Major genomic mitochondrial lineages delineate early human expansions

BMC Genetics

BioMed Central

BMC 22001,Genetics Research article

:13

Major genomic mitochondrial lineages delineate early human expansions Nicole Maca-Meyer, Ana M González, José M Larruga, Carlos Flores and Vicente M Cabrera* Address: Department of Genetics, Faculty of Biology, University of La Laguna, Tenerife, 38271, Spain E-mail: Nicole Maca-Meyer - [email protected]; Ana M González - [email protected]; José M Larruga - [email protected]; Carlos Flores - [email protected]; Vicente M Cabrera* - [email protected] *Corresponding author

Published: 13 August 2001 BMC Genetics 2001, 2:13

Received: 9 July 2001 Accepted: 13 August 2001

This article is available from: http://www.biomedcentral.com/1471-2156/2/13 © 2001 Maca-Meyer et al; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any noncommercial purpose, provided this notice is preserved along with the article's original URL. For commercial use, contact [email protected]

Abstract Background: The phylogeographic distribution of human mitochondrial DNA variations allows a genetic approach to the study of modern Homo sapiens dispersals throughout the world from a female perspective. As a new contribution to this study we have phylogenetically analysed complete mitochondrial DNA(mtDNA) sequences from 42 human lineages, representing major clades with known geographic assignation. Results: We show the relative relationships among the 42 lineages and present more accurate temporal calibrations than have been previously possible to give new perspectives as how modern humans spread in the Old World. Conclusions: The first detectable expansion occurred around 59,000–69,000 years ago from Africa, independently colonizing western Asia and India and, following this southern route, swiftly reaching east Asia. Within Africa, this expansion did not replace but mixed with older lineages detectable today only in Africa. Around 39,000–52,000 years ago, the western Asian branch spread radially, bringing Caucasians to North Africa and Europe, also reaching India, and expanding to north and east Asia. More recent migrations have entangled but not completely erased these primitive footprints of modern human expansions.

Background Human mtDNA is a non-recombining molecule with maternal inheritance and practically haploid genetics. Differences between mtDNA sequences are only due to mutation. As time passes, mutations accumulate sequentially along less and less related molecules that constitute independent lineages known as haplotypes. Relationships among lineages can be estimated by phylogenetic networks [1] where mutations are classified in hierarchical levels. Basal mutations are shared for clusters of lin-

eages, defined as haplogroups, whereas those at the tips characterize individuals. Major haplogroups [2] are continental or ethnically specific. Three of them (L1, L2, and L3) group sub-Saharan African lineages, nine (H, I, J, K, T, U, V, W and X) encompass almost all mtDNAs from European, North African and Western Asian Caucasians. Finally, haplogroups A, B, C, D, E, F, G and M embrace the majority of the lineages described for Asia, Oceania and native Americans. The geographic distribution of derived branches of these haplogroups has shed light on

5046

5393

5655

10589

10664

10915

16293

16320

73

16278

16188A

16184

15317

12967C

11172

10984g

8460

8272d9

8251

7561

6607

6257

5711

5581

5147

4596

3808

L1a

185

152

16519

16293

L2

513

325

303i

198

195

150

16390

15849

15236

15217

15110

14061A

13958C

13590

12236

12026

11944

10115

9221

8206

7624A

6437

5988

5201

3200A

2416

2332

1442

709

680

L3d

458

152

16519

16256

15061

14284

13886

13752

8618

7424

7389

5147

4688

1719

L3b

16362

16278

15824

15314

15311

13914A

11002

10424T

10373

10086

9449

9305

8393

6221

5773

4164

3492

3450

16124

13105

C

493

286dd

263

248d

16327

16325

16298

14318

13263

12574

12454

11914

9557

9545

8584

7196A

7013

4715

3552A

100

M2

15431 152 195A 204

12234

9824

8059

6455

5442

4850

4071

489

15043

14783

10400

M1

303ii

199

16362

16295

15236

12091

11719

11152g

146

11946

12007

8870

84

247

16311

16189

16187

15301

13506

13105

10810

10688

8655

8468

7146

2885

2758

825A

D

303ii

152

151

150

16519

16362

16316

16311

16190i

16184i

15737

15622

15562

14927

13984

12810

11902C

10397

8580

7673

7669

7129

5554

5442

5301

5178A

4883

4317

4216

4200T

3254

1106

16278

M3

303i

G

215

152

16362

16256

146

16399

16197g

16195g

16194

14569

14200

14173

13563

11553g

9575

9377

7600

5601

5108

4833

3243

709 6680

6446

M12

466

303ii

16185

15670

15247g

14645

813

16129

15884

15172

14127

M11

513

311ii

152

16183C

16182C

195

16311

16249

14110

12403

8868

96

16189

182

152

16278

13650

7521

7256

4104

16320

16274

16262

16209

16140

15915

15422

12771

11143

10730

9824

7337

6455

4958

4386

2772

2626

1047

514dd

93

769 3594

1018

A

437

303i

235

16362

16319

16290

16209

16111

15205

13448

12007

8794

8027

5480

4824

4248

1736

663

6371

6221

4722

1719

X

227

225

195

16278

16189

16129

15927

14470

13708

11908

8393

7400A

153

W

204

195

189

16292

12574

12414

11947

8994

7864

5460

5046

709

N1b

514dd

152

16390

16180

16176g

16145

12822

12372

11362

9335

8836

5471

4960

3921A

2639

1703

1598

68

8251

9540

8701

10398

10034

IF

16184A

I

250

16148

15452A

14233

13602

12961

8272d9

16391

15758

6515

16129

15924

100 13780 15043

12501 4529T

15301

10873

10398

10238

1719

78

B

499

303i

207

202

199

16284

16217

16189

16183C

16136

15535

15236

13590

8272d9

8206

6455

6413

6023

4117

3816

961

827

J2

248d

152

16300

16193

15924

9380

8633

7963

7789

11251

12633A

16519

T1

195

152

16186

16163

9899

303i 736

T5

150

16153

14233

14180

2308

16294

16189

15928

15607

14905

13368

10463

8697

4917

1888

709

16261

16222

16145

14180

1733

16126

15452A

J1b

489

462

295

16069

13708

12612

10398

3010

80

4216

514dd

HV

150

16519

16311

16278

9667

9137

V

303i

72

16519

16298

15904

14629

12237d

9656

8279

8277

4928

4580

2387

14766

H2

303i

73

12358

12245

5189

73

RCRS

311i

263

16519

15326

8860

4769

3010

1438

7028

3010

68

16223

12705

11719

62

HF

16189

16183d

16093

9066

7309

4452

2706

H1

14233

7669

61

U6

16219

16172

15632

15530

15043

14179

14034

7805

5471

2706

1692T

16399

16311

16256

16153

16093

15924

15218

U5a

195

U5b

303i

150

16192

16189

14182

12618

10927

7768

7385

5656

16270

14861

152

9477

3197 13617

14793

5495

3316

1780

1700

709

78

16519

U2

471

16294

16234

14926

13789

11893

10398

9545T

6962

5460

5147

4025

1700

15907

13020

10876

6152

6045

5426

5390

3720

U22

340

217

16319

12346

11176

7759

7151

508

U21

150

16368

16092

13734

6917

3849

16362

16129C

709

100

16189

16051

152

12372

12308

11467

81

65

U32

514dd

152

150

U31

16356

15734

10724

3010

16390

16343

15454

14139

10352

867

100

13934

10506

9266

7256

6518

4703

1811

U7

514dd

16318T

16248

15601

13500

10382

10142

9698

9055

5913

1189

K

514iCA

195

152

146

16311

16224

16145

15301

14167

12738g

11299

10550

10398

10154

303i

14798

10084

8137

5360

3741

980

52

Figure 1 Phylogenetic network based on complete mtDNA genome sequences. Nomenclature of individuals is as in Table 1. Numbers along the links refer to nucleotide positions; suffixes are transversions; underlining indicates recurrent mutations; the order of the mutations on a path not interrupted by any branching or distinguished nodes is arbitrary. The same topology was supported by bootstraps, using NJ and 1000 replicates; the bootstrap values higher than 50% are shown over the branches. The star shows the position where the chimpanzee sequence roots in the network.

L1aA

514dd

171

89

16168

16129

14308

14007

8191

7258

5911

L1c

316

5628

5231

186A

189C

L1b

151

16360

16294

16187

16129

15978

15905

15622

14911

14000A

12810

12616

12501

12019

11719

11396

11302

10586

9966

9377

9370

9072

8027

7789

7498

6917

6071

5951

5096

1539A

16188g

236

357

16270

16230

189

16264

16172

195

16126

16148

185T

15115

15431

16519

14812

15136

93

14769

13276

182

14203

12720

95C

8248

12519

12007

7867

5036

9818

11914

3308

9755

7055

2768

9347

6827

2352

11641

1738

11176

1406

9042

2394d

14560

14178

13789

8566

3565

64

7389

3666

13293

8428

95

709

5603

5460

5442

4586

4312

6185

2857

100

2245

1048

3516A

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crucial aspects of human history, such as the probable origin and approximate dating of migrations into the New World [3] and Polynesia [4,5], and quantitative estimations of the relative Paleolithic and Neolithic contributions to the extant European mtDNA diversity [2]. At the other end of the phylogenetic tree, the ultimate coalescence of all worldwide mtDNA lineages into Africa has favored, since the beginning, the recent African origin hypothesis for all modern humans [6]. The analyses of the complete mtDNA sequence of 53 humans of diverse origins [7] have added statistical support to this hypothesis. However, as the current definition of the major haplogroups is not based on total genomic sequences, there is not yet a clear resolution of their basal relationships. This genomic phylogenetic reconstruction is necessary to infer the early human dispersal routes after the African exodus. We present the phylogenetic network of 42 complete mtDNA sequences including representatives of the major haplogroups. Based on their relative clustering and coalescence ages we propose a tentative model of the way the Old World could have been colonized by modern humans.

Results and Discussion The phylogenetic network of the 42 mtDNA sequences (Fig. 1) was free of reticulations when mutations [8] 150, 152, 303i and 16519 were omitted in its construction. The tree topology was the same as the bootstrap supporting neighbor joining tree. We detected 35 parallel substitutions from 124 variable positions (28%) in the non-coding region (1,122 bp in length), and 45 from 409 (11%) in the coding region (15,447 bp in length). Shared mutations in basal branches of the tree relate haplogroups,

U5

A 39.000-53.000

U6

U2 L3

M

B C D G

59.000-69.000 L2 L1b/c L1a

Figure 2 Geographic dispersal routes and minimal estimated ages of major human expansions in the Old World, deduced from the age and geographic localisation of main mtDNA haplogroups.

however, parallel mutations should be avoided in their global affiliations. As can be expected from haplotypes of well-differentiated haplogroups the majority of mutations are in the external branches of the tree, including those that specifically define them [2]. Nevertheless, it is well known that in population studies these main lineages sprout into several sub-clusters sometimes with interesting geographic localization. In the cases where representatives of these sub-clusters have also been analyzed, it is evident that the African ones are at the same level of divergence as non-African clusters. More information of cluster structure in Africa is necessary. In non African groups, two haplotypes belonging to sub-haplogroup U2 have a divergence similar to that found between other sub-clusters of the Caucasian U haplogroup. One of them, lacking mutations 16129C and 15907, that are present in all western Eurasian representatives, resembles haplotypes found in India [9]. The proposed inclusion of haplogroup K into the U cluster [10] is confirmed, being U7 its most probable related sub-clade. Main Asian haplogroups belong to two different major clusters, whereas A and B rooted with Caucasoid haplogroups, C, D, G and M constitute a monophyletic cluster. Likewise, African haplogroup L3 is more related to Eurasian haplogroups than to the most divergent African clusters L1 and L2. Chimpanzee rooting shows that the oldest lineage of extant modern humans is the African L1a cluster. In addition, the significant bootstrap values on the deep African branches reinforce the statistical support that the out of Africa hypothesis has obtained through a parallel genomic mtDNA study [7]. We have estimated a minimum total coalescence for modern human lineages from 156,000 to 169,000 years before present (yr BP). The two subsequent ancient splits also happened inside Africa, originating the L1b/c and L2 haplogroups with ages of 122,000–132,000 yr BP and 85,000–95,000 yr BP respectively. These three clades still have an overwhelming sub-Saharan African implantation. The next branching (Fig. 2), dated between 59,000–69,000 yr BP, also occurred in Africa but comprising clades currently found only in this continent (L3), and others with a first expansion out of Africa. Today, L3 derivatives are present in nearly all the African populations. This ancient spread inside Africa has been directly detected by the ages of several sub-clade expansions [11] and indirectly confirmed by genetic admixture, involving archaic and modern autosomal gene alleles, detected only in Africa [12]. The coexistence in African populations of very divergent non-recombining lineages may erroneously bias demographic estimations based on pair-wise nucleotide differences [11]. Two hypothetical routes for the Asian colonization have been proposed [13], one through Central Asia and one through South Asia. Coincidentally, we detect at least two independent lineages spreading out of Africa. One comprises all M de-

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Table 1: HVS I motifs

Ref.a

Sample

HVS I motif

Haplogroup

Origin

K U7 U31 U32 U21 U22 U2 U5b U5a U6 H1 HF RCRS H2 V HV T5 T1 J1b J2 B I IF N1b W X A M11 M12 G M3 D M1 M2 C L3b L3d L2 L1c L1b L1a

145 224 311 248 318T 343 356 390 343 390 051 092 129C 189 362 368 051 129C 189 319 362 051 189 234 294 189 192 270 093 153 256 270 311 399 172 219

K U7 U3 U3 U2 U2 U2 U5b U5a1a U6 H H H H V HV T5 T1 J1b J2 B I I N1b W X A M1 M1 G M D M M C L3b L3d L2 L1c L1b

Iberian Iberian Canarian Moroccan Jordanian Iberian Jordanian Berber Swede Moroccan Mauritanian

Jordanian Iberian Moroccan Canarian Moroccan Jordanian Japanese Japanese Japanese Filipino Indian Canarian Mauritanian Jordanian Mauritanian Mauritanian Mauritanian

1 1 1 1 1 1 1 1 2 1 1 3 4 1 1 1 1 1 1 1 5 1 3 1 1 1 1 1 1 6 7 6 1 1 1 1 1 1 1 1

L1a

Moroccan

1

L1a

African

8

L1aA

093 183d 189

298 278 311 126 153 189 294 126 163 186 189 294 069 126 145 222 261 069 126 193 300 136 183C 189 217 284 129 148 223 391 129 184A 223 391 145 176G 180 223 390 223 292 129 189 223 278 111 209 223 290 319 362 129 182C 183C 189 223 249 311 185 189 223 249 311 189 194 195G 197G 223 256 278 362 140 209 223 262 274 320 399 184iC 190iC 223 311 316 362 223 295 362 223 223 298 325 327 124 223 278 362 124 223 256 223 278 390 129 189 223 278 294 311 360 126 187 189 223 264 270 278 293 311 129 148 168 172 187 188G 189 223 230 278 293 311 320 148 172 184 187 188A 189 223 230 311 320

European Iberian Berber Jordanian Moroccan Iberian Moroccan Iberian Japanese Iberian

a

1, This work; 2, GenBank accession number X93334; 3, H and I references [34], we have added for the comparisons the 263, 311i and 16519 mutations in both sequences and 00073 in the I sequence; 4, revised Cambridge reference, GenBank accession number NC 001807; 5, Positive control [35], for comparisons we added 1438; 6, MELAS, P-1 (G) and FICM (D) [36]; 7, (ref [37]); 8, GenBank accession number D38112, for comparisons we added 311i.

rivatives that radiated 30,000–57,600 yr BP. Subsequent expansions of this clade have been found in India [9] and Eastern Asia where it possibly originated and expanded as haplogroups C, D, G and others [14]. The star-

like radiation of these clades suggests that this wide geographic colonization could have happened in a relatively short time. Genetic support for this southern spread of M through Ethiopia and the Arabian Peninsula along South

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Asia has been recently proposed due to the presence of subclade M1 in Eastern Africa [15]. However, a posterior return from Asia to Africa of these lineages is a more plausible explanation because the genetic diversity of M is much greater in India [9] than in Ethiopia [15]. In fact, M1 could be a branch of the Indian cluster M as ancestral motifs of the African M1 are found in M*, M3 and M4 Indian subclusters [16]. Furthermore, one of the most derived M3 haplotypes in India (10398, 10400, 16086, 16129, 16223, 16249, 16259, 16311) has all the basic substitutions that defined the Ethiopian clade, excepting the highly variable 16189 [9]. This supposed Indian expansion to the west also reached northern areas since evolved representatives of M4 have been also detected in Central Asia [17]. We may consider the upper bound for this return to Africa 25,000–47,000 yr BP, the age calculated for M1 in Eastern Africa based on HVSI sequences or 33,000–63,000 obtained using RFLPs [15]. The other major branch that left Africa gave rise mainly to Caucasoid lineages which is congruent with a northern route through the Levant. With a lower bound of 43,000–53,000 yr BP this branch spread into at least three main clusters. One comprises haplogroups X and A with only a shared mutation between them and different geographic distributions. Whereas A is widespread in Asia, X is mainly restricted to Europe. Curiously, representatives of both clusters have been detected in native Americans raising the possibility that some American Indian could have European ancestry [18]. Nevertheless, X haplotypes have recently been detected in Central Asia. These Asian X haplotypes lack the 225A mutation, as the majority of the American X, pointing to this area as the most probable source for the dispersal of the New World founders [19]. The second cluster groups minor haplogroups W, I and N1b, the three are present although in low frequencies in Europe, Near East and Caucasus but only I and N1b have been also detected in Egypt and Arabia [2]. The last group radiated around 39,000–52,000 yr BP, giving at least four ancestral clusters. One of them originated haplogroup B that expanded to Eastern Asia, reaching Japan and southeastern Pacific Archipelagos [20,21]. In early studies, this clade was defined by the 9bp COII-tRNALys deletion but after that it has been found with independent origins on other haplogroup backgrounds [22–24]. In this study we have detected this deletion on an Iberian haplotype belonging to haplogroup I. Curiously, it was also found in an Italian haplotype I [25]. However, the 9-bp deletion was absent in a wide screen that we carried out on Iberian and Northwest African I haplotypes. The detection in two Mediterranean populations of I haplotypes harboring the 9-bp deletion points to the existence in this area of a subset of I haplotypes that share a recent common ancestor. As happens with A, haplogroup B has not been found in

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northern India [9] but is present in Mongolia [26], favoring a Central Asian route for the expansion of these prominent Asian haplogroups. Two additional clades join haplogroups J and T and haplogroups H, V and HV respectively. Derivatives of at least some of them are found in Europe, North Africa, Central Asia and even India, but the most probable origin for all these expansions is the Near East-Caucasus area [2,17,27]. Finally, cluster U seems to have suffered a radial spread (Fig. 2), giving subsequent diversification in different geographic areas. Three sub-haplogroups, U2, U5 and U6 had their major expansions in India, Europe and North Africa respectively. U2 split in two branches, one, characterized by mutations 16129C and 15907, is geographically scattered from Western Europe to Mongolia [2,26] but has not been detected in North Africa. The other reached India where it gave origin to several sub-clusters with global frequencies around 10% being, after its predecessor haplogroup M (53%), the second most abundant haplogroup in India [9]. U7 with a minor implantation in Europe but third in frequency in India [9] and also not detected in North Africa might have had a similar expansion as U2. The main radiation of haplogroup U5 occurred in Europe. It has been stated that this lineage entered Europe during the Upper Paleolithic [2], most probably from the Middle East-Caucasus area. The great divergence found here for the two U5 representatives is in agreement with the old age proposed for this haplogroup. Finally, U6 traces the first detectable Paleolithic return to Africa of ancient Caucasoid lineages. It has been mostly found in Northwest Africa, with a global estimated age of 47,000 years [28] reflecting an old human continuity in that rather isolated area. The fact that in Europe it has only been detected in the Iberian Peninsula [29] rules out a possible European route, unless a total lineage extinction in all the path is invoked. On the other hand, its presence in Northeast Africa [30], albeit in low frequencies, reinforces its way through North Africa. A third possibility could be that this lineage never went out of Africa but its coalescence with clades which all had prominent expansions in Eurasia weakens this option. U3 has also been found with a comparatively higher frequency in Northwest Africa [29] and might have followed the same route as U6, however, as its star-like expansion in the Caucasus has been dated around 30,000 yr BP [30], it most probably reached Africa in a posterior expansion. This out of Africa and back again hypothesis has also been suggested for Y-chromosome lineages [31]. Subsequent Neolithic and historic expansions have doubtlessly reshaped the human genetic pool in wide geographic areas but mainly as limited gene flow, not admixture, between populations. Consequently, the continental origin of the major haplogroups can still be detected and the earliest human routes inferred through them.

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Table 2: Oligonucleotide pairs used in the amplification and sequencing

Name

CRS reference

Sequence (5'–3')

L16340 H408 L382 H945 L923 H1487 L1466 H2053 L2025 H2591 L2559 H3108 L3073 H3670 L3644 H4227 L4210 H4792 L4750 H5306 L5278 H5832 L5781 H6367 L6337 H6899 L6869 H7406 L7379 H7918 L7882 H8345 L8299 H8861 L8799 H9397 L9362 H9928 L9886 H10462 L10403 H10975 L10949 H11527 L11486 H12076 L12028 H12603 L12572 H13124 L13088 H13666 L13612 H14186 L13612 H14186 L14125

(16318–16340) (429–408) (362–382) (964–945) (902–923) (1508–1487) (1445–1466) (2073–2053) (2004–2025) (2612–2591) (2538–2559) (3128–3108) (3051–3073) (3690–3670) (3625–3644) (4247–4227) (4189–4210) (4813–4792) (4729–4750) (5327–5306) (5259–5278) (5851–5832) (5762–5781) (6387–6367) (6318–6337) (6918–6899) (6850–6869) (7427–7406) (7358–7379) (7937–7918) (7861–7882) (8366–8345) (8280–8299) (8882–8861) (8779–8799) (9416–9397) (9342–9362) (9950–9928) (9865–9886) (10481–10462) (10383–10403) (10994–10975) (10930–10949) (11546–11527 (11467–11486 (12095–12076 (12008–12028 (12623–12603 (12553–12572 (13143–13124 (13068–13088 (13685–13666 (13593–13612 (14206–14186 (13593–13612 (14206–14186 (14104–14125

AGCCATTTACCGTACATAGCACA TGTTAAAAGTGCATACCGCCA CAAAGAACCCTAACACCAGCC GGGAGGGGGTGATCTAAAAC GTCACACGATTAACCCAAGTCA GTATACTTGAGGAGGGTGACGG GAGTGCTTAGTTGAACAGGGCC TTAGAGGGTTCTGTGGGCAAA GCCTGGTGATAGCTGGTTGTCC GGAACAAGTGATTATGCTACCT CACCGCCTGCCCAGTGACACAT TCGTACAGGGAGGAATTTGAA AAAGTCCTACGTGATCTGAGTTC GGCGTAGTTTGAGTTTGATGC GCCACCTCTAGCCTAGCCGT ATGCTGGAGATTGTAATGGGT CCACTCACCCTAGCATTACTTA ACTCAGAAGTGAAAGGGGGCTA CCAATACTACCAATCAATACTC GGTGATGGTGGCTATGATGGTG TGGGCCATTATCGAAGAATT GACAGGGGTTAGGCCTCTTT AGCCCCGGCAGGTTTGAAGC TGGCCCCTAAGATAGAGGAGA CCTGGAGCCTCCGTAGACCT GCACTGCAGCAGATCATTTC CCGGCGTCAAAGTATTTAGC GGGTTCTTCGAATGTGTGGTAG AGAAGAACCCTCCATAAACCTG AGATTAGTCCGCCGTAGTCG TCCCTCCCTTACCATCAAATCA TTTCACTGTAAAGAGGTGTTGG ACCCCCTCTAGAGCCCACTG GAGCGAAAGCCTATAATCACTG CTCGGACTCCTGCCTCACTCA GTGGCCTTGGTATGTGCTTT GGCCTACTAACCAACACACTA AACCACATCTACAAAATGCCAGT TCCGCCAACTAATATTTCACTT AATGAGGGGCATTTGGTAAA AAAGGATTAGACTGAACCGAA CCATGATTGTGAGGGGTAGG CTCCGACCCCCTAACAACCC CAAGGAAGGGGTAGGCTATG AAAACTAGGCGGCTATGGTA GGAGAATGGGGGATAGGTGT GGCTCACTCACCCACCACATT ACGAACAATGCTACAGGGATG ACAACCCAGCTCTCCCTAAG ATTTTCTGCTAGGGGGTGGA AGCCCTACTCCACTCAAGCAC AGGGTGGGGTTATTTTCGTT AAGCGCCTATAGCACTCGAA TGGTTGAACATTGTTTGTTGG AAGCGCCTATAGCACTCGAA TGGTTGAACATTGTTTGTTGG TCTTTCTTCTTCCCACTCATCC

Fragment size (pb)

Annealing temp.(°C)

681

52

603

56

607

56

629

58

609

52

591

56

640

52

623

58

625

55

599

52

593

58

626

58

601

58

578

58

580

56

506

56

603

56

638

58

609

56

617

56

612

56

617

58

629

56

615

58

591

56

618

58

614

56

614

56

602

58

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Table 2: Oligonucleotide pairs used in the amplification and sequencing (Continued)

H14685 L14650 H15211 L15162 H15720 L15676 H16157 L15996 H16401

(14705–14685 (14629–14650 (15232–15211 (15143–15162 (15739–15720 (15657–15676 (16180–16157 (15975–15996 (16420–16401

CATTGGTCGTGGTTGTAGTCC CCCCATTACTAAACCCACACTC TTGAACTAGGTCTGTCCCAATG CTCCCGTGAGGCCAAATATC GTCTGCGGCTAGGAGTCAAT TCCCCATCCTCCATATATCC TGATGTGGATTGGGTTTTTATGTA CTCCACCATTAGCACCCAAAGC TGATTTCACGGAGGATGGTG

Conclusions After coming out of Africa, modern humans first spread to Asia following two main routes. The southern one is represented by haplogroup M and related clades that are overwhelmingly present in India and eastern Asia. The northern one gave a posterior radiation that, through Central Asia, again reached North and East Asia carrying, among others, the prominent lineages A and B. Later expansions, can be detected by the presence of subclades of haplogroup U in India and Europe. There were also returns to Africa, most probably from the same two routes. The return from India could be detected by the presence of derivatives of M in Northeast Africa, and the arrival of Caucasoids by the existence of a subclade of haplogroup U that, today, is mainly confined to Northwest Africa.

597

58

524

56

446

58

Accesion numbers Sequences are available in GenBank (accession nos. AF381981-AF382013)

References 1. 2.

Lineages We have manually sequenced 33 complete mtDNA genomes from available samples previously assigned to major haplogroups. To include lacking haplogroups we added 9 published sequences to the analyses (Table 1).

3.

4.

Statistic analyses Sequences were aligned manually. Phylogenetic relationships were estimated using median-joining networks [32] as implemented in Network 2.0d [http://www.fluxus-engineering.com] and refined by hand. The same topology was obtained using the neighbor-joining method [33]. A chimpanzee sequence (GenBank accession n° D38113) was added to root the networks. Statistical significance of the branches were accomplished by boot-

58

strap resampling with 1000 replications (PHYLIP Package 3.5c, [http://evolution.genetics.washington.edu/phylip.html] ). Minimum estimates of coalescence ages, and 95% confidence intervals, were based on mean divergence among lineages for the coding region and a constant evolutionary rate of 1.7 × 10-8 per site per year that has been inferred for this region on the basis of 53 complete mtDNA sequences [7].

Materials and Methods

Complete mtDNA sequences Complete mtDNA were amplified in 32 overlapping fragments with primers and PCR conditions described in Table 2. The same primers were utilized to directly sequence both strands of the fragments using the Promega fmol® DNA Cycle Sequencing System and the Usb Thermo Sequenase Radiolabelled Terminator Cycle Sequencing Kits.

604

5. 6. 7. 8.

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