding region estimates we employed the rate calibrated by Kivisild et al. [32] con- BMC Evolutionary Biology 2008, 8:227 http://www.biomedcentral.com/1471-2148/8/227 Authors' contributions GC, MK, EM, DS–R, VKS, AS, BPN, AK, NA, CBM, BT, SP, RR, PS, SB, SVe, SVa, IK, AB, DS, AS, MR, VC and AGR car- ried out the mtDNA genotyping. GC, MK, EM, DS–R, VKS, AS, and BPN carried out the mtDNA sequencing analysis. AT, KT and LS contributed to the analysis and interpreta- tion of the data. AT provided complete sequence informa- tion of Sindhi sample. GC, MK, EM, MM and TK analyzed the data. TK, GC, MM and RV were responsible for con- ceiving and designing the study. GC, MK, MM, TK, RV, AT, RF, KT and LS wrote the paper. All authors read and approved the final manuscript. Additional material Acknowledgements We thank Jaan Lind, Ille Hilpus and Tuuli Reisberg for technical assistance. This work was supported by Estonian Basic Research grant SF0182474 and Estonian Centre of Excellence Grant TK10 (to RV), Tartu University grant PBGMR06901 (to TK), Estonian Science Foundation Grant 5807 (To EM), and UKIERI grant RG47772 (to TK and KT). LS and KT were supported by CSIR, Government of India. References 1. Papiha SS: "Genetic variation in India". Hum Biol 1996, 68(5):607-28. 2. Singh KS, Ed: The Scheduled Tribes. In People of India Oxford, Oxford University Press; 1997. 3. Singh KS, Ed: The Scheduled Castes. In People of India New Dehli, Oxford University Press; 2002. 4. Palanichamy MG, Sun C, Agrawal S, Bandelt HJ, Kong QP, Khan F, Wang CY, Chaudhuri TK, Palla V, Zhang YP: Phylogeny of mito- chondrial DNA macrohaplogroup N in India, based on com- plete sequencing: implications for the peopling of Indian subcontinent. Am J Hum Genet 2004, 75(6):966-978. 5. Forster P, Matsumura S: Evolution. Did early humans go north or south? Science 2005, 308(5724):965-966. 6. Macaulay V, Hill C, Achilli A, Rengo C, Clarke D, Meehan W, Black- burn J, Semino O, Scozzari R, Cruciani F, Taha A, Shaari NK, Raja JM, Ismail P, Zainuddin Z, Goodwin W, Bulbeck D, Bandelt HJ, Oppenhe- imer S, Torroni A, Richards M: Single, rapid coastal settlement of Asia revealed by analysis of complete mitochondrial genomes. Science 2005, 308(5724):1034-1036. 7. Thangaraj K, Sridhar V, Kivisild T, Reddy AG, Chaubey G, Singh VK, Kaur S, Agarawal P, Rai A, Gupta J, Mallick CB, Kumar N, Velavan TP, Suganthan R, Udaykumar D, Kumar R, Mishra R, Khan A, Annapurna C, Singh L: Different population histories of the Mundari- and Mon-Khmer-speaking Austro-Asiatic tribes inferred from the mtDNA 9-bp deletion/insertion polymorphism in Indian populations. Hum Genet 2005, 116(6):507-517. 8. Thangaraj K, Chaubey G, Singh VK, Vanniarajan A, Thanseem I, Reddy AG, Singh L: In situ origin of deep rooting lineages of mito- chondrial Macrohaplogroup 'M' in India. BMC Genomics 2006, 7:151. 9. Thangaraj K, Chaubey G, Kivisild T, Selvi Rani D, Singh VK, Ismail T, Carvalho-Silva D, Metspalu M, Bhaskar LV, Reddy AG, Chandra S, Pande V, Prathap Naidu B, Adarsh N, Verma A, Jyothi IA, Mallick CB, Shrivastava N, Devasena R, Kumari B, Singh AK, Dwivedi SK, Singh S, Rao G, Gupta P, Sonvane V, Kumari K, Basha A, Bhargavi KR, Lalrem- ruata A, Gupta AK, Kaur G, Reddy KK, Rao AP, Villems R, Tyler- Smith C, Singh L: Maternal footprints of Southeast Asians in North India. Hum Hered 2008, 66(1):1-9. Additional file 1 Phylogenetic tree of 22 Indian complete mtDNA sequences of superhaplo- group R. The tree includes data reported [[4] and references there in] Suf- fixes A, C, G, and T indicate transversions, "d" signifies a deletion; recurrent mutations are underlined. 16182C, 16183C and 16519 poly- morphisms are omitted in phylogenetic reconstruction. The sample code, geographic and linguistic affiliations are described in Table 1. The sub- tree of haplogroup R7 sequences is displayed in Fig. 2. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-8-227-S1.jpeg] Additional file 2 Haplogroup R5-8, R30 and R31 frequency plots with 95% credible regions. Data calculated from the posterior distribution of the proportion of a haplogroup/sub-haplogroup in the population. Linguistic affiliations of the populations are indicated by colors. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-8-227-S2.jpeg] Additional file 3 Map of Indian subcontinent depicting the spatial frequency distribution of mtDNA haplogroup R7. Isofrequency maps were generated by using Surfer7 Golden software (Golden Software Inc., Golden, Colorado), fol- lowing the Kriging procedure. The spread of R7 in India is centered around the AA "heartland" (Bihar, Jharkhand, and Chhattisgarh). Dots indicate the sampling locations. Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-8-227-S3.jpeg] Additional file 4 Spatial Autocorrelation Analyses Correlograms of haplogroup R7 in Indian subcontinent. The Moran's I coefficient was calculated with five distance classes in binary weight matrix. Significant values are shown as black (p < .05) whereas nonsignificant values as blank circles. Distances are given in Kilometers (KM's). Click here for file [http://www.biomedcentral.com/content/supplementary/1471- 2148-8-227-S4.jpeg] Additional file 5 Map of India showing the frequency distribution (%) of haplogroup R7 at the district level. Only 2,200 samples were available at this resolution. Nevertheless, it is still evident that the frequency peak of R7 is observed in Bihar, Jharkhand, Chhattisgarh, Madhya-Pradesh and the northern dis- tricts of Andhra-Pradesh (Adilabad, Warangal and Khammam). 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