letters to nature 13. Pfanner, N. & Geissler, A. Versatility of the mitochondrial protein import machinery. Nature Rev. Mol. Cell. Biol. 2, 339­349 (2001). 14. Winzeler, E. A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901­906 (1999). 15. Bukau, B. & Horwich, A. L. The Hsp70 and Hsp60 chaperone machines. Cell 92, 351­366 (1998). 16. Lill, R. & Kispal, G. Maturation of cellular Fe-S proteins: an essential function of mitochondria. Trends Biochem. Sci. 25, 352­356 (2000). 17. Hollister, W. S. et al. Development and ultrastructure of Trachipleistophora hominis n.g., n.sp. after in vitro isolation from an AIDS patient and inoculation into athymic mice. Parasitology 112, 143­154 (1996). 18. Huynen, M. A., Snel, B., Bork, P. & Gibson, T. J. The phylogenetic distribution of frataxin indicates a role in iron-sulfur cluster protein assembly. Hum. Mol. Genet. 10, 2463­2468 (2001). 19. Akhmanova, A. et al. A hydrogenosome with a genome. Nature 396, 527­528 (1998). 20. van Der Giezen, M. et al. Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles. EMBO J. 21, 572­579 (2002). 21. Dyall, S. D. & Johnson, P. J. Origins of hydrogenosomes and mitochondria: evolution and organelle biogenesis. Curr. Opin. Microbiol. 3, 404­411 (2000). 22. Tovar, J., Fischer, A. & Clark, C. G. The mitosome, a novel organelle related to mitochondria in the amitochondrial parasite Entamoeba histolytica. Mol. Microbiol. 32, 1013­1021 (1999). 23. Mai, Z. et al. Hsp60 is targeted to a cryptic mitochondrion-derived organelle (`crypton') in the microaerophilic protozoan parasite Entamoeba histolytica. Mol. Cell. Biol. 19, 2198­2205 (1999). 24. Canning, E. U. in Handbook of Protoctista (eds Margulis, L., Corliss, J. O., Melkonian, M. & Chapman, D. J.) 53­72 (Jones and Bartlett, Boston, 1990). 25. Roger, A. J. et al. A mitochondrial-like chaperonin 60 gene in Giardia lamblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria. Proc. Natl Acad. Sci. USA 95, 229­234 (1998). 26. Cheney, S. A., Lafranchi-Tristem, N. J. & Canning, E. U. Serological differentiation of microsporidia with special reference to Trachipleistophora hominis. Parasite 8, 91­97 (2001). 27. Lockhart, P. J., Steel, M. A., Hendy, M. D. & Penny, D. Recovering evolutionary trees under a more realistic model of sequence evolution. Mol. Biol. Evol. 11, 605­612 (1994). 28. Wilbanks, S. in Guidebook to the Molecular Chaperones and Protein-Folding Catalysts (ed. Gething, M.-J. H.) 13­18 (Oxford Univ. Press, New York, 1997). 29. Buchberger, A. & Bukau, B. in Guidebook to the Molecular Chaperones and Protein-Folding Catalysts (ed. Gething, M.-J. H.) 22­25 (Oxford Univ. Press, New York, 1997). Acknowledgements We thank E. Canning for the T. hominis culture system and advice on growing microsporidia; M. Duchen and A. Ball for help with confocal microscopy; C. Thomson for help with electron microscopy; and C. Danpure and G. Birdsey for comments on the manuscript and help with heterologous transfection experiments. J.M.L. was supported by a Wellcome Trust Research Leave Fellowship and Tenovus Scotland. B.A.P.W. was supported by a Wellcome Trust Biodiversity studentship, R.P.H. was supported by a Wellcome Trust University award. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to T.M.E. (e-mail: tme@nhm.ac.uk). The sequence of T. hominis mtHsp70 has been deposited in GenBank under accession code AF492453. .............................................................. Molecular evolution of FOXP2, a gene involved in speech and language Wolfgang Enard*, Molly Przeworski*, Simon E. Fisher, Cecilia S. L. Lai, Victor Wiebe*, Takashi Kitano*, Anthony P. Monaco & Svante Pa¨a¨bo* * Max Planck Institute for Evolutionary Anthropology, Inselstrasse 22, D-04103 Leipzig, Germany Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK ............................................................................................................................................................................. Language is a uniquely human trait likely to have been a prerequisite for the development of human culture. The ability to develop articulate speech relies on capabilities, such as fine control of the larynx and mouth1 , that are absent in chimpanzees and other great apes. FOXP2 is the first gene relevant to the human ability to develop language2 . A point mutation in FOXP2 co-segregates with a disorder in a family in which half of the members have severe articulation difficulties accompanied by linguistic and grammatical impairment3 . This gene is disrupted by translocation in an unrelated individual who has a similar disorder. Thus, two functional copies of FOXP2 seem to be required for acquisition of normal spoken language. We sequenced the complementary DNAs that encode the FOXP2 protein in the chimpanzee, gorilla, orang-utan, rhesus macaque and mouse, and compared them with the human cDNA. We also investigated intraspecific variation of the human FOXP2 gene. Here we show that human FOXP2 contains changes in amino­ acid coding and a pattern of nucleotide polymorphism, which strongly suggest that this gene has been the target of selection during recent human evolution. FOXP2 (forkhead box P2) is located on human chromosome 7q31, and its major splice form encodes a protein of 715 amino acids belonging to the forkhead class of transcription factors2 . It contains a glutamine-rich region consisting of two adjacent polyglutamine tracts, encoded by mixtures of CAG and CAA repeats. Such repeats are known to have elevated mutation rates. In the case of FOXP2, the lengths of the polyglutamine stretches differed for all taxa studied. Variation in the second polyglutamine tract has been observed in a small family affected with speech and language impairment, but this did not co-segregate with disorder, suggesting that minor changes in length may not significantly alter the function of the protein4 . If the polyglutamine stretches are disregarded, the human FOXP2 protein differs at only three amino-acid positions from its orthologue in the mouse (Fig. 1). When compared with a collection of 1,880 human­rodent gene pairs5 , FOXP2 is among the 5% most-conserved proteins. The chimpanzee, gorilla and rhesus macaque FOXP2 proteins are all identical to each other and carry only one difference from the mouse and two differences from the human protein, whereas the orang-utan carries two differences from the mouse and three from humans (Fig. 1). Thus, although the FOXP2 protein is highly conserved, two of the three amino-acid differences between humans and mice occurred on the human lineage after the separation from the common ancestor with the chimpanzee. These two amino-acid differences are both found in exon 7 of the FOXP2 gene and are a threonine-to-asparagine and an asparagine-to-serine change at positions 303 and 325, respectively. Figure 2 shows the amino-acid changes, as well as the silent changes, mapped to a phylogeny of the relevant primates. We compared the FOXP2 protein structures predicted by a variety of methods6 for humans, chimpanzees, orang-utans and mice. Whereas the chimpanzee and mouse structures were essen­ tially identical and the orang-utan showed only a minor change in secondary structure, the human-specific change at position 325 creates a potential target site for phosphorylation by protein kinase C together with a minor change in predicted secondary structure. Several studies have shown that phosphorylation of forkhead transcription factors can be an important mechanism mediating transcriptional regulation7,8 . Thus, although the FOXP2 protein is extremely conserved among mammals, it acquired two amino-acid changes on the human lineage, at least one of which may have functional consequences. This is an intriguing finding, because Table 1 Variation at the FOXP2 locus in humans No. of chromosomes sequenced 40 Length covered (double stranded, all individuals) 14,063 bp Divergence from the chimp sequence* 0.87% No. of variable positions 47 Singletons (no. of variable sites occurring 31 at frequency 1 and 39) vW (nucleotide diversity based on the no. 0.079% of polymorphic sites) vp (mean nucleotide diversity) 0.03% vH (nucleotide diversity with more weight given 0.117% to alleles at high frequency17 ) D (P , 0.01) 22.20 H (P , 0.05) 212.24 ............................................................................................................................................................................. *The corresponding value for the orang-utan is 2.5. A negative D value indicates a relative excess of low-frequency alleles15 . A negative H value indicates a relative excess of high-frequency derived alleles17 . NATURE | VOL 418 | 22 AUGUST 2002 | www.nature.com/nature 2002 NaturePublishing Group 869 letters to nature FOXP2 is the first gene known to be involved in the development of speech and language. To investigate whether the amino acids encoded in exon 7 are polymorphic in humans, we sequenced this exon from 44 human chromosomes originating from all major continents. In no case was any amino-acid polymorphism found. Further, a study that ana­ lysed the complete coding region of FOXP2 in 91 unrelated individuals of mainly European descent found no amino-acid replacements except for one case of an insertion of two glutamine codons in the second polyglutamine stretch4 . Because the two amino-acid variants specific to humans occur in 226 human chromosomes, this suggests that they are fixed among humans. The evolutionary lineages leading to humans and mice diverged about 70 million years (Myr) ago9,10 . Thus, during the roughly 130 Myr of evolution that separate the common ancestor of humans and chimpanzees from the mouse, a single amino-acid change occurred in the FOXP2 protein. By contrast, since the human and chimpanzee lineages diverged about 4.6­6.2 Myr ago11 , two fixed amino-acid changes occurred on the human lineage whereas none occurred on the chimpanzee and the other primate lineages, except for one change on the orang-utan lineage. We used a likelihood ratio12 to test for constancy of the ratio of amino-acid replacements over nucleotide changes that do not cause amino-acid changes among the evolutionary lineages in Fig. 2. Whereas a significant increase in this ratio was observed on the human lineage (P , 0.001), no such increase was seen on any other lineage. This finding is consistent with the action of positive selection on amino­ acid changes in the human lineage. However, the alternative hypothesis of a relaxation of constraints on FOXP2 specific to the human lineage cannot be excluded on the basis of these data alone. If these two changes in amino-acid encoding (or some other feature of the human FOXP2 gene) were positively selected recently during human evolution, traces of a selective sweep should be detectable in the pattern of variation found among humans13,14 . To investigate this possibility, we sequenced a segment of 14,063 base pairs (bp) covering introns 4, 5 and 6 of the FOXP2 gene in seven individuals from Africa, four from Europe, one from South Amer­ ica, five from mainland Asia and three from Australia and Papua New Guinea. In addition, we sequenced the same segment in a chimpanzee from central Africa, a chimpanzee from western Africa and an orang-utan (Table 1). One hallmark of a recent selective sweep is that more low-frequency alleles should be observed than expected under a neutral model of a random-mating population of constant size. To test this prediction, we calculated Tajima's D statistic15 . The value is 22.20 for our sample, indicating a sharp excess of rare alleles. Under the standard neutral model outlined above, the probability of such an excess by chance is 0.002. Population growth can also lead to negative D values throughout the genome. However, the value of D at FOXP2 is unusually low compared with other loci. For example, among 313 human genes16 0K= 3-5)6-61 5553/ 56553,)/ 54,/455/,6 55-8568- 03333)3) )433336 5/5255, 34238285 8)623816 +DEF /HE= H=C 8 4DAIKI KIA - 0K= +DEF /HE= H=C 4DAIKI KIA 23333133 3852333) 3333)8 33333-.; 33-303 3302/3)- 333333333 3333333333 333333333 3333333333 3 3 0K= 3333333333 ))338.33 333333 3305343 /15122/3) )283523) /52)-133 9-86/805 -,/10// ,665556 +DEF /HE= H=C 4DAIKI KIA 0K= +DEF /HE= H=C 4DAIKI KIA 65565)52 6 6 6 6 6 21600518/ 35585)44, 5550--6/)5 06;/0/8+ 92/+-51+-, ./3.0 -0),,456) 3+4833883 3-135-4 0K= +DEF /HE= H=C 4DAIKI KIA -43)60 0425-225 228558 65-65 235236266 26)2862163 /258162)58 28/)14440 5,;1255 -1)2;-.; 0K= +DEF /HE= H=C 4DAIKI KIA ;.44))69 ),8422.6; )6143)1- 55,436- 1;59.646.) )84050 +.848-8 /)8968,-8- ;3445316 /526812 0K= +DEF /HE= H=C 4DAIKI KIA 0K= +DEF /HE= H=C 4DAIKI KIA 65/;/)) -,,4-1---2 )53)))-5 5-,- 5252/ 1)55/3 )80-,/5 ,01,5/55 2/+5232010 5108--281 )-,-,+25 866)052- Figure 1 Alignment of the amino-acid sequences inferred from the FOXP2 cDNA sequences. The polyglutamine stretches and the forkhead domain are shaded. Sites that differ from the human sequence are boxed. 2002 NaturePublishing Group NATURE | VOL 418 | 22 AUGUST 2002 | www.nature.com/nature870 20 letters to nature sequenced in a sample of 164 chromosomes, only one has a more negative value (22.25). A second prediction for a selective sweep at a recombining locus is that more derived (that is, non-ancestral) alleles at high frequency are expected than under the standard neutral model, a feature reflected in a negative H value17 . To estimate H, we inferred the ancestral states of variable positions seen among the humans by using the chimpanzee and orang-utan DNA sequences. The H value of 212.24 deviates significantly from the neutral expectation of zero (P 0.042) and would be even less likely by chance under a model with population growth13 . The strongly negative D and H reflect an extreme skew in the frequency spectrum of allelic variants at FOXP2 towards rare and high-frequency alleles. Because we considered a worldwide sample of humans, population structure might contribute to the negative D value. However, this type of sampling scheme is highly unlikely to produce a significantly negative H value. In contrast to demographic explanations, a selective sweep affecting the FOXP2 gene can account for both aspects of the frequency spectrum. We do not observe a reduced diversity at human FOXP2 relative to its divergence from the chimpanzee, as expected under a simple selective-sweep model. However, the magnitude of the reduction in variability expected after a selective sweep depends crucially on the rate of recombina­ tion. Estimates of recombination between intronic polymorphisms taken from a study of FOXP2 (ref. 4) suggest that this region of the gene experiences rates of genetic exchange roughly five times the genome-wide average. If we assume that a selective sweep at a linked site does account for the patterns of variability recovered at FOXP2, it is noteworthy that the next gene is located 286 kilobases (kb) away from the sequenced segment. A selective sweep is not expected to lead to an excess of high-frequency derived alleles at sites that are 286 kb distant from the target of selection13,17 . Thus, the best candidates for the selected sites are the two amino-acid substi­ tutions specific to humans in exon 7. Individuals with disruption of FOXP2 have multiple difficulties with both expressive and receptive aspects of language and gram­ mar, and the nature of the core deficit remains a matter of debate18­ . Nevertheless, a predominant feature of the phenotype of affected individuals is an impairment of selection and sequencing of fine orofacial movements18 , an ability that is typical of humans and not present in the great apes. We speculate that some human-specific feature of FOXP2, perhaps one or both of the amino-acid substi­ tutions in exon 7, affect a person's ability to control orofacial movements and thus to develop proficient spoken language. If this speculation is true, then the time when such a FOXP2 variant became fixed in the human population may be pertinent with regard to the evolution of human language. We estimated this time point using a likelihood approach. Under a model of a randomly mating population of constant size, the most likely date since the fixation of the beneficial allele is 0, with approximate 95% confidence intervals of 0 and 120,000 years. Our point-estimate of 0 2/0*** 0/7 1/2 0/5 0/5 0/2 0/20/2 Human Gorilla Orang-utan Rhesus Chimp 1/131 Mouse Figure 2 Silent and replacement nucleotide substitutions mapped on a phylogeny of primates. Bars represent nucleotide changes. Grey bars indicate amino-acid changes. reflects the fact that high-frequency alleles rapidly drift to fixation, so an excess is most likely immediately after a selective sweep. However, if population growth soon succeeds the fixation of the advantageous allele, the rate of drift will be decreased and high­ frequency alleles may persist longer in the population. Thus, the inclusion of population growth may push this time estimate back by at most the time since the onset of human population growth, some 10,000­100,000 years ago21 . In any case, our method suggests that the fixation occurred during the last 200,000 years of human history, that is, concomitant with or subsequent to the emergence of anatomically modern humans22 . This is compatible with a model in which the expansion of modern humans was driven by the appearance of a more-proficient spoken language22 . However, to establish whether FOXP2 is indeed involved in basic aspects of human culture, the normal functions of both the human and the chimpanzee FOXP2 proteins need to be clarified. A Methods Isolation of cDNA sequences For all analysed species, we amplified by polymerase chain reaction (PCR) and sequenced overlapping fragments of the FOXP2 coding region from first-strand cDNA. Details are available in Supplementary Information. Genomic sequencing Full details are available in Supplementary Information. In brief, we designed primers from a human bacterial artificial chromosome (BAC) sequence (accession number AC020606), PCR-amplified fragments of 6­14 kb, re-amplified 2.2-kb fragments from these products that were then sequenced with internal primers. For each individual, each nucleotide position was read from both strands. Sequence traces were manually analysed for polymorphic positions using the program Seqman of the DNAStar package (see also Supplementary Information). Data analysis We aligned sequences with the help of the program ClustalW23 and calculated most statistics with DnaSP 3.51 (ref. 24). P values for D and H were obtained by coalescent simulations implemented for a fixed number of segregating sites, and assuming no recombination. If we take into account recombination within the 14 kb, the P values decrease (for example, P , 0.01 for H and P , 1024 for D if one assumes an effective population size of 104 and a recombination rate of 5 centimorgans (cM) per Mb). Because the chimpanzee and orang-utan do not differ at any polymorphic position compared with humans, we assumed no back mutations when estimating the P value for H. The likelihood ratio tests for non-silent and silent substitutions were performed using the PAML package12 as described25 (see Supplementary Information). We predicted the structure of human, chimpanzee, mouse and orang-utan FOXP2 using the program PredictProtein (http://www.embl-heidelberg.de/predictprotein/predictprotein.html)6 , which includes prediction of sites of protein kinase C phosphorylation by PROSITE26 . The orang-utanspecific alanine-to-valine change at position 6 results in the prediction of a b-sheet at positions 8­10 in the orang-utan, and the human-specific change at position 325 results in the prediction of a b-sheet in positions 323­326. However, these are not reliable and may not be relevant. We used the University of California at Santa Cruz Human Genome Project Working Draft, 22 December 2001 assembly (http://genome.cse.ucsc.edu), to estimate distances to the closest genes. The middle of the sequenced region is 220 kb away from the known 59 end and 54 kb away from the 39 end of FOXP2, respectively. The next gene (supported by the cDNA sequence with GenBank accession number AF054589) is located 286 kb distant in the 39 direction. Modelling the selective sweep A summary likelihood method (compare with ref. 27) was used to estimate the time, T, since the fixation of the beneficial allele in the population. The polymorphism data was summarized as vH (ref. 17) and p (ref. 28). We then ran coalescent simulations of a selective sweep with recombination as in ref. 13. These simulations assume that we have polymorphism data for a neutral locus, at some distance from a selected site, and that selection acted on a newly arising variant. The likelihood of T is estimated as the proportion of n simulated data sets, where vHobs 2 vHsim , 1 and pobs 2 psim , 1j j j j (here, n 3 106 and 1 0.2). The likelihood of T was evaluated over a grid of points spaced every 1,000 generations. We then chose the T value that maximizes the probability of obtaining the observed (vH, p) values. In addition to T, several additional parameters are in this selective sweep model: the distance to the selected site, the effective population size of humans, the strength of selection, the mutation rate and the recombination rate. It is not computationally feasible to co-estimate all of these parameters, and we proceeded by assuming that the values of most nuisance parameters are known exactly. We hypothesized that one of the substitutions on the human lineage was the selected site and used a point estimate of the population mutation rate (assuming 5 Myr to the common ancestor of a human and chimpanzee DNA sequence). We modelled uncertainty in the recombination rate per megabase by choosing the rate for each simulation from a g distribution with parameters (5, 1); the mean was set to the recombination rate estimated from two polymorphic markers in introns 2 and 16, respectively, of the FOXP2 gene4 . The effective NATURE | VOL 418 | 22 AUGUST 2002 | www.nature.com/nature 2002 NaturePublishing Group 871 letters to nature population size was taken to be 104 , on the basis of estimates for other loci29 . We tried three different values for the selection coefficient: s 5%, 1% and 0.5%. For these parameters, an s of 1% resulted in the highest likelihoods, so we reported the results for s 1%. If we use the chi-squared approximation with one degree of freedom for the log-likelihood ratio statistic 2ln Lik T^ =Lik T, we obtain an approximate 95% confidence interval for T of [0, 4,000 generations]. However, this approximation may not be appropriate in this context. Thus, we also ran 100 simulations to examine the distribution of T^ when the true T is equal to our maximum likelihood estimate of T 0 (here, n 5 105 and 1 0.2). These simulations suggested an approximate 95% confidence interval of [0, 6,000 generations]. We assumed a generation time of 20 years for converting T into years. Received 11 November 2001; accepted 29 July 2002; doi:10.1038/nature01025. Published online 14 August 2002. 1. Liebermann, P. The Biology and Evolution of Language (Harvard Univ. Press, Cambridge, Massachusetts, 1984). 2. Lai, C. S. L., Fisher, S. E., Hurst, J. A., Vargha-Khadem, F. & Monaco, A. P. A forkhead-domain gene is mutated in a severe speech and language disorder. Nature 413, 519­523 (2001). 3. Fisher, S. E., Vargha-Khadem, F., Watkins, K. E., Monaco, A. P. & Pembrey, M. E. Localisation of a gene implicated in a severe speech and language disorder. Nature Genet. 18, 168­170 (1998). 4. Newbury, D. F. et al. Foxp2 is not a major susceptibility gene for autism or specific language impairment. Am. J. Hum. Genet. 70, 1318­1327 (2002). 5. Makalowski, W. & Boguski, M. S. Evolutionary parameters of the transcribed mammalian genome: An analysis of 2,820 orthologous rodent and human sequences. Proc. Natl Acad. Sci. USA 95, 9407­9412 (1998). 6. Rost, B. Phd: Predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol. 266, 525­539 (1996). 7. Kops, G. J. et al. Control of cell cycle exit and entry by protein kinase b-regulated forkhead transcription factors. Mol. Cell. Biol. 22, 2025­2036 (2002). 8. Brunet, A. et al. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 96, 857­868 (1999). 9. Kumar, S. & Hedges, S. B. A molecular timescale for vertebrate evolution. Nature 392, 917­920 (1998). 10. Eizirik, E., Murphy, W. J. & O'Brien, S. J. Molecular dating and biogeography of the early placental mammal radiation. J. Hered. 92, 212­219 (2001). 11. Chen, F. C. & Li, W. H. Genomic divergences between humans and other hominoids and the effective population size of the common ancestorof humans and chimpanzees. Am. J. Hum. Genet. 68, 444­456 (2001). 12. Yang, Z. Paml: A program package for phylogenetic analysis by maximum likelihood. Comput. Appl. Biosci. 13, 555­556 (1997). 13. Przeworski, M. The signature of positive selection at randomly chosen loci. Genetics 160, 1179­1189 (2002). 14. Simonsen, K. L., Churchill, G. A. & Aquadro, C. F. Properties of statistical tests of neutrality for DNA polymorphism data. Genetics 141, 413­429 (1995). 15. Tajima, F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123, 585­595 (1989). 16. Stephens, J. C. et al. Haplotype variation and linkage disequilibrium in 313 human genes. Science 293, 489­493 (2001). 17. Fay, J. C. & Wu, C. I. Hitchhiking under positive darwinian selection. Genetics 155, 1405­1413 (2000). 18. Vargha-Khadem, F., Watkins, K., Alcock, K., Fletcher, P. & Passingham, R. Praxic and nonverbal cognitive deficits in a large family with a genetically transmitted speech and language disorder. Proc. Natl Acad. Sci. USA 92, 930­933 (1995). 19. Gopnik, M. & Crago, M. B. Familial aggregation of a developmental language disorder. Cognition 39, 1­50 (1991). 20. Watkins, K. E., Dronkers, N. F. & Vargha-Khadem, F. Behavioural analysis of an inherited speech and language disorder: Comparison with acquired aphasia. Brain 125, 452­464 (2002). 21. Wall, J. D. & Przeworski, M. When did the human population size start increasing? Genetics 155, 1865­1874 (2000). 22. Klein, G. The Human Career, Human Biological and Cultural Origins (Univ. Chicago Press, Chicago, 1989). 23. Thompson, J. D., Higgins, D. G. & Gibson, T. J. ClustalW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673­4680 (1994). 24. Rozas, J. & Rozas, R. Dnasp version 3: An integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15, 174­175 (1999). 25. Yang, Z. Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution. Mol. Biol. Evol. 15, 568­573 (1998). 26. Bairoch, A., Bucher, P. & Hofmann, K. The prosite database, its status in 1997. Nucleic Acids Res. 25, 217­221 (1997). 27. Jensen, M. A., Charlesworth, B. & Kreitman, M. Patterns of genetic variation at a chromosome 4 locus of Drosophila melanogaster and D. simulans. Genetics 160, 493­507 (2002). 28. Tajima, F. Evolutionary relationship of DNA sequences in finite populations. Genetics 105, 437­460 (1983). 29. Harpending, H. & Rogers, A. Genetic perspectives on human origins and differentiation. Annu. Rev. Genom. Hum. Genet. 1, 361­385 (2000). 30. Watterson, G. A. On the number of segregating sites in genetical models without recombination. Theor. Popul. Biol. 7, 256­276 (1975). Supplementary Information accompanies the paper on Nature's website (http://www.nature.com/nature). Acknowledgements We thank F. Heissig for help with the cDNA sequencing; A. von Haeseler, G. Weiss and S. Zo¨llner for help with the data analysis on an earlier version of the manuscript; J. Wickings at the Centre International de Recherches Medicales for DNA samples of central chimpanzees; and the Bundesminsterium fu¨r Bildung und Forschung, the Max Planck Society and the Wellcome Trust for financial support. M.P. was supported by a National Science Foundation postdoctoral research fellowship in bioinformatics. S.E.F. is a Royal Society Research Fellow and A.P.M. is a Wellcome Trust Principal Research Fellow. Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to S.P. (e-mail: paabo@eva.mpg.de). FOXP2 cDNA sequences of the mouse, rhesus macaque, orang-utan, gorilla, chimpanzee and human have GenBank accession numbers AY079003, AF512950, AF512949, AF512948, AF512947 and AF337817, respectively. Accession numbers for genomic sequences for the twenty humans, two chimpanzees and one orang-utan are AF515031­AF515050, AF515051­AF515052 and AF515053, respectively. .............................................................. A corollary discharge maintains auditory sensitivity during sound production James F. A. Poulet & Berthold Hedwig Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK ............................................................................................................................................................................. Speaking and singing present the auditory system of the caller with two fundamental problems: discriminating between self­ generated and external auditory signals and preventing desensi­ tization. In humans1 and many other vertebrates2­7 , auditory neurons in the brain are inhibited during vocalization but little is known about the nature of the inhibition. Here we show, using intracellular recordings of auditory neurons in the singing cricket, that presynaptic inhibition of auditory afferents and postsynaptic inhibition of an identified auditory interneuron occur in phase with the song pattern. Presynaptic and postsyn­ aptic inhibition persist in a fictively singing, isolated cricket central nervous system and are therefore the result of a corollary discharge from the singing motor network. Mimicking inhibition in the interneuron by injecting hyperpolarizing current sup­ presses its spiking response to a 100-dB sound pressure level (SPL) acoustic stimulus and maintains its response to sub­ sequent, quieter stimuli. Inhibition by the corollary discharge reduces the neural response to self-generated sound and protects the crickeťs auditory pathway from self-induced desensitization. We have examined auditory information processing in the sing­ ing cricket, Gryllus bimaculatus. Males attract females or warn off rival males with songs that are generated by rubbing their forewings together. Calling song can be produced for many hours on end with a sound intensity greater than 100 dB SPL measured 50 mm from the ear8 . It consists of a 250-ms series of chirps that is separated by a 300-ms chirp interval (Fig. 1a). A chirp itself comprises four syllables of 21-ms duration, which are generated by the closing movements of the forewings. Crickets' ears are located on their forelegs and are therefore exposed fully to the self-generated sounds. About 60 auditory afferent neurons project from the ear along the fifth prothoracic nerve and terminate in the auditory neuropile of the prothoracic ganglion9 . Two local, mutually inhibitory omega 1 neurons (ON1s) are located in the prothoracic ganglion10 . This identified pair of bilaterally symmetrical interneurons receives auditory information from the ear ipsilateral to their soma. These interneurons are most sensitive to the carrier frequency (4.5 kHz) of the male calling song. Crickets must maintain auditory sensitivity during bouts of singing because they respond behaviourally to auditory stimulation 2002 NaturePublishing Group NATURE | VOL 418 | 22 AUGUST 2002 | www.nature.com/nature872