ORIGINAL INVESTIGATION

Kumarasamy Thangaraj Æ Vempati Sridhar

Toomas Kivisild Æ Alla G. Reddy Æ Gyaneshwer Chaubey

Vijay Kumar Singh Æ Suminder Kaur Æ Pooja Agarawal

Amit Rai Æ Jalaj Gupta Æ Chandana Basu Mallick

Niraj Kumar Æ Thrimulaisamy P. Velavan

Rajanbabu Suganthan Æ Divi Udaykumar

Rashmi Kumar Æ Rachana Mishra

Arif Khan Æ Chitikineni Annapurna Æ Lalji Singh

Different population histories of the Mundari- and Mon-Khmer-speakingAustro-Asiatic tribes inferred from the mtDNA 9-bp deletion/insertionpolymorphism in Indian populations

Received: 18 August 2004 / Accepted: 13 January 2005 / Published online: 17 March 2005� Springer-Verlag 2005

Abstract Length variation in the human mtDNA in-tergenic region between the cytochrome oxidase II(COII) and tRNA lysine (tRNAlys) genes has been

widely studied in world populations. Specifically,Austronesian populations of the Pacific and Austro-Asiatic populations of southeast Asia most frequentlycarry the 9-bp deletion in that region implying theirshared common ancestry in haplogroup B. Further-more, multiple independent origins of the 9-bp deletionat the background of other mtDNA haplogroups hasbeen shown in populations of Africa, Europe, Austra-lia, and India. We have analyzed 3293 Indian individ-uals belonging to 58 populations, representing differentcaste, tribal, and religious groups, for the lengthvariation in the 9-bp motif. The 9-bp deletion (onecopy) and insertion (three copies) alleles were observedin 2.51% (2.15% deletion and 0.36% insertion) ofthe individuals. The maximum frequency of the dele-tion (45.8%) was observed in the Nicobarese inassociation with the haplogroup B5a D-loop motif thatis common throughout southeast Asia. The lowpolymorphism in the D-loop sequence of the Nicoba-rese B5a samples suggests their recent origin and afounder effect, probably involving migration fromsoutheast Asia. Interestingly, none of the 302 (exceptone Munda sample, which has 9-bp insertion) fromMundari-speaking Austro-Asiatic populations fromthe Indian mainland showed the length polymorphismof the 9-bp motif, pointing either to their independentorigin from the Mon-Khmeric-speaking Nicobareseor to an extensive admixture with neighboring Indo-European-speaking populations. Consistent withprevious reports, the Indo-European and Dravidicpopulations of India showed low frequency of the9-bp deletion/insertion. More than 18 independentorigins of the deletion or insertion mutation could beinferred in the phylogenetic analysis of the D-loop se-quences.

K. Thangaraj Æ V. Sridhar Æ A. G. Reddy Æ G. ChaubeyV. K. Singh Æ S. Kaur Æ L. Singh (&)Centre for Cellular and Molecular biology,Uppal Road, Hyderabad, 500 007, IndiaE-mail: lalji@ccmb.res.inTel.: +91-40-27160789Fax: +91-40-27160591

T. KivisildEstonian Biocenter, Riia23, Tartu, 51010, Estonia

P. Agarawal Æ T. P. Velavan Æ D. UdaykumarInstitute of Microbiology & Biotechnology,Barkatullah University, Bhopal, India

A. Rai Æ N. KumarDepartment of Biotechnology, VBS Purvanchal University,Jaunpur, India

J. Gupta Æ R. Suganthan Æ R. KumarDepartment of Biotechnology,Bundelkhand University, Jhansi, India

C. B. MallickDepartment of Biotechnology,APS University, Rewa, India

R. MishraDepartment of Biotechnology,Allahabad Agricultural University,Allahabad, India

A. KhanDepartment of Biosciences,Sardar Patel University,Vallabh Vidyanagar, India

C. AnnapurnaDepartment of Biotechnology,Kandaswamy Kandars College, P. Velur, India

Hum Genet (2005) 116: 507–517DOI 10.1007/s00439-005-1271-6

Introduction

Indian populations, consisting of approximately 4635culturally and anthropologically differentiated groups,can broadly be divided into four different linguisticfamilies, namely, Austro-Asiatic, Dravidian, Tibeto-Burman, and Indo-European. Each population, affili-ated to one of these linguistic families, has its own socialand cultural identity, and practices endogamy, whichhelps to maintain its genetic architecture. Several studieshave been carried out with maternally inherited mito-chondrial DNA (mtDNA) markers to understand thepopulation structure and genetic origins of Indian pop-ulations (Torroni et al. 1998; Watkins et al. 1999;Kivisild et al. 1999, 2002, 2003; Clark et al. 2000;Roychoudhury et al. 2001; Cordaux et al. 2003;Thangaraj et al. 2003). These studies have shown thatIndians are readily distinguishable from Europeans andeast Asians and interestingly, despite the linguistic(Austro-Asiatic languages) and cultural (the spread ofrice-based economies) links, share few lineages in com-mon with neighboring southeast Asian populations(Kumar and Reddy 2003). For example, haplogroup B,which is defined by a 9-bp deletion in the intergenic re-gion V of mtDNA, is characteristically frequent insoutheast Asia, reaching almost fixation in some Poly-nesian populations and, in contrast, is totally absent inmost populations of India examined so far.

Most humans have inherited two tandem copies ofthe 9-bp motif (CCCCCTCTA), located in the non-coding region V, between the cytochrome oxidase II(COII) and tRNA lysine (tRNAlys) genes, from themitochondrial ‘‘Eve’’. Initially, deletion of one copy of9-bp was characterized as an Asian-specific marker,since it was reported only in Asian populations and washypothesized to have originated in China (Horai et al.1987; Stoneking and Wilson 1989; Ballinger et al. 1992;Harihara et al. 1992; Yao et al. 2000) and to have spreadfrom there to America (Schurr et al. 1990; Ward et al.1991, 1993; Torroni et al. 1992) and Polynesia (Hertz-berg et al. 1989; Hagelberg and Clegg 1993; Hagelberget al. 1994; Redd et al. 1995). However, several sub-sequent studies have reported variable frequencies of the9-bp deletion in other non-Asian populations, such asAfrican Pygmies (Vigilant et al. 1991), primarily Bantu-speaking sub-Saharan Africans (Soodyall et al. 1996),and aboriginal Australians (Betty et al. 1996).

Although Majumder (2001) has indicated the absenceof the 9-bp deletion in most Indian populations (prob-ably because of the few groups studied and the smallsample size), some recent studies have found it to bepresent in some tribal and caste groups with frequenciesreaching up to 50% in some small south Indian tribalgroups (Watkins et al. 1999; Clark et al. 2000; Prasadet al. 2001; B. M. Reddy, personal communication).However, there is no comprehensive study of the 9-bppolymorphism in Indian populations of differentlinguistic and ethnic groups, distributed throughout

India. Therefore, we have carried out a phylogeographicanalysis of the 9-bp deletion/insertion polymorphism inIndian populations, covering the entire Indian subcon-tinent, including 350 samples from Austro-Asiaticpopulations, represented both by the Mundari andMon-Khmer linguistic branches. Previous studies ofIndian populations have suggested that the presence of9-bp deletion variants in India is likely through multipleindependent occurrences of the mutation (Watkins et al.1999; Clark et al. 2000). In order to gain more insightsinto the phylogenetic affiliation of the samples with 9-bplength variation, we have analyzed the first hypervari-able segment (HVS-I) of the D-loop and coding regionsingle-nucleotide polymorphism (SNPs; Rieder et al.1998; Quintana-Murci et al. 1999; Kivisild et al. 2003;Kong et al. 2003a, 2003b) to compare them againstexisting data sets for south and southeast Asia (Kivisildet al. 1999, 2003; Roychoudhury et al. 2001; Prasad et al.2001; Comas et al. 1998; Melton et al. 1998; Pfeifferet al. 1998; Seo et al. 1998; Lee et al. 1999; Nishimakiet al. 1999; Yao et al. 2000, 2002a, 2002b; Fucharoenet al. 2001; Tsai et al. 2001; Oota et al. 2002; Kong et al.2003a, 2003b).

Materials and methods

We have analyzed a total of 3293 individuals from 58endogamous populations of India (Table 1). A bloodsample (5–10 ml) was collected from each individualwith informed written consent. DNA was isolated byusing protocols described elsewhere (Thangaraj et al.2002). The non-coding intergenic region between mito-chondrial genes COII and tRNALys was amplified(Thomas et al. 1998) and size-fractionated in an 8%polyacrylamide gel. The 9-bp deletion and insertion werefurther confirmed by direct sequencing (Thangaraj et al.2003). We sequenced the first hypervariable segment(HVS-I) of the D-loop and determined informativecoding region SNPs (Rieder et al. 1998; Quintana-Murciet al. 1999; Kivisild et al. 2003; Kong et al. 2003a,2003b). Phylogenetic relationships between the observedhaplotypes were reconstructed by using the NETWORKprogram (Bandelt et al. 1999; http://www.fluxus-engi-neering.com).

Results

Of the 3293 individuals studied, the deletion or insertionof 9-bp was observed in 71 (2.15%) and 12 (0.36%)samples, respectively (Table 1). Both deletion andinsertion frequencies varied highly between populations;their distribution among populations is depicted inFig. 1. Out of 58 endogamous populations analyzed, 18populations showed the 9-bp deletion (single copy of theCCCCCTCTA motif). The maximum frequency of the9-bp deletion was observed in the Nicobarese (45.8%),an Austro-Asiatic tribe of Andaman and Nicobar Is-

508

lands, followed by Yanadi (20%), a Dravidian tribalpopulation of Andhra Pradesh. Only five populationsshowed the presence of the 9-bp insertion (three copies

of CCCCCTCTA repeats), out of which Kathodi ofGujarat showed the highest frequency of insertion(6.6%), and only one population of Uttar Pradesh

Table 1 Frequencies of the 9-bp deletion/insertion in studied tribal and caste populations (LC lower caste, MC middle caste, T tribe, UCupper caste)

State Populations Linguisticaffiliation

Samplesize

9-bp deletion 9-bp insertion

Number Frequency Number Frequency

Andaman and Nicobar Islands Nicobarese (T) Mon-Khmer (AA) 48 22 0.458 - -Onge (T) Andamanese 46 - - - -Great Andamanese (T) Andamanese 20 - - - -Jarawa (T) Andamanese 8 - - - -

Tamil Nadu Sakkili (T) Dravidian 65 2 0.030 - -Baduga (T) Dravidian 60 2 0.033 - -Irula (T) Dravidian 72 1 0.013 - -Kani (T) Dravidian 50 - - - -Malayan (T) Dravidian 100 - - - -Oorali (T) Dravidian 60 3 0.05 - -

Andhra Pradesh Velama (UC) Dravidian 49 - - - -Gowda (MC) Dravidian 49 - - - -Erukala (T) Dravidian 80 2 0.025 - -Sugali (T) Indo-European 64 - - - -Muslim ——– 39 - - - -Brahmin (UC) Dravidian 48 - - - -Kamma (UC) Dravidian 46 - - - -Yanadi (T) Dravidian 45 9 0.2 - -

Kerala Kuruman (T) Dravidian 30 - - - -Kurucha (T) Dravidian 33 - - - -Paniyan (T) Dravidian 34 - - - -Kattunaikan (T) Dravidian 87 - - - -

Madhya Pradesh Bharia (T) Indo-European 82 10 0.121 - -Gond (T) Dravidian 147 2 0.014 - -Keer (LC) Indo-European 29 - - - -Bhil (T) Indo-European 73 - - - -Baiga (T) Indo-European 40 - - - -Oraon (T) Dravidian 56 - - - -Kanwar (T) Indo-European 88 - - - -Koli (LC) Indo-European 96 - - - -Mawasi (T) Austro-Asiatic 111 - - - -

Uttar Pradesh Rajput (UC) Indo-European 256 4 0.015 - -Tiwari (UC) Indo-European 99 1 0.010 - -Yadava (MC) Indo-European 64 1 0.015 1 0.015Munda (T) Austro-Asiatic 11 - - - -Muslim —— 45 - - - -Saharia (LC) Indo-European 12 - - - -Kabutharein (LC) Indo-European 24 - - - -Kori (LC) Indo-European 50 1 0.020 - -Rohidas (LC) Indo-European 50 - - 1 0.020Lodhe (UC) Indo-European 26 1 0.038 - -

Gujarat Koli (T) Indo-European 50 1 0.020 - -Kathodi (T) Indo-European 15 - - 1 0.066Bhil (T) Indo-European 15 - - - -Dhungri Bhil (T) Indo-European 50 - - - -Dhungiri Garasia (T) Indo-European 51 - - - -Vasava (T) Indo-European 49 - - - -Rabari (T) Indo-European 34 - - - -

West Bengal Random samples 75 3 0.040 - -Arunachal Pradesh Nishi (T) Tibeto-Chinese 42 2 0.048 - -Bihar Oraon (T) Dravidian 50 - - 3 0.06Jharkhand Santhal (T) Austro-Asiatic 108 - - - -

Oraon (T) Dravidian 89 - - 5 0.057Munda (T) Austro-Asiatic 40 - - 1 .025

Chhattisgarh Kanwar (T) Dravidian 31 3 0.097 - -Satnami (LC) Indo-European 30 1 0.033 - -Santhal (T) Austro-Asiatic 32 - - - -

Jammu and Kashmir Kashmiri Brahmins (UC) Indo-European 40 - - - -Total —— 3293 71 —— 12 ——Average Frequency —— —— — 0.022 —- 0.004

509

(Yadava) showed both the deletion and insertion(Table 1).

By using HVS-I sequence information, 35 differenthaplotypes were observed among the 83 samples thatshowed length variation in the COII-tRNALys region(Table 2). Of these, nine haplotypes occurred in two ormore individuals. All 22 Nicobarese samples with the 9-bp deletion, except for two that differed from the othersonly by an extra C in the C-stretch at nucleotide posi-tions 16184–16193, shared one common haplotype(H25; Table 2). HVS-I sequences exactly matching tothe type observed in the Nicobarese have also beenfound in Austro-Asiatic, Daic, Austronesian, and Ti-beto-Burman populations of southeast Asia and Ocea-

nia (Sykes et al. 1995; Redd et al. 1995; Yao et al. 2002a,2002b; Oota et al. 2001, 2002; Fucharoen et al. 2001).Interestingly, however, none of the other 302 samplestaken from five Mundari group of Austro-Asiatic pop-ulations on mainland India showed the presence of the9-bp deletion (Table 1). Partial sequencing of the codingregion of the Nicobarese samples with the 9-bp deletionrevealed their close phylogenetic relationship (as indi-cated in Fig. 3) with the B5a complete sequence fromChina (Kong et al. 2003a, 2003b).

The frequency of the 9-bp polymorphism in mainlandDravidic and Indo-European-speaking populations wasrelatively low (Table 1). Analysis of the HVS-I se-quences and coding region SNP information indicated atleast six independent origins of the deletion in haplo-group M, at the M2a, M3, M3a, M4, M5, and M*background. Furthermore, four sub-clades of M—M4,

Fig. 1 Frequencies and distribution of the 9-bp deletion/insertionamong Indian populations

510

Table

2Haplogroupaffiliationofthe9-bpdeletion/insertionsamplesbasedontheHVS-I

andcodingregionSNPs.Nucleotidechangeisspecified

only

fortransversions(C

RSCambridgereference

sequence,

asterisknucleotideinsertion,spotsamenucleotideasCRS,dash

data

notanalyzed)

HaplotypeSample

size/

population

HVS-I

(�16000)

Haplogroup4474897093537354045805252696070288584870195409950103981040010873114671171911881123081237212705133951478315607

CRS

CT

GA

TG

GC

CC

AT

TA

CT

AG

CA

GC

AT

A

9bpdeletion

H1

1 Rajput/4

Bharia/1

Kori

223

M*

.C

––

–.

.–

––

GC

–G

TC

.A

–.

.T

–C

–

H2

1 Rajput/1

wBengali

129-223

M5

.C

––

–.

.–

T–

.C

..

TC

.A

–.

.T

–C

–

H3

1 Rajput

223-156-274-

320-360

M*

.C

––

–.

.–

––

GC

.G

TC

.A

–.

.T

–C

–

H4

1 Rajput

129-140-189-

261-266A

B5a

..

––

–.

.–

T–

..

CG

..

.A

–.

..

–.

–

H5

1 Erukula/9

Yanadi/3

Oorali/1

Baduga

184-223-256G-

311-362

M4

.C

––

–.

.–

––

–C

–G

TC

.A

–.

.T

–C

–

H6

1 Kanwar

218-223-231-

311-362-356

M4

.C

––

–.

.–

––

.C

–G

TC

.A

–.

.T

–C

–

H7

1 Erukula

223-233-290

M*

.C

––

–.

.–

T–

.C

–G

TC

.A

–.

.T

–C

–

H8

1 Satnami

179-223

M*

.C

––

–.

.–

T–

.C

–G

TC

.A

–.

.T

–C

–

H9

1 Nishi

270-319-352

M2a

GC

––

–.

A–

––

–C

–G

TC

.A

–.

.T

–C

–

H10

1 Kanwar

093-126-223-290

M3a

.C

––

–A

.–

T–

–C

–G

TC

.A

–.

.T

–C

–

H11

6 Bharia

126-223

M3a

.C

––

–A

.–

––

–C

–G

TC

.A

–.

.T

–C

–

H12

1 Sakkili

126-223-234-290

M3

.C

––

–.

.–

––

–C

–G

TC

.A

–.

.T

–C

–

H13

1 Sakkili

119-126-223-234-290M3

.C

––

–.

.–

––

–C

–G

TC

.A

–.

.T

–C

–

H14

1 Koli

145

R.

.–

––

..

–T

––

.–

G.

..

A–

..

.–

.–

H15

1 Gond

126

H.

.–

––

..

–.

–.

.–

G.

..

.–

..

.–

.–

H16

1 Nishi

181del-197

R.

.–

––

..

–T

–.

.–

G.

..

A–

..

.–

.–

H17

1 Lodhe

311-318T

U7

..

––

–.

.–

––

–.

–G

..

GA

–G

A.

–.

–

H18

1 Kanwar

075-260-261-319-362R7

..

––

–.

.–

T–

–.

–G

..

.A

–.

..

–.

–

H19

1 Yadava

129-318T

U7

..

––

–.

.–

––

–.

–.

..

GA

–G

A.

–.

–

511

Table

2(C

ontd.)

HaplotypeSample

size/

population

HVS-I

(�16000)

Haplogroup

4474897093537354045805252696070288584870195409950103981040010873114671171911881123081237212705133951478315607

CRS

CT

GA

TG

GC

CC

AT

TA

CT

AG

CA

GC

AT

A

H20

1 Baduga/1

Irula

CRS

U.

.–

––

..

–T

––

.–

..

.G

A–

GA

.–

.–

H21

1 wBengali

129-189

B.

.–

––

..

––

–.

.–

..

..

A–

..

.–

.–

H22

1 Tiwari

147-217-235

B4

..

––

–.

.–

––

–.

–.

..

.A

–.

..

–.

–

H23

1 wBengali

147-189-235

B4

..

––

–.

.–

––

–.

–.

..

.A

–.

..

–.

–

H24

2 Nicobarese

140-183C-266A

B5a1

..

AG

C.

.T

–A

–.

CG

..

.A

T.

..

G.

–

H25

20

Nicobarese

140-183C-189-266A

B5a1

..

AG

C.

.T

–A

–.

CG

..

.A

T.

..

G.

–

H26

1 Gond

129-140-189-243

B5b

..

––

–.

.–

––

–.

–G

..

.A

–.

..

––

–

9bpinsertion

H27

1 Oraon

050-190-223-311-357M4a

.C

––

–.

.–

––

–C

–G

TC

.A

–.

.T

–C

–

H28

2Oraon

048-223-311-357

M4a

.C

––

–.

.–

––

–C

–G

TC

.A

–.

.T

–C

–

H29

2Oraon/1

Yadava

129-223-291

M5a

.C

––

–.

.–

––

–C

–G

TC

.A

–.

.T

–C

–

H30

1Oraon

223-295-318T-325

M18

.C

––

–.

.–

––

.C

–G

TC

.A

–.

.T

–C

–

H31

1Oraon

223-318T-325

M18

.C

––

–.

.–

––

–C

–G

TC

.A

–.

.T

–C

–

H32

1Oraon

093-223-256-261

M.

C–

––

..

––

––

C–

GT

C.

A–

..

T–

C–

H33

1Munda

111-223-311-316-362M4

.C

––

–.

.–

––

–C

–G

TC

.A

–.

.T

–C

–

H34

1Kathodi

192-311

R.

.–

––

..

–T

–G

.–

G.

..

A–

..

.–

.–

H35

1Rohidas

126-294-296-325

T.

.–

––

..

–T

–G

.–

G.

..

A–

..

.–

.G

512

M4a, M5a, and M18 showed the presence of three copiesof the 9-bp motif (insertion). One independent event ofthe 9-bp insertion has also been found on the unresolvedM background (Table 2). Altogether, haplogroup Msamples covered 46 out of the 61 individuals with lengthvariation in the COII-tRNALys region in India, exclud-ing the Nicobarese (Fig. 2). In addition to haplogroupB, six additional independent origins of the 9-bp deletion

[twice at different branches of haplogroups R* and U,and one each in R7 (Palanichamy et al. 2004) and H] andtwo of the insertions were inferred in phylogeneticanalysis of haplogroup N lineages (one in the R and onein the T lineage; Fig. 3). A high proportion of sampleshaving 9-bp deletion (22 Nicobarese, two West Bengali,one Tiwari, one Rajput, and one Gond) fell in haplo-group B. All the Nicobarese with one Rajput fell intohaplogroup B5a, whereas one Gond sample fell intohaplogroup B5b. One West Bengali and one Tiwarisample fell into haplogroup B4, whereas one WestBengali sample was defined as unresolved haplogroup B(Fig. 3).

Discussion

Our comprehensive study of Indian populations fromvarious geographic zones with respect to the lengthvariation in the COII-tRNALys region has revealed that

Fig. 2 The phylogenetic placement of haplotypes of haplogroup Massociated with length variation in the COII-tRNALys intergenicregion in India. Substitutions in HVS-I or in the coding region areshown along the branches. Nucleotide change is specified only fortransversions (circle with solid color 9-bp deletion, stripes insertion,white circles control samples with two copies of the 9-bp repeat).The sample names marked in red are the control samples in thisstudy, whereas the sample names marked in black are the samples ofKivisild et al. 1999 (Ind;Lam;Lob;Bog;Bv;By;Bn;K;Ktk;J;Ks;M;ML;VS;WB;Y) and our unpublished data (San;Lm;Nai;KO).M*-del is a potentially paraphyletic grouping of all 9-bp deletedlineages in haplogroup M and did not resolve into sub-cladesdefined by stable coding region markers. Nucleotide positions (boldand underlined) are the haplogroup-defining mutation sites

513

2.51% (2.15% deletion and 0.36% insertion) of 3293individuals have the 9-bp length variation, and that thefrequency of the 9-bp deletion/insertion does not varysignificantly across the different ethnic and linguisticpopulations. The sharing of a common haplotype foundin all of the 9-bp deleted samples of Yanadi (nine), in thethree Oorali, and one each in the Baduga and Erukula inM4, together with the Koya and Irula samples (Watkinset al. 1999; Kivisild et al. 2003), all of which belong toDravidian linguistic group and inhabit southern India,suggest either a common origin or maternal gene flowbetween these populations. Further, the sharing of one

of the haplotypes of Sakkili with the Dravidian MariaGond and Chenchu populations (Watkins et al. 1999;Kivisild et al. 2003) in addition to the sharing of the 9-bpdeleted haplotype on an M3a background by two Indo-European populations of central India (Kanwar ofChhattisgarh; Bharia of Madhya Pradesh) suggest that,although there is maternal gene flow between thesepopulations, it is low and limited by geographic dis-tance, ethnicity, and language.

Consistent with the high frequency of the haplogroupM in India, the analysis of the coding markers in indi-viduals with length variation in the COII-tRNALys re-gion has shown that most of the individuals (46 out of83) belong to this haplogroup (Fig. 2). Interestingly,both samples with the ancestral (M3) and derived (M3a)allele at nucleotide position 4580 include lineages withthe 9-bp deletion (Fig. 2). As the majority consensus ofboth M3 and M3a includes individuals without thedeletion or insertion (two copies of the 9-bp motif;Kivisild et al. 2003; unpublished), the deletion haspresumably occurred at least twice on the haplogroupM3 background. Relatively high sequence variation of

Fig. 3 Phylogenetic placement of haplotypes of haplogroup Nassociated with length variation in the COII-tRNALys intergenicregion in India. Substitutions in HVS-I or in the coding region areshown along the branches. Nucleotide change is specified only fortransversions (circle with solid color 9-bp deletion, stripes insertion,white circles represent the control samples with two copies of the9-bp repeat). The sample names marked in red are the controlsamples in this study, whereas the samples name marked in blackare the samples taken from Kivisild et al. 1999 (Ind;Lam;Ktk;Pak)and our unpublished data (Ao;Rm;Pn). Nucleotide positions (boldand underlined) are the haplogroup-defining mutation sites

514

the 9-bp deletion in the paraphyletic cluster M*, whichincludes all unclassified branches of M, probably reflectthe lack of adequate phylogenetic resolution and there-fore might be inflated by several additional independentoccurrences of the 9-bp deletions. Seven independentorigins of the 9-bp deletion and two occurrences of theinsertion were inferred in the background of haplogroupN (Fig. 3). Taken together, these results in Indian pop-ulations indicate that the length variation in the COII-tRNALys intergenic region can be highly recurrent, evenwithin a single sub-continent.

Based on the coding region SNPs, a large proportionof Nicobarese (22), two West Bengali samples, and oneTiwari, one Gond, and one Rajput sample having the9-bp deletion could be classified within two knownbranches of haplogroup B. All the Nicobarese with oneRajput and one Gond sample fell into haplogroups B5aand B5b, whereas the West Bengali and the Tiwari (anIndo-European-speaking high caste group from UttarPradesh) samples fell into haplogroup B4. Although, nostudy until now has reported haplogroup B in mainlandIndia, the presence of southeast-Asian-specific haplo-group B4 and B5b lineages at marginally low frequency(Fig. 3) on the Indian mainland suggest low levels ofadmixture with populations of southeast Asian origin.Intriguingly, among the Austro-Asiatic groups, only theNicobarese harbor haplogroup B5a at significant pro-portions, and none of the 302 mainland Mundari-speaking groups possess either haplogroup B or the9-bp length polymorphism (except for one sample thathas the 9-bp insertion). Thus, one may postulate thatMundari-speaking Austro-Asiatic populations frommainland India have independently migrated/originatedfrom the Mon-Khmeric-speaking Nicobarese or exten-sive admixture occurred with the neighboring Indo-European and Dravidian populations.

Low haplotype diversity in Nicobarese B5a individu-als suggests a recent founder effect. This observationconcurs with the hypothesis that the 9-bp deletion in theNicobarese is probably rooted in China and expandedvia Cambodia and Thailand to the Nicobar Islands (Yaoet al. 2000). This migration could have occurred in theNeolithic era during the demic expansion of Neolithicagriculturalists in southeast Asia as inferred in the studiesof Thangaraj et al. (2003) and Underhill et al. (2000)based on results from Y-chromosome markers. Codingregion analysis also supports the theory, based on mor-phometric and linguistic data, that the Nicobarese are ofsoutheast Asian origin. The presence of haplogroup R10and F1a in the Nicobarese (Prasad et al. 2001; unpub-lished) and in the Thailand samples (Oota et al. 2001;Fucharoen et al. 2001) further strengthens the view thatthe Nicobarese, Thai, and Chinese might have a commonorigin. Further analysis of the coding region of B5asamples from Burma, Myanmar, and Thailand and fromother adjoining south Asian countries may provide uswith further insights into the origin of the Nicobarese.

In conclusion, we provide here a complete geneticreview of the 9-bp polymorphism in Indian populations;

this suggests a total of 20 independent origins of the 9-bpdeletion and insertion. These events are not population-specific and its frequency varies between the popula-tions. Although the caste and tribes of India are wellknown for strictly adhering to and practicing endogamy,the sharing of 9-bp-deleted haplotypes suggests maternalgene flow, albeit low, between populations of the samelinguistic family and geographic region and not acrosslinguistic or geographic groups. Further, our resultsstrongly corroborate the existing hypothesis that theNicobarese may have arrived on the Andaman andNicobar islands of India from southeast Asia during theNeolithic period, unlike the Mundari groups, whichhave been conjectured to have arrived in India fromAfrica around 50,000 years ago (Kumar and Reddy2003). Intriguingly, haplogroup B5a, which bears wit-ness to the link between the Nicobarese and the south-east Asian population, is totally absent in the mainlandAustro-Asiatic tribes indicating that this genetic tag doesnot relate all existing Austro-Asiatic speakers.

Electronic Database Information

Accession numbers and URL for data presented hereinare as follows: GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for mtDNA HVS-I, accession numbersAY255133–AY255180; for mtDNA HVS-II, accessionnumbers AY370779-AY370829)

Acknowledgements We thank Peter A. Underhill, V. Kumar, V. R.Rao, and M. W. Pandit for their valuable comments during thepreparation of this manuscript. We are grateful to the students andstaff of various colleges and universities, who actively participatedin this study by collecting samples from the various ethnic groups.This study was supported by the Council of Scientific and Indus-trial Research (CSIR), Government of India, New Delhi.

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