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Mitochondrial genome variation and the origin of modern humans
Author: Max Ingman, Henrik Kaessmann,Ulf Gyllensten,Svante Paabo
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"10. Russell, D. A. & Dong, Z.-M. A nearly complete skeleton of a new troodontid dinosaur from the Early Cretaceous of the Ordos Basin, Inner Mongolia, People?s Republic of China. Can. J. Earth Sci. 30, 2163?2173 (1994). 11. Currie, P. J. Bird-like characteristics of the jaws and teeth of troodontid theropods (Dinosauria, Saurischia). J. Vert. Paleont. 7, 72?81 (1987). 12. Clark, J. M., Perle, M. & Norell, M. A. The skull of Erlicosaurus andrewsi, a Late Cretaceous ??Segnosaur?? (Theropoda: Therizinosauridae from Mongolia). Am. Mus. Novit. 3115, 1?39 (1994). 13. Ostrom, J. H. Osteology of Deinonychus antirrhopus, an unusual theropod dinosaur from the Lower Cretaceous of Montana. Peabody Mus. Nat. Hist. Bull. 30, 1?165 (1969). 14. Wellnhofer, P. Das siebte Exemplar von Archaeopteryx aus den Solnhofener Schichten. Archaeopteryx 11, 1?47 (1993). 15. Currie, P. J. New information on the anatomy and relationships of Dromaeosaurus albertensis (Dinosauria: Theropoda). J. Vert. Paleont. 15, 576?591 (1995). 16. Martin, L. D. in Origins of the Higher Groups of Tetrapods (eds Schultze, H.-P. & Trueb, L.) 485?540 (Comstock, Ithaca and London, 1991). 17. Zhou, Z. & Martin, L. D. Feathered dinosaur or bird??a new look at the hand of Archaeopteryx. Smithson. Contrib. Paleobiol. 89, 289?293 (1999). 18. Forster, C. A., Sampson, S. D., Chiappe, L. M. & Krause, D. W. The theropod ancestry of birds: new evidence from the Late Cretaceous of Madagascar. Science 279, 1915?1919 (1998). 19. Makovicky, P. & Sues, H.-D. Anatomy and phylogenetic relationships of the theropod dinosaur Microvenator celer from the Lower Cretaceous of Montana. Am. Mus. Novit. 3240, 1?27 (1998). 20. Holtz, T. R. Jr. The phylogenetic position of the Tyrannosauridae: implications for theropod systematics. J. Paleont. 68, 1100?1117 (1994). 21. Norell, M. A. & Makovicky, P. J. Important features of the dromaeosaur skeleton: information from a new specimen. Am. Mus. Novit. 3215, 1?28 (1997). 22. Currie, P. J. & Peng, J.-H. A juvenile specimen of Saurornithoides mongoliensis from the Upper Cretaceous of northern China. Can. J. Earth Sci. 30, 2224?2230 (1994). 23. Currie, P. J., Rigby, J. K. & Sloan, R. E. in Dinosaur Systematics: Perspectives and Approaches (eds Carpenter, K. & Currie, P. J.) 108?125 (Cambridge Univ. Press, Cambridge, 1990). 24. Wellnhofer, P. A. A new specimen of Archaeopteryx from the Solnhofen Limestone. Nat. Hist. Mus. Los. Angeles County Sci. Ser. 36, 3?23 (1992). 25. Zhou, Z.-H. & Hou, L.-H. Confuciusornis and the early evolution of birds. Vertebrata PalAsiatica 36(2), 136?146 (1998). 26. Buffetaut, E., Suteethorn, V. & Tong, H.-Y. The earliest know tyrannosaur from the Lower Cretaceous of Thailand. Nature 381, 689?691 (1996). 27. Xu, X., Tang, Z.-L. & Wang, X.-L. A therizinosaurid dinosaur with integumentary structures from China. Nature 399, 350?354 (1999). 28. Clark, J. M., Hopson, J. A., Hernandez, R., Fastovsky, D. E. & Montellano, M. Foot posture in a primitive pterosaur. Nature 391, 886?889 (1998). 29. Chiappe, L. M. Climbing Archaeopteryx? A response to Yalden. Archaeopteryx 15, 109?112 (1997). 30. Chatterjee, S. The Rise of Birds (John Hopkins Univ. Press, Baltimore, 1997). Supplementary information is available on Nature?s World-Wide Web site (http://www.nature.com) or as paper copy from the London editorial office of Nature. Acknowledgements We thank D. Unwin, L. Chiappe, X.-C. Wu, J. Clark and L. Witmer for comments; M.-M. Chang and Y.-Q. Wang for assistance during the course of the work; H.-J. Wang for preparing the specimen; R.-S. Li for making the drawings; and J. Zhang and H.-L. You for taking the photographs. This work was supported by the Special Funds for Major State Basic Research Projects of China, and research grants from the National Geographic Society of the United States, Chinese Natural Science Foundation, and the Chinese Academy of Sciences. Correspondence and requests for materials should be addressed to X.X. (e-mail: xxu@midwest.com.cn). letters to nature 708 NATURE | VOL 408 | 7 DECEMBER 2000 | www.nature.com ................................................................. Mitochondrial genome variation and the origin of modern humans Max Ingman*, Henrik Kaessmann?, Svante Pa�a�bo? & Ulf Gyllensten* * Department of Genetics and Pathology, Section of Medical Genetics, Rudbeck Laboratory, University of Uppsala, S-751 85 Uppsala, Sweden ? Max Planck Institute for Evolutionary Anthropology, Inselstrasse 22, D-04103 Leipzig, Germany .............................................................................................................................................. The analysis of mitochondrial DNA (mtDNA) has been a potent tool in our understanding of human evolution, owing to char- acteristics such as high copy number, apparent lack of recombination 1 , high substitution rate 2 and maternal mode of inheritance 3 . However, almost all studies of human evolution based on mtDNA sequencing have been confined to the control region, which constitutes less than 7% of the mitochondrial genome. These studies are complicated by the extreme variation in substitution rate between sites, and the consequence of parallel mutations 4 causing difficulties in the estimation of genetic dis- tance and making phylogenetic inferences questionable 5 . Most comprehensive studies of the human mitochondrial molecule have been carried out through restriction-fragment length poly- morphism analysis 6 , providing data that are ill suited to estima- tions of mutation rate and therefore the timing of evolutionary events. Here, to improve the information obtained from the mitochondrial molecule for studies of human evolution, we describe the global mtDNA diversity in humans based on analyses of the complete mtDNA sequence of 53 humans of diverse origins. Our mtDNA data, in comparison with those of a parallel study of the Xq13.3 region 7 in the same individuals, provide a concurrent view on human evolution with respect to the age of modern humans. The molecular clock hypothesis postulates that DNA sequence evolution is roughly constant over time in all evolutionary lineages. We used a test 8 that compares the log likelihoods of trees recon- structed with and without the molecular clock assumption to examine the supposition that the mtDNA lineages evolve at ?clock-like? rates. The human mtDNA sequences, excluding the D-loop, have evolved at roughly constant rates (P 0:094), and a relative rates test 9 , using a gorilla sequence as an outgroup, demonstrates that there is also no significant difference between the evolutionary rate of human and chimpanzee mtDNAs (P 0:123), excluding the D-loop. In contrast, the D-loop has not evolved at a constant rate across all human lineages (P , 0:001), and is consequently less suitable for dating evolu- tionary events. Therefore, unless specifically mentioned, we have excluded the D-loop from the analyses that follow. 1,500 4,000 6,500 Distance 0.1 0.3 0.5 0.7 0.9a | D '| Figure 1 The relationship between linkage disequilibrium, measured by |D9| versus distance between nucleotide sites for all 53 complete human mtDNA genomes. Values of 61.0 have been removed. a, Individuals of African descent (n 1;719 comparisons), R 2 = 0.001; b, only non-African individuals (n 741 comparisons), R 2 0:005. 1,500 4,000 6,500 Distance 0.1 0.3 0.5 0.7 0.9b | D '| � 2000 Macmillan Magazines Ltd letters to nature NATURE | VOL 408 | 7 DECEMBER 2000 | www.nature.com 709 From the mean genetic distance between all the humans and the one chimpanzee sequence (0.17 substitutions per site) and the assumption, based on palaeontological 10 and genetic 11 evidence, of a divergence time between humans and chimpanzees of 5 Myr, the mutation rate (m) for the mitochondrial molecule, excluding the D-loop, is estimated to be 1:70 3 10 2 8 substitutions per site per year. On the basis of the correlation between linkage disequilibrium and distance between sites, it has been claimed that mtDNA sequences show signs of recombination 12,13 . These analyses can be criticized for the methodology used 14 , particularly for the use of a linkage disequilibrium measure (Hill and Robertson measure, r 2 ) that doesn?t take allele frequency into account. We examined linkage Table 1 Summary of statistical parameters for the mtDNA Data set Length nSMPSD p ............................................................................................................................................................................. Total All humans 16,553 53 657 61.1 3:7 3 10 2 3 Non-Africans 16,555 32 358 38.5 2:3 3 10 2 3 Africans 16,556 21 367 76.7 4:6 3 10 2 3 D-Loop All humans 1,118 53 141 17.2 1:5 3 10 2 2 Non-Africans 1,118 32 103 12.8 1:1 3 10 2 2 Africans 1,121 21 77 19.7 1:8 3 10 2 2 Rest All humans 15,435 53 516 43.9 2:8 3 10 2 3 Non-Africans 15,437 32 255 25.7 1:7 3 10 2 3 Africans 15,448 21 290 57.0 3:7 3 10 2 3 ............................................................................................................................................................................. mtDNA data are given as entire sequences, or separated into D-loop and rest (all but the D-loop), and further separated into groups of African and non-African individuals. Length, aligned sequence length excluding gaps; n, number of sequences; S, number of segregating sites; MPSD, mean pairwise sequence difference; and p, genetic diversity. Chimp 39 Mandenka 40 Effik 41 Effik 33 Mkamba 34 Ewondo 35 Bamileke 36 Lisongo 37 Yoruba 38 Yoruba Non-African African 98 98 100 82 100 98 1 Chukchi 2 Australian 3 Australian 4 Piman 5 Italian 6 PNG Highland 7 PNG coast 8 PNG Highland 9 Georgian 10 German 13 Crimean Tatar 12 Saam 11 Uzbek 14 Dutch 15 French 16 English 17 Samoan 18 Korean 19 Chinese 20 Asian Indian 21 Chinese 22 PNG coast 23 Australian 24 Evenki 25 Buriat 26 Khirgiz 27 Warao 28 Warao 29 Siberian Inuit 30 Guarani 31 Japanese 32 Japanese 42 Ibo 43 Ibo 44 Mbenzele 45 Biaka 46 Biaka 47 Mbenzele 48 Kikuyu 49 Hausa 50 Mbuti 51 Mbuti 52 San 53 San 0.0005 * Figure 2 Neighbour-joining phylogram based on complete mtDNA genome sequences (excluding the D-loop). Data was constructed using PAUP*4.0 Beta (Sinauer Associates) and bootstrapped with 1,000 replicates (bootstrap values shown on nodes). The population origin of the individual is given at the twigs. Branches have been colour coded as in Fig. 4. Individuals of African descent are found below the dashed line and non- Africans above. The node marked with an asterisk refers to the MRCA of the youngest clade containing both African and non-African individuals. � 2000 Macmillan Magazines Ltd letters to nature 710 NATURE | VOL 408 | 7 DECEMBER 2000 | www.nature.com disequilibrium among all informative sites in our set of complete mtDNA genomes (including the D-loop) using a standard estimate (D9) that allows all variable positions to be examined 15 . For this analysis, we studied the sequences of Africans and non-Africans separately. There is no correlation between D9 and nucleotide distance between sites in the 53 sequences (African correlation coefficient, R 2 1 3 10 2 3 ; non-African R 2 5 3 10 2 3 ) (Fig. 1). We also analysed the association between r 2 and distance, and again there is no evidence of a correlation (R 2 2:23 3 10 2 6 , and 1 3 10 2 3 , respectively; data not shown). Thus, it does not appear to be necessary to consider recombination as contributing to the evolution of mtDNA. The two main hypotheses for the evolution of modern humans agree that Homo erectus spread from Africa around 2 Myr ago. The ?recent African origin? hypothesis 16,17 states that anatomically modern humans originated in Africa 100,000?200,000 years ago and subsequently spread to the rest of the world, replacing archaic human forms with little or no genetic mixing. The alternative, ?multi-regional? hypothesis proposes that the transformation to anatomically modern humans occurred in different parts of the world, and supports this with fossil evidence of cultural and morphological continuity between archaic and modern humans outside Africa 18 . There are, of course, variants of these two basic hypotheses that introduce additional assumptions, such as gene flow, that make the hypotheses increasingly difficult to test. Support for a recent African origin of modern humans has been provided by a number of mtDNA studies 16,17,19,20 ; however, these results have been troubled by the lack of statistical support for tree topology, especially the deep African branches 21,22 . Lacking suffi- ciently strong empirical data, it is impossible to confidently place the root of modern human mtDNA lineages in sub-Saharan Africa. The neighbour-joining 23 (NJ) tree constructed from our mtDNA sequences has a strongly supported basal branching pattern (Fig. 2). The three deepest branches lead exclusively to sub-Saharan mtDNAs, with the fourth branch containing both Africans and non-Africans. The deepest, statistically supported branch (NJ bootstrap 100) provides compelling evidence of a human mtDNA origin in Africa. The amount of mtDNA sequence diversity (p) among Africans (3:7 3 10 2 3 nucleotide differences per site) is more than twice that among non-Africans (1:7 3 10 2 3 ) (Table 1), corroborating earlier studies of the D-loop 16 and nuclear loci 24 . Also notable is the contrast between the deep branches of African mtDNAs and the ?star-like? phylogeny of non-African mtDNAs (Fig. 2). This high African diversity might result from either a considerably larger effective population size or a significantly longer genetic history. The ?star-like? phylogeny of the non-African sequences suggests a 20 40 60 80 100 10 20 30 40 Pairwise differences Pairwise differences Fr equency 0 0 10 20 30 40 50 10 30 50 70 90 Fr equency a b Figure 3 Mismatch distributions of pairwise nucleotide differences between mtDNA genomes (excluding the D-loop). a, African; b, non-African. 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C --- -- --- -- - - AT T A C --- - - - -- -- --- -- --- - - - -- -- --- - - - -- -- --- -- A -- -- --- - - - -- -- --- - - --- -- --- - - - -- -- --- - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- T - --- -- --- -- - - AT T A C --- - - - -- -- --- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - - --- C - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- T - --- -- --- -- - A - - -- -- - - - - - - - TC A -- -- --- - - - -- -- --- - - - -- -- --- -- - A - - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - - --- -- --- -- --- -- --- -- --- -- --- C - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- G A - - -- -- - - - - - - - TC A -- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- - CC -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- - A - - -- -- - - - - - - - TC A -- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - A --- - - --- -- --- - - - -- -- --- - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- -- T -- --- -- --- -- - A - - -- -- - - - - - - - TC A A -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- -- - - - - - - -- -- A - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- - C -- -- - - A - --- -- - A - - -- -- - - - - - - AT CA -- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - - CT A G A A G G -- --- - - - -- -- --- - - --- -- --- -- --- -- --- -- AC - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- - C - - - --- -- --- -- - A - - -- -- - - - - - - AT CA -- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - - CT A G A A G G -- --- - - - -- -- --- - - --- -- --- -- --- -- --- -- AC - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- G -- -- --- -- --- -- - A - - -- -- - - - - - - AT CA -- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - - CT A G A A G G - - - - -- -- - - - - - - -- -- A - - - --- -- --- -- --- -- AC - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- G -- -- --- -- --- -- - A - - -- -- - - - - - - AT CA -- -- --- - - - A - - - - -- -- --- - - --- -- --- - - - -- -- --- - - CT A G A A G G -- --- - - - -- -- --- - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- AA --- -- --- -- --- -- - A - - -- -- - - - - - - AT CA -- -- --- - - - A - - - - -- -- --- - - --- -- --- - - - -- -- --- - - CT A G A A G G -- --- - - - -- -- --- - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- AA --- -- --- -- --- -- - A - - -- -- - - - - - - - TC A -- -- --- - - - A -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- TA TT -- --- - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- A -- -- - A - - -- -- - - - - - - AT CA -- -- --- - - - A -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- TA TT -- --- - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- - A - - -- -- - - - - - - - TC A -- -- --- - - - A -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- TA TT -- --- - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- -- A -- - A - - -- -- - - - - - - - TC A - -- -- - - - - - A -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- TA TT -- --- - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- -- T -- --- -- --- -- G A --- - - - -- -- --- - - --- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - - --- -- --- -- --- -- --- -- --- -- - A - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- - AC -- - C -- -- A - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- -- - - CG C -- -- --- -- --- -- --- -- --- -- --- -- - A - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- - C --- -- - A - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- -- - - CG C -- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- - A - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- -- - - CG C -- -- --- -- --- -- --- -- --- -- --- -- A -- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- - A - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- -- - - - - - - -- -- - - - - - - - - - - - -- -- - - CG C -- -- --- -- --- -- --- -- --- -- --- -- A -- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- G -- A - - -- -- - - - - - - -- -- - - - - - - -- -- - C -- --- - - - -- -- --- -- --- - - - -- -- --- - - - -- -- --- -- --- - - - -- -- --- - - --- -- --- -- --- -- --- -- -- A -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- - A Consensus Chimp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 Figure 4 Data matrices showing all informative nucleotide positions, in decreasing order of frequency. Left, the whole mtDNA genome, excluding the D-loop. Right, the D-loop. The � 2000 Macmillan Magazines Ltd letters to nature NATURE | VOL 408 | 7 DECEMBER 2000 | www.nature.com 711 population bottleneck, potentially associated with the colonization of Eurasia from Africa. The date of this exodus from Africa can be estimated if the departing group subsequently experienced a popu- lation expansion. The mtDNA mismatch distributions for Africans and non-Africans indicate a marked difference in population history for the two groups 25 (Fig. 3). Mitochondrial DNAs from individuals of African origin show a ragged distribution consistent with constant population size, whereas the bell-shaped distribution of the non-African comparisons clearly indicates a recent popula- tion expansion. The assumption of constant population size can be verified by tests of selective neutrality that examine the correlation between the mean pairwise sequence difference (MPSD) and the number of segregating sites (S) 26,27 . In the African group, we cannot reject this assumption (Fu and Li?s D 21:17 (ref. 26); Tajima?s D 21:22 (ref. 27)), consistent with the premise that the popula- tion has been of roughly constant size . However, it can be rejected in the non-African group (D 24:02; D 22:28), indicating that this group has experienced a period of population growth 25 . The trees on the left are cladograms with the same topology and numbering of individuals as the tree in Fig. 2. Individuals of African descent are found below the dashed line and non-Africans above. The four major groups of sequences have been colour coded as in Fig. 2. Blocks denote groups of nucleotides that are identical in several sequences. - C C C C - -T A - - T -- -- --- -- G - - T - --- -- --- -- --- G C --- -- --- -- --- -- --T -- --- -- --- -- --- -- --- - - C C C C - -T A - - T -T A - --- -A C A T T G --T -- --- T - --- G - --- -- --- -- --- -- --- -- --- -- --- -- --- -- -- - - - C C C C C -T A - - T -T A - --- -A C A T - G --- -- A - - T - T T - -- --- -- C C - -- --- -- --- -- --- -- --- -- --- -- --- T - C C C C C -T A - - --T A - --- -A C A T - G --- -- A - - T - T T - -- --- -- C C - -- --- -- --- -- --- -- --- -- --- C - --- T - C C C -- -T A - - T -T A - --G -A C A T T G --- C - A - - - - --- G - --- -- --C -- --- -- --- -- --- -- --- -- --- -- --- - - C C C -- -T A - - T -T A - --G -A C A T T G --- C - A - - - - --- G - --- -- --C -- --- -- --- -- --- -- --- -- --- -- --- - T C C C C - -T A - - T -T A - --- -A C A T T G --T -- --- T - -T - - - --- -- --- -- -T - - - --- -- --- -- --- -- --C -- -A - - - C -C -- --A T - T C - A A C -G -- G - - - - --- C G -T - - G --- -- T - - - - --- T - --- -- --- -A --- -- --- -- --- -- --- - - C -C -- - T -- -A C - G - - --- -- A - - C G - T - -- --- -- T - - - - --- T - --- -- --- -- --- -- --- -A T - - - - --- - - C -C -- - T -- -A C - G - - --- -- A - - C G - T - -- --- -- T - - - - --- T - --- -- --- -- --- -- --- -A T - - - - --- - - C C C C - C T -- -- C - - - - G -- -- --T -G --- -- T - - - - --- -- --- -- --- -- --- C - --A C G A - - - - --- -- --- - - C C C C - C --- -- C - - - - G -- -- --T -G --- -- T - - - - --- -- C - - - - --- -- --- C - --A C G A - - - - --- -- --- - T --- C C --- -- --T -- --- -- --- -- --- -- --- -- --C -- --C -- --- -- --- -- -T - - - --- -- --- -- --- -- --- - T --- -C --- -- --- -- --- -- --- -- --- -- --G -- --C -- --C -- --- -- --- -- -T - - - --- -- --T -- --- -- --- - T C -- -- --- -- --- -- --- C - --- -- --- -- --G -- --C -- --C -- --- -- --- -- -T T - - --- -- --T -- --- -- --- - - --- -C --- T - --T -- --- -- --- -- --- -- --- -- --- -C --- -- --- -- --- -- --- -- --- -- --- -- --- -- --A - T --- -C C - - - - --- -- --- -- --- -- --- -- --- -- --- -C --- -- --- -- --- -- --- -A --- -- -A - - - --- -- --- - - C -- -C --- -- --- -- --- C - --- -- --- -- --G -- --- -- --- -- --- -- --- C - --- -- --- -- --- -- --- -- --- - C TTT TT TC G C T C TC G G TTA A C A G C C A G C C TA G C A C A C C T A T C C T C T TTA C A G C A T T C C C TG TG G A C G G C C C C C A TG A G G C - A C - -C C - A T C T C - A - C - - - - G : - -- A - - C G --- G - -A C C - T -- -C --- -G -T - - A --- C C --- T - --- -- A - C C A - A A - T --- -- --- -C - C - - - --- -- --- -- --- -- --- -- --- -- -T - - - --- -- --- -- --- -- --- -- -A - - - --- -A --- - T --- C - --- T - --- -- --- -- --- -- --- -- --G -- --- -- --- -- --- -- A - - - - --- C - --- -- --- -- --- -- --- - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- T - --- -- --- - - C -- -C -T - - - --- -- --- C - G - - - - --- -- --- -- --- -- -T - - - --- -- --C -- --- -- --- -- --- -- --- -- --- - - --- -C --- T - --- -- --- -- --- -- --- -- --G -- --- -- --- -- --- -- --- -- --T -- --- -- --- -- --- -- -A - - T --- -C --- -- --- -A -C G - - --- -- --- -- --- -- --- -- --- -- --C -- --- -- --- -- C - - - - --- -- --- -- --A - T --- -C --- T - --- -A --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -C T - - - - --- -- --- -- --- -- --- - T --- -- --- -- --- -A --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -C T - - - - --- -- --- T - --- -- --- - - --- -C --- -- --- -A --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- - - - C - -C --- -- --- -A -C - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- C - - - - --- -- --- -- --- - - C -- C C --- -C --- -- --- C - --- -- --- -- --- -- --- -- --- -- --- -- --C C - --- -- --- -- --- -- -T - - - --- - - C -C -C --- -- --- -- --- C - --- -- --- -- --- -- --- -- --- -- --- -- --C C - --- -- --- -- --- -- -T - - - --- - T --- -C --- -C -C - - - --- -- --- -- --- -- --- -- --- -C --- -- --- -G --- -C --- -- --- -- --- -- --- -- --- - T - C - -- C - - T - - C - -- --- -- --- -- --- -- A - - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- - T --- -- C - - - - - C - -- --- -- --- -- --- -- --- -- -T - - - --- -- --- -- -T - - - --- -- --- -- --- -- --- -- --- - T --C -- C - - - C --- A - C - - - - --- -- --- -- --- -- --- -- T - - - - --- -- --- -- --- -- --- -- --- -- --C -- --- - - --C C - C - - - - --- -- --- -- --- -- A - - - - --- -- --- -- --- -- --- -G --- -- --- -- --- -- --- -- --- -- --- - - --- -- C - - - - --- -- -C - - - --- -- -T - - - --- -- --C -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- - - --- -- C - - - - --- -- -C - - - --- -- -T - - - --- -- --- -- --- -- -C - - - --- -- --- -- --- -- --- -- --- -- --- - - --- -- C - - - C --- -- -C - - - --- -- -T - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- - T C -- -- C - - - - --- -- -C - - - --- -- -T - - - --- -G --- -- --- T C --- -- A - - - - --- -- --- -- --- -- --- -- G - - - T C -- -- C - - - - --- -- -C - - - --- -- -T - - - --- -G --- -- --- T C --- -- A - - - - --- -- --- -- --- -- --- -- G - - - - --- -- C - - - - - C - A - --- -- --- -- --- -- --- -- --- -- -T - - - --- -- --- -- --- -- --- -- --- -- --- C - --- - T --- -- C - - - - - C - -- --- -- --- -- --- -- --- -- --- -- --- -C --- -- --- -- --- -- -A - - - --- -- --- -- --- - - --- C - C - - - - - C - A - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- - - --- -- C - - - - - C - -- --- -- --- -- --- -- --- -- --- -- -T - T - --- -- --- -- --- -- --- -- --- -- --- -- --- - T --C -- --- -C --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -G --- -- --- -- --- -- --- -- --- -- --- - T --- -- --- T - --- -- --- -- G - - - - - T - -- --- -G --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- - - - C - -- --- T - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- -T - - - --- -- --- -- --- -- --- -- --- - - C C C -- --- T - --- -- C - - C - --- -- --- -- -T - - - --- -- --- -- --- -- --- -- T - - - - --- -- --- -- --- -- --- - - C C - -- --- T - --- -- C - - C - --- -- --- -- -T - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- - T --- C - --- T - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -A --- - T - C - C - -T - T C T C - -- --G -- --- T - A - T - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- - - - C - C - -T - - C --T -- --- -- --- -- A - - - - --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- -- --- - T C C - C - -T - - C - C T -- --- -- --- -- A - - - - --- -- --- -- --- -- --- -- --- -- --- -- -A - - - --- -- --- -- --- - Consensus Chimp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 --A - --A - -T A - -T A - � 2000 Macmillan Magazines Ltd letters to nature 712 NATURE | VOL 408 | 7 DECEMBER 2000 | www.nature.com time when the expansion began was estimated (t 20:23) to be about 1,925 generations ago 28 . Assuming a generation time of 20 yr this equates to 38,500 yr BP, a date that coincides with the onset of a period of cultural change about 35,000?40,000 years ago 29 . This involves, for example, the first appearance of regional cultural variation and the acceleration of artefactual change. The age of the most recent common ancestor (MRCA) for mtDNA, on the basis of the maximum distance between two humans (5:82 3 10 2 3 substitutions per site between the Africans Mkamba and San), is estimated to be 171;500 6 50;000 yr BP.We can also estimate the age of the MRCA for the youngest clade that contains both African and non-African sequences (Fig. 2, asterisk) from the mean distance of all members of that clade to their common node (8:85 3 10 2 4 substitutions per site) as 52; 000 6 27; 500 yr BP. Because genetic divergence is expected to precede the divergence of populations, this date can be considered as the lower bound for an exodus from Africa. Notably, a group of six African sequences (Fig. 4a, sequences 33? 38) are genetically distant to those of other Africans, but share a common ancestor with non-Africans. These lineages represent descendants of a population that evidently gave rise to all the non-African lineages. Whether the ancestors of these six extant lineages originally came from a specific geographic region is not possible to determine, but we note that these sequences are from five populations that are now geographically unrelated. Our study of the entire mitochondrial genome has some sig- nificant distinctions from previous studies of the D-loop. Most notably, the sequences outside of the D-loop evolve in a roughly ?clock-like? manner, enabling a more accurate measure of mutation rate, and therefore improved estimates of times to evolutionary events. Also of importance is the strong statistical support for the tree topology that has been lacking in earlier investigations. The difference between the D-loop and the rest of the molecule is visually evident in the contrast between the jumbled arrangement of polymorphic sites in the D-loop and the clear haplotypes defined by the sites in the rest of the molecule (Fig. 4). The use of largely the same individuals in the this study, as in that of the nuclear Xq13.3 (ref. 7) region, provides a unique opportunity to compare the information gained from the two genetic systems. Because the X chromosome has an effective population size three times that of mitochondria, the MRCA of an X-chromosomal locus is expected to be three times higher. Thus, the age of the MRCA of Xq13.3 is in agreement with the mtDNA data (mtDNA: 171,500; Xq13.3: 479,000 yr BP). The results from Xq13.3 also concur with the mtDNA data with respect to the greater genetic diversity found among African individuals relative to non-Africans, but Xq13.3 shows a considerably lower difference in diversity between the two groups (mtDNA: African, 3:9 3 10 2 3 , non-African, 1:7 3 10 2 3 ; Xq13:3:3:5 3 10 2 4 ,3:05 3 10 2 4 ). Owing to its lower substitution rate, only 33 segregating sites were present in 69 sequences of 10.2 kilobases (kb) at Xq13.3, as compared with the 657 segregating sites in the mitochondrial dataset. Comparisons of the mtDNA and Xq13.3 sequences carried by specific individuals show little correla- tion between the two loci, as expected from the different modes of inheritance. For example, the two Warao indians showing the highest similarity in mtDNA have, in fact, two of the most divergent sequences studied at the Xq13.3 locus. Others, such as the Saami and Mandenka sequences, are closely related at Xq13.3 but have relatively high mitochondrial divergence. Our results indicate that the field of mitochondrial population genomics will provide a rich source of genetic information for evolutionary studies. Nevertheless, mtDNA is only one locus and only reflects the genetic history of females. For a balanced view, a combination of genetic systems is required. With the human genome project reaching fruition, the ease by which such data may be generated will increase, providing us with an evermore detailed understanding of our genetic history. M Methods Sampling strategy and mtDNA sequences To assess the global genetic diversity in humans, while analysing a restricted number of samples, we studied 53 individuals representing 14 of the major linguistic phyla. This sampling strategy attempts to avoid the bias inherent in selecting individuals on the basis of current world demographics, such as current population size or geographic location 5 . To provide an opportunity for comparison, we selected, where possible, the same individuals as those used by a previous study 7 for the analysis of the Xq13.3 region. All the complete mtDNA sequences are unique and vary in length from 16,558 to 16,576 base pairs (bp). From nearly 900 kb sequenced, 5 heteroplasmic sites were confidently identified. We identified a total of 657 segregating sites (141 in the D-loop; 516 outside) among these 53 individuals, of which 283 (80 in the D-loop; 203 outside) showed the same polymorphism in at least 2 individuals (Fig. 4). The pairwise sequence distances between mtDNAs, corrected for multiple substitutions 4 , vary from 6:0 3 10 2 5 substitutions per site between two South American indians (Warao) to 6:8 3 10 2 3 substitutions per site between two Africans (Mbenzele pygmy and San). The average distance between mtDNA genomes is 3:8 3 10 2 3 substitutions per site. PCR primers and sequencing The primers we used for polymerase chain reaction (PCR) amplification have been described 30 . Sequencing was performed on the PCR products directly using BigDye (Applied Biosystems) chemistry. Separation of sequencing ladders was performed on the ABI 377 instrument for automated fragment analysis. We sequenced both forward and reverse strands. Sequence analysis was performed using Sequencing Analysis 3.3 (Applied Biosystems), and sequence alignment was made with Sequencher 3.1.1 (Gene Codes). Accession numbers The chimpanzee sequence (GenBank accession no. D38113 and the gorilla sequence (accession no. 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H. in The Human Revolution: Behavioural and Biological Perspectives on the Origins of Modern Humans (eds Mellars, P. & Stringer, C.) 62?108 (Princeton Univ. Press, Princeton, New Jersey, 1989). 19. Horai, S., Hayasaka, K., Kondo, R., Tsugane, K. & Takahata, N. Recent African origin of modern humans revealed by complete sequences of hominoid mitochondrial DNAs. Proc. Natl Acad. Sci. USA 92, 532?536 (1995). 20. Ruvolo, M. et al. Mitochondrial COII sequences and modern human origins. Mol. Biol. Evol. 10, 1115?1135 (1993). 21. Templeton, A. R. Human origins and analysis of mitochondrial DNA sequences. Science 255, 737 (1992). 22. Nei, M. Age of the common ancestor of human mitochondrial DNA. Mol. Biol. Evol. 9, 1176?1178 (1992). 23. Saitou, N. & Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406?425 (1987). � 2000 Macmillan Magazines Ltd letters to nature NATURE | VOL 408 | 7 DECEMBER 2000 | www.nature.com 713 24. Zietkiewicz, E. et al. Genetic structure of the ancestral population of modern humans. J. Mol. Evol. 47, 146?155 (1998). 25. Rogers, A. R. & Harpending, H. Population growth makes waves in the distribution of pairwise genetic differences. Mol. Biol. Evol. 9, 552?569 (1992). 26. Fu, Y. X. & Li, W. H. Statistical tests of neutrality of mutations. Genetics 133, 693?709 (1993). 27. Tajima, F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123, 585?595 (1989). 28. Rozas, J. & Rozas, R. DnaSP, DNA sequence polymorphism: an interactive program for estimating population genetics parameters from DNA sequence data. Comput. Appl. Biosci. 11, 621?625 (1995). 29. Klein, R. G. The Human Career: Human Biological and Cultural Origins (Univ. Chicago Press, Chicago, 1989). 30. Reider, M. J., Taylor, S. L., Tobe, V. O. & Nickerson, D. A. Automating the identification of DNA variations using quality-based fluorescence re-sequencing: analysis of the human mitochondrial genome. Nucleic Acids Res. 26, 967?973 (1998). Acknowledgements We thank M. Stoneking for his advice regarding the analysis of recombination, and L. Cavalli-Sforza, G. Destro-Bisol, L. Excoffier, T. Jenkins, K. Kidd, J. Kidd, G. Klein, R. Mahabeer, V. Nasidze, E. Poloni, H. Soodyall, M. Stoneking, M. Voevoda and S. Wells for scmples. This work was supported by grants from Swedish Natural Sciences Research Council and Beijer Foundation. Correspondence and requests for materials should be addressed to U.G. (e-mail: ulf.gyllensten@genpat.uu.se). ................................................................. The HIC signalling pathway links CO 2 perception to stomatal development Julie E. Gray*, Geoff H. Holroyd?, Frederique M. van der Lee?, Ahmad R. Bahrami*, Peter C. Sijmons�, F. Ian Woodwardk, Wolfgang Schuch� & Alistair M. Hetherington? * Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK ? Department of Biological Sciences, University of Lancaster, Lancaster LA1 4YQ, UK ? Zeneca Mogen, PO Box 628, 2300 AP Leiden, The Netherlands � Cellscreen, PO Box 134, 6700 AC Wageningen, Netherlands kDepartment of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, UK � Zeneca Wheat Improvement Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK .............................................................................................................................................. Stomatal pores on the leaf surface control both the uptake of CO 2 for photosynthesis and the loss of water during transpiration. Since the industrial revolution, decreases in stomatal numbers in parallel with increases in atmospheric CO 2 concentration have provided evidence of plant responses to changes in CO 2 levels caused by human activity 1,2 . This inverse correlation between stomatal density and CO 2 concentration also holds for fossil material from the past 400 million years 3 and has provided clues to the causes of global extinction events 4 . Here we report the identification of the Arabidopsis gene HIC (for high carbon dioxide), which encodes a negative regulator of stomatal devel- opment that responds to CO 2 concentration. This gene encodes a putative 3-keto acyl coenzyme A synthase?an enzyme involved in the synthesis of very-long-chain fatty acids 5 . Mutant hic plants exhibit up to a 42% increase in stomatal density in response to a doubling of CO 2 . Our results identify a gene involved in the signal transduction pathway responsible for controlling stomatal numbers at elevated CO 2 . As part of a promoter trap screen aimed at isolating tissue- specific genes in Arabidopsis thaliana 6,7 , we identified an individual plant designated hic in which the b-glucuronidase (GUS) reporter gene was expressed only in the guard cells (Fig. 1) and nowhere else in the plant. Southern analysis of hic genomic DNA using a GUS gene probe detected a single band, suggesting that there was only one GUS gene insertion in this line (data not shown). We next investigated the phenotype of the hic mutant and found it to be different from previously described Arabidopsis stomatal development mutants 8,9 . hic stomata had no obvious phenotype and the plants were not wilting, suggesting that guard-cell function was not impaired. hic guard cells increased turgor in response to light and reduced turgor in response to the addition of the plant hormone abscisic acid in stomatal bioassays (data not shown). hic plants were indistinguishable from the parental ecotype when analysed by infrared thermography (data not shown); however, analysis of plants that had been grown under differing CO 2 concentrations showed that the HIC gene affects stomatal development. We grew hic and C24, the parental ecotype, at elevated and ambient concentrations of CO 2 , and measured the stomatal index and density on the leaf surface. A reduction in stomatal index and density in response to elevated CO 2 is well characterized in some species, including three ecotypes of Arabidopsis 1,2 , and indicates that the plant has the potential for increased water-use efficiency with increasing CO 2 . Table 1 shows that growth of hic plants under elevated CO 2 induced marked increases in both stomatal index and Table 1 Effect of ambient and elevated CO 2 on stomatal and epidermal cell density and stomatal index Stomatal density (n mm -2 ) Epidermal cell density (n mm -2 ) Stomatal index (%) Experiment Line Ambient Elevated Difference (P) % change Ambient Elevated Difference (P) % change Amnbient Elevated Difference (P) % change ................................................................................................................................................................................................................................................................................................................................................................... 1 C24 280 (9) 233 (8) ,0.001 -17.0 1,047 (9) 902 (8) ,0.001 -13.8 21.1 (9) 20.5 (8) NS -3.1 hic 231 (8) 320 (8) ,0.001 +38.0 891 (8) 902 (8) NS +1.2 20.8 (8) 26.0 (8) ,0.005 +25 2 C24 265 (7) 245 (9) NS -7.8 1,056 (7) 1,028 (9) NS -2.7 20.0 (7) 19.1 (9) NS -4.4 hic 257 (9) 324 (10) ,0.001 +25.2 1,047 (9) 1,061 (10) NS +1.3 19.8 (9) 23.2 (10) ,0.001 +17.8 3 C24 324 (9) 245 (7) ,0.001 -24.5 1,322 (9) 1,058 (7) ,0.005 -20 19.6 (9) 18.7 (7) NS -4.7 hic 273 (7) 387 (9) ,0.001 +41.8 1,053 (7) 1,092 (9) NS +3.7 20.5 (7) 26.2 (9) ,0.001 +27.6 4 C24 273 (12) 283 (12) NS +3.1 1,052 (12) 1,049 (12) NS -0.3 20.6 (12) 21.2 (12) NS +3.2 hic 266 (12) 351 (12) ,0.001 +31.8 1,035 (12) 1,062 (12) NS +2.6 20.6 (12) 24.9 (12) ,0.001 +21.2 ................................................................................................................................................................................................................................................................................................................................................................... Data are presented for four separate experiments in the hic mutant and the parental ecotype (C24). The average density or stomatal index is presented for each treatment. The numbers in brackets indicate the number of individual plants in each treatment. Treatments were compared using the Mann?Whitney Rank Sum Test (SigmaStat 2.03). NS, not significant. Figure 1 GUS expression in hic Arabidopsis plants is specific to guard cells. Part of a hic leaf histochemically stained for GUS activity shows guard-cell-specific expression at low (a; scale bar, 100 mm) and higher magnification (b; scale bar, 10 mm). � 2000 Macmillan Magazines Ltd "
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