Annotated Bibliography

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5. Charlesworth, B. The effect of background selection against deleterious mutations on weakly selected, linked variants. Genet. Res. 63, 213±227 (1994).

6. Fay, J., Wycoff, G. J. & Wu, C.-I. Positive and negative selection on the human genome. Genetics 158, 1227±1234 (2001).

7. McDonald, J. H. & Kreitman, M. Adaptive evolution at the Adh locus in Drosophila. Nature 351, 652± 654 (1991).

8. Charlesworth, B., Morgan, M. T. & Charlesworth, D. The effect of deleterious mutations on neutral molecular variation. Genetics 134, 1289±1303 (1993).

9. Maynard Smith, J. & Haigh, J. The hitch-hiking effect of a favourable gene. Genet. Res. 23, 23±35 (1974). 10. Begun, D. J. & Aquadro, C. F. levels of naturally occuring DNA polymorphism correlate with

recombination rates in D. melanogaster. Nature 356, 519±520 (1992). 11. Begun, D. The frequency distribution of nucleotide variation in Drosophila simulans. Mol. Biol. Evol.

18, 1343±1352 (2001). 12. Kliman, R. Recent selection on synonymous codon usage in Drosophila. J. Mol. Evol. 49, 343±351 (1999). 13. Adams, M. D. et al. The genome sequence of Drosophila melanogaster. Science 287, 2185±2195 (2000). 14. Powell, J. R. & DeSalle, R. Drosophila molecular phylogenies and their uses. Evol. Biol. 28, 87±138

(1995). 15. Haldane, J. B. S. The cost of natural selection. J. Genet. 55, 511±524 (1957). 16. Kimura, M. Evolutionary rate at the molecular level. Nature 217, 624±626 (1968). 17. Thompson, J. D., Higgins, D. G. & Gibson, T. J. ClustalWÐimproving the sensitivity of progressive

multiple alignment through sequence weighting, position-speci®c gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673±4680 (1994).

18. Xia, X. Data Analysis in Molecular Biology and Evolution (Kluwer Academic, London, 2000). 19. Rozas, J. & Rozas, R. DnaSP version 3: an integrated program for molecular population genetics and

molecular evolution analysis. Bioinformatics 15, 174±175 (1999). 20. Yang, Z. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput. Appl.

Biosci. 13, 555±556 (1997).

Supplementary Information accompanies the paper on Nature's website (http://www.nature.com).

Acknowledgements We thank B. Charlesworth, C.-I. Wu, S. Otto, M. Whitlock, T. Johnson, P. Awadalla, J. Gillespie, G. McVean and P. Keightley for helpful discussions, and E. Moriyama for help with data collection. N.G.C.S. was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and A.E.-W. is funded by the Royal Society and the BBSRC.

Competing interests statement The authors declare that they have no competing ®nancial interests.

Correspondence and requests for materials should be addressed to A.E.-W. (e-mail: [email protected]).

................................................................. Testing the neutral theory of molecular evolution with genomic data from Drosophila Justin C. Fay*², Gerald J. Wyckoff*² & Chung-I Wu*³

* Committee on Genetics, University of Chicago, Chicago, Illinois 60637, USA ³ Department of Ecology and Evolution, University of Chicago, Chicago, Illinois 60637, USA

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Although positive selection has been detected in many genes, its overall contribution to protein evolution is debatable1. If the bulk of molecular evolution is neutral, then the ratio of amino-acid (A) to synonymous (S) polymorphism should, on average, equal that of divergence2. A comparison of the A/S ratio of polymorphism in Drosophila melanogaster with that of divergence from Drosophila simulans shows that the A/S ratio of divergence is twice as highÐa difference that is often attributed to positive selection. But an increase in selective constraint owing to an increase in effective population size could also explain this observation, and, if so, all genes should be affected similarly. Here we show that the differ- ence between polymorphism and divergence is limited to only a

² Present addresses: Department of Genome Sciences, Lawrence Berkeley National Laboratory, Berkeley, California 94720 (J.C.F.); Department of Human Genetics, University of Chicago, Chicago, Illinois 60637, USA (G.J.W).

fraction of the genes, which are also evolving more rapidly, and this implies that positive selection is responsible. A higher A/S ratio of divergence than of polymorphism is also observed in other species, which suggests a rate of adaptive evolution that is far higher than permitted by the neutral theory of molecular evolution. The neutral theory holds that the bulk of DNA divergence

between species is driven by mutation and drift, rather than by positive darwinian selection3. But because the effect of positive selection is often masked by negative selection4, detecting positive selection is a challenging task. A rate of amino-acid substitution greater than that of synonymous substitution can be explained only by positive selection5, but such a criterion is very stringent as negative selection lowers the rate of amino-acid substitution. A high rate of amino-acid substitution is limited mostly to genes that are involved in resistance to disease or in sexual reproduction, where there is continual room for improvement6,7. The McDonald±Kreitman test can detect positive selection even

in the presence of negative selection through a ratio of amino-acid divergence to synonymous divergence greater than that of polymorphism2. The A/S ratio of divergence is in¯ated above polymorphism by advantageous amino-acid mutations, which quickly sweep through a population but have a cumulative effect on divergence. The McDonald±Kreitman test has been applied to many genes individually, but only a few have yielded a signi®cant excess of amino-acid divergence (Drosophila genes are reviewed in refs 8, 9). This may in part be caused by a lack of power in detecting positive selection in individual genes unless a large number of adaptive substitutions have occurred. For those genes that have yielded a signi®cant McDonald±

Kreitman test result, the A/S ratio of divergence is more than twice as great as polymorphism10±12 . The effects of positive selection may also be obscured by slightly deleterious amino-acid mutations that in¯ate the A/S ratio of polymorphism but not divergence. The effects of slightly deleterious mutations can be removed by comparing common polymorphism with divergence, because dele- terious amino-acid mutations are kept at low frequency in the population4. This can only be done when the data from a large number of genes are combined; individual genes rarely contain more than a few common amino-acid polymorphisms. An important but rarely appreciated assumption of the

McDonald±Kreitman test is that the selective constraint on a gene remains constant over time. The selective constraint on a gene is determined by the proportion of amino-acid mutations that are deleterious3, 2Ns , -1, so both a change in the selection coef®cient (s) and a change in effective population size (N) can result in a change in selective constraint. Although it is well known that selective constraint is not static across phylogenetic lineages13,14, this assumption is rarely justi®ed in applications of the McDonald± Kreitman test. Whereas the strength of selection on each gene might ¯uctuate over time depending on the genetic or environmental background, a genome-wide change in constraint, such as that caused by a change in effective population size, should produce a consistent increase or decrease in the A/S ratio across all genes. Alternatively, under positive selection each gene might be affected to a different degree and some genes might not be affected at all. To compare genomic patterns of amino-acid and synonymous

Table 1 Polymorphisms in D. melanogaster and divergence from D. simulans

Gene* Class Amino-acid Synonymous A/S polymorphism, A polymorphism, S

Common (.12.5%) 6 46 0.13 Divergence 42 189 0.22

Autosomal Rare 79 126 0.63 Common 44 118 0.37 Divergence 421 521 0.81

............................................................................................................................................................................. * There are 5 X-linked and 31 autosomal genes with a sample size of eight or greater (see text for the data from all 45 genes).

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Table 2 African and non-African common polymorphism and divergence

Class Population Amino-acid Synonymous A/S polymorphism, A polymorphism, S

African 40 159 0.25 Divergence 413 663 0.62 .............................................................................................................................................................................

site evolution, we tabulated polymorphism in D. melanogaster and divergence from D. simulans from 45 gene surveys (Methods). If all amino-acid and synonymous variation is neutral, then the A/S ratio of polymorphism and divergence should be constant. The A/S ratio of divergence (598/950 = 0.63) is signi®cantly greater than that of common polymorphism (65/224 = 0.29; P , 10 -6). We compared divergence with the common rather than the total polymorphism because deleterious mutations at low frequency in¯ate the A/S ratio of polymorphism. For the 36 genes with sample sizes of eight or greater, there is a signi®cant excess of rare over common amino-acid variation in autosomal genes (P = 0.022; Table 1), as is observed in humans4. The absence of a difference in X-linked genes suggests that the deleterious mutations are partially recessive and are more readily eliminated from the X chromosome. Both positive selection and an increase in selective constraint on

amino-acid changes can produce a higher A/S ratio of divergence than of polymorphism. But only under certain restrictive conditions is a genome-wide change in constraint possible. One such condition is an increase in effective population size that is neither too distant nor too recent in the evolutionary past. If this possibility can be ruled out, positive selection may be the only viable explanation for the high rate of amino-acid divergence. If an increase in selective constraint resulted from a population

size increase associated with the spread of D. melanogaster outside Africa15, it might be more appropriate to compare the A/S ratio of the African population with that of divergence. Table 2, which includes the 32 genes for which both African and non-African populations were surveyed, shows that there is a signi®cantly larger A/S ratio of divergence than of polymorphism in either population. If a recent increase in effective population size increased constraint on amino-acid polymorphism in both African and non-African populations, then patterns of synonymous polymorphism might be skewed towards rare variants. Neither African or non-African populations show this pattern16. Finally, if there has been a decrease in effective population size along the D. melanogaster lineage17,18, the A/S ratio of polymorphism should be greater than that of divergence between the two species.

–12 –8 –4 0 4 8 >10

ka < 0.02

ka > 0.02

Excess of amino-acid divergence

N u m

b e r

o f

g e n e s

Figure 1 The distribution of the excess of amino-acid divergence contributed by each gene. For reference, fast and slowly evolving genes are denoted by a rate of amino-acid substitution (ka) greater than (®lled bars) or less than (open bars) 2%.

Table 3 Polymorphism and divergence in neutral and fast genes

Genes* Class Amino-acid Synonymous A/S polymorphism, A polymorphism, S

............................................................................................................................................................................. Neutral Rare 31 90 0.34

Common 16 69 0.23 Divergence 65 247 0.26

Fast Rare 48 36 1.33 Common 28 49 0.57 Divergence 356 274 1.30

If an increase in effective population size has produced a genome- wide increase in selective constraint, the A/S ratio of all genes should be affected. In Fig. 1, the distribution of each gene's contribution to the excess of amino-acid divergence suggests that there are two classes of gene: neutral and rapidly evolving. The neutral class comprises 34 genes that deviate by less than 10 amino-acid sub- stitutions from that expected on the basis of the A/S ratio of all common polymorphism. The remaining 11 genes all have a higher A/S ratio of divergence than of polymorphism, and account for the whole difference in the A/S ratio of polymorphism and divergence. These genes are Acp26Aa, Acp29Ab, anon1A3, anon1E9, anon1G5, ci, est-6, Ref2P, Rel, tra and Zw. As expected under positive selection, which increases the rate of protein evolution, these 11 genes have a high rate of amino-acid substitution (Fig. 1). Can the pattern in Fig. 1 be explained by selection or demogra-

phy? Table 3 shows that, in the rapidly evolving genes, the A/S ratios of divergence and of rare polymorphism are much higher than the A/S ratio of the common polymorphism. This is expected if the genes are under positive selection. Although a large increase in population size in the recent past could account for the difference between the A/S ratio of divergence and that of common poly- morphism, this explanation is incompatible with the very small difference found in the 26 neutral genes. Because both the neutral and rapidly evolving genes have a higher A/S ratio of rare poly- morphism than of common polymorphism, both should have been affected by an increase in effective population size. If positive selection is common, other species should also have an

A/S ratio of divergence greater than that of polymorphism. In addition, any demographic scheme is not likely to be shared by several species. In a study of eight genes in D. simulans, Drosophila mauritiana and Drosophila sechellia, the A/S ratio of polymorphism (A/S = 32/183) is 34% that of divergence (28/55)19. In a study of 42 genes with polymorphism in both D. melanogaster and D. simulans, the A/S ratio of polymorphism is 65% that of divergence (N. G. C. Smith and A. Eyre-Walker, personal communication). In another study of 23 genes, the A/S ratio of polymorphism (45/305) is 30% that of divergence along the D. simulans lineage (65/133)20. In humans, the A/S ratio of common polymorphism (70/122) found in 181 genes is 65% that of divergence (3,660/4,151) found in a different set of 182 human and Old World monkey genes4. Although these genomic patterns of variation are not explained

easily by the neutral theory, slightly deleterious mutations must clearly be accounted for in attempting to measure positive selection. In humans, 38% of amino-acid polymorphism was estimated to be slightly deleterious4, and in D. melanogaster the estimate is 26%, (0.63 - 0.37) ́ 126/123, from the combined neutral and rapidly evolving genes (Table 3). These slightly deleterious mutations, which are emphasized by the nearly neutral theory21, could become effectively neutral and ®xed during a population bottleneck of suf®cient severity, providing a burst of amino-acid substitutions and an increase in the A/S ratio of divergence. We control for the impact of these slightly deleterious mutations by comparing the rapidly evolving class of gene to the neutral class (Fig. 1, Table 3). Additional genomic data from other species will be needed to estimate the general impact of these slightly deleterious mutations on protein evolution. M

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Methods Data A literature search yielded 45 genes for which polymorphism had been surveyed in D. melanogaster and for which an outgroup sequence was available. Of these, 36 had a sample size of eight or greater, 32 had been surveyed in at least two African and two non- African individuals and 10 were of X-linked genes. The 45 genes and their references are listed in Supplementary Information.

Analysis Polymorphism data was tabulated by hand or from GenBank accession numbers using SITES21 or DNASP22. For each polymorphic site, the minor allele was classi®ed as rare (# 12.5%) or common (. 12.5%). The cutoff of 12.5% was chosen to exclude deleterious mutations from the common frequency class and to include those genes with samples of eight or more in the analysis of rare compared to common polymorphism. Cutoffs of 10 and 15% produce similar results. We treated three alleles segregating at a single nucleotide as two segregating sites and excluded complex variations. Divergence data was obtained by comparing a randomly chosen sequence of D. melanogaster with that of D. simulans or, if unavailable, either D. mauritiana or D. sechellia. The number of amino-acid and synonymous substitutions between species was estimated using Kimura's two-parameter model to correct for multiple hits.

The contribution of each gene to the excess number of amino-acid substitutions was calculated as the excess number of amino-acid substitutions minus the excess number of amino-acid polymorphisms found in each gene. The excess for polymorphism and divergence is A - S ́ (65/224), where A and S are the number of amino-acid and synonymous substitutions, respectively, and 65/224 is the total number of amino-acid polymorphisms divided by synonymous polymorphisms. (Ideally, the excess of amino- acid divergence in each gene should be calculated using only polymorphism and divergence in that gene but there is rarely suf®cient polymorphism in a single gene for comparison with divergence.) We also calculated the contribution to the excess separately for three groups of genes sorted by their rate of amino-acid divergence. The two methods produced a similar distribution so the simpler method using a single group of genes was used.

Received 27 June; accepted 4 December 2001.

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351, 652±654 (1991). 3. Kimura, M. The Neutral Theory of Molecular Evolution (Cambridge Univ. Press, Cambridge, 1983). 4. Fay, J. C., Wyckoff, G. J. & Wu, C.-I. Positive and negative selection on the human genome. Genetics

158, 1227±1234 (2001). 5. Kimura, M. Preponderance of synonymous changes as evidence for the neutral theory of molecular

evolution. Nature 267, 275±276 (1977). 6. Yang, Z. & Bielawski, J. P. Statistical methods for detecting molecular adaptation. Trends Ecol. Evol. 15,

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846 (1997). 23. Rozas, J. & Rozas, R. DnaSP version 3: an integrated program for molecular population genetics and

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Supplementary Information accompanies the paper on Nature's website (http://www.nature.com).

Acknowledgements This work was supported by grants from the NIH and NSF to C.-I.W. and a Genetics Training Grant and a Department of Education PhD fellowship to J.C.F.

Competing interests statement The authors declare that they have no competing ®nancial interests.

Correspondence and requests for materials should be addressed to J.C.F. (e-mail: [email protected]).

................................................................. Brain potential and functional MRI evidence for how to handle two languages with one brain Antoni Rodriguez-Fornells*, Michael Rotte², Hans-Jochen Heinze², ToÈmme NoÈsselt² & Thomas F. MuÈ nte*

* Department of Neuropsychology, Otto von Guericke University, UniversitaÈtsplatz 2, GebaÈude 24, 39106 Magdeburg, Germany ² Klinik fuÈr Neurologie 2, Otto von Guericke University, Leipzigerstrasse 44, 39120 Magdeburg, Germany

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Bilingual individuals need effective mechanisms to prevent inter- ference from one language while processing material in the other1. Here we show, using event-related brain potentials and functional magnetic resonance imaging (fMRI), that words from the non- target language are rejected at an early stage before semantic analysis in bilinguals. Bilingual Spanish/Catalan and monolingual Spanish subjects were instructed to press a button when presented with words in one language, while ignoring words in the other language and pseudowords. The brain potentials of bilingual subjects in response to words of the non-target language were not sensitive to word frequency, indicating that the meaning of non-target words was not accessed in bilinguals. The fMRI activation patterns of bilinguals included a number of areas previously implicated in phonological and pseudoword process- ing2±5 , suggesting that bilinguals use an indirect phonological access route to the lexicon of the target language to avoid interference6. High-pro®ciency bilingual subjects manage to understand and

speak one of their languages without apparent interference from the other. This is a remarkable ability in the face of the fact that neuro- imaging studies have revealed, at least for high-pro®ciency bilin- guals, that neuro-anatomical representations of both languages are

Monolinguals

–2 m V

400 800 1,200 ms

Bilinguals

Spanish Catalan Pseudo

Figure 1 Lateralized readiness potentials (LRPs) from the main experiment indicating the preparation of motor responses. The onset latency of the LRP to Spanish words, estimated by the time at which the amplitude was signi®cantly different from zero for at least 4 consecutive time points (sequential t-tests)14, was 408 ms in the monolingual and 520 ms in the bilingual group. No LRP activity is observed for Catalan words, indicating an effective blocking of `word' (go) responses in the bilingual group.

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