ARTICLES. A role for a neo-sex chromosome in stickleback speciation

Transcription

1 Vol Octoer 9 doi:1.138/nture8441 A role for neo-sex chromosome in stickleck specition Jun Kitno 1 {, Joseph A. Ross 1,2 {, Seiichi Mori 3, Mnu Kume 4, Felicity C. Jones 5, Yinggung F. Chn 5, Devin M. Asher 6 {, Jne Grimwood 6 {, Jeremy Schmutz 6 {, Richrd M. Myers 6 {, Dvid M. Kingsley 5 & Ctherine L. Peichel 1 Sexul ntgonism, or conflict etween the sexes, hs een proposed s driving force in oth sex-chromosome turnover nd specition. Although closely relted species often hve different sex-chromosome systems, it is unknown whether sex-chromosome turnover contriutes to the evolution of reproductive isoltion etween species. Here we show tht newly evolved sex chromosome contins genes tht contriute to specition in threespine stickleck fish (Gsterosteus culetus). We first identified neo-sex chromosome system found only in one memer of symptric species pir in Jpn. We then performed genetic linkge mpping of mle-specific trits importnt for reproductive isoltion etween the Jpnese species pir. The neo-x chromosome contins loci for mle courtship disply trits tht contriute to ehviourl isoltion, wheres the ncestrl X chromosome contins loci for oth ehviourl isoltion nd hyrid mle sterility. Our work not only provides strong evidence for lrge X-effect on reproductive isoltion in verterte system, ut lso provides direct evidence tht young neo-x chromosome contriutes to reproductive isoltion etween closely relted species. Our dt indicte tht sex-chromosome turnover might hve greter role in specition thn ws previously pprecited. Sexully ntgonistic selection hs een proposed s mjor driving force in the evolution of sex chromosomes. Specificlly, nturl selection is expected to fvour linkge etween genes with sexully ntgonistic effects (tht is, eneficil in one sex nd detrimentl in the other) nd the sex-determintion locus, resulting in reduction of recomintion etween sex chromosomes 1. In n XY sex-chromosome system, reduction in recomintion ultimtely leds to the degenertion of the Y chromosome, therey exposing lleles on the hemizygous X chromosome to selection in mles 2. Thus, mle-eneficil lleles, mnifested s sexully dimorphic nd/or sexully selected trits, re predicted to ccumulte on the X chromosome 3,4. When these mle-eneficil trits or lleles re importnt for reproductive isoltion etween species, the X chromosome is lso predicted to hve key role in specition. A disproportiontely lrge effect of the X chromosome hs een demonstrted for hyrid mle sterility 5,6, lthough the dt supporting lrge X-effect for other isolting rriers hs een less consistent 6. Sexully ntgonistic selection is lso predicted to drive the divergence of sex-chromosome systems etween closely relted species 7,8. Sex-chromosome turnover hs een oserved cross mny tx, ut it is prticulrly striking in fishes 9,1. Mny independent groups of fishes show evidence for the rpid evolution of sex chromosomes through severl different mechnisms, including the trnsposition of n existing mle-determintion locus to n utosome 11, the evolution of new mle-determintion locus on n utosome 12, nd fusions etween n utosome nd n existing Y chromosome 13,14. It hs een suggested tht the rpid turnover of sex chromosomes driven y sexul conflict might lso hve role in the high specition rtes seen in some groups of fishes However, direct role for sex-chromosome turnover in specition hs not een empiriclly investigted in ny system. Here we demonstrte tht there is newly formed sex-chromosome system in threespine stickleck popultion found in Jpn. This Jpn Se form diverged from the Pcific Ocen threespine stickleck during periods of geogrphicl isoltion etween the Se of Jpn nd the Pcific Ocen out million yers go,19. Becuse we cn cross the derived Jpn Se form to the ncestrl Pcific Ocen form, we hve een le to tke dvntge of the genetic tools ville for the threespine stickleck to investigte whether the evolution of neo-sex chromosome hs contriuted to the evolution of mle trits tht ct s isolting rriers etween the derived Jpn Se nd the ncestrl Pcific Ocen popultions. Jpn Se stickleck neo-sex chromosome In ll threespine stickleck popultions exmined previously, including the Pcific Ocen form, linkge group 19 (LG19) is the sex chromosome nd LG9 is n utosome 14,21,22. However, in the course of mking linkge mp from Jpn Se cross, we noticed tht severl LG9 mrkers co-segregted with LG19 mrkers previously found to e tightly linked to the sex-determintion locus in region of reduced recomintion nd rerrngements on the Y chromosome 21,22. This ssocition ws oserved when mle meioses, ut not femle meioses, were nlysed (Fig. 1). These dt indicted tht one copy of LG9 might e fused to one copy of LG19 (the Pcific Ocen Y chromosome), forming neo-y chromosome in Jpn Se sticklecks. Therefore, we performed fluorescence in situ hyridiztion (FISH) with LG19 nd LG9 proes on metphse chromosome spreds from two different popultions of the Jpn Se form. We found tht the Jpn Se mles (n 5 4) from two different popultions hve n odd numer of chromosomes (2n 5 41), with one lrge 1 Division of Humn Biology, Fred Hutchinson Cncer Reserch Center, 11 Firview Avenue North, Settle, Wshington 9819, USA. 2 Grdute Progrm in Moleculr nd Cellulr Biology, University of Wshington, Settle, Wshington 98195, USA. 3 Biologicl Lortory, Gifu-keizi University, Ogki, Gifu , Jpn. 4 Aqu Restortion Reserch Center, Pulic Works Reserch Institute, Kkmighr, Gifu , Jpn. 5 Deprtment of Developmentl Biology nd Howrd Hughes Medicl Institute, Stnford University, Stnford, Cliforni 9435, USA. 6 Deprtment of Genetics nd Stnford Humn Genome Center, Stnford University, Stnford, Cliforni 9434, USA. {Present ddresses: Deprtment of Ecology nd Evolutionry Biology, Grdute School of Life Sciences, Tohoku University, Sendi, Miygi , Jpn (J.K.); Deprtment of Biology, University of Mrylnd, College Prk, Mrylnd 742, USA (J.A.R.); HudsonAlph Institute for Biotechnology, Huntsville, Alm 3586, USA (D.M.A., J.G., J.S. nd R.M.M.) 9 Mcmilln Pulishers Limited. All rights reserved 179

2 NATURE Vol Octoer 9 unpired chromosome tht hyridized to the LG19 nd LG9 proes, providing evidence for the LG9 Y chromosome fusion (Fig. 1 nd Supplementry Fig. 1). In contrst, the Jpn Se femles (n 5 4) from oth popultions hve n even numer of chromosomes (2n 5 42), nd the LG9 nd LG19 proes hyridize to seprte chromosome pirs (Fig. 1 nd Supplementry Fig. 1). Becuse the fused copy of LG9 segregtes with the Y chromosome in Jpn Se mles, the other copy segregtes s n X chromosome; this neo-sex chromosome system is defined s n X 1 X 2 Y system 13, in which X 1 is the ncestrl X chromosome (LG19) nd X 2 is the neo-x chromosome (LG9). Thus, the Jpn Se form hs unique neo-sex chromosome system, which hs proly evolved within the pst million yers of isoltion etween the Pcific Ocen nd Jpn Se sticklecks,19. Components of reproductive isoltion We hve the opportunity to test the role of this neo-sex chromosome in reproductive isoltion etween the Jpn Se nd the Pcific Ocen forms ecuse oth re ndromous nd migrte into Lke Akkeshi nd the Bekneushi mrsh during the reeding seson. In this loction, they co-occur, long with hyrid dults nd juveniles, within hyrid zone in the Bekneushi River (Mid2 in Fig. 2, nd Supplementry Fig. 2). We found tht temporl isoltion, ehviourl isoltion nd hyrid mle sterility contriute to reproductive isoltion etween the forms in the hyrid zone (Supplementry Fig. 3 nd Supplementry Discussion). We previously demonstrted tht ehviourl isoltion nd hyrid mle sterility re symmetric, ut ct in complementry directions 19. Pcific Ocen femles mte exclusively with Pcific Ocen mles, wheres the Jpn Se femles mte with oth types of mles in lortory mte-choice trils 19. Although Jpn Se femles do mte with Pcific Ocen mles, hyrid mles resulting from this cross hve severely reduced fertility, wheres reciprocl hyrid mles nd ll hyrid femles re fertile 19. To test whether reproductive isoltion is linked to sex-chromosome divergence, we first investigted which mle mting trits contriute to symmetric ehviourl isoltion. First, we nlysed the reltionship etween finl femle choice nd differences in mle ody size nd found tht oth types of femles tend to choose lrger mles (Fig. 3; Pcific Ocen femles, n 5 3, coefficient estimte 5.254, Z , P 5.228, logistic regression; Jpn Se femles, n 5 29, coefficient estimte 5.122, Z , P 5.8, logistic regression). Becuse Jpn Se mles (stndrd length mm (men 6 s.e.m.), n 5 59) re smller thn Pcific Ocen mles (stndrd length mm, n 5 45; nlysis of vrince (ANOVA), F 1, , P, ), divergence in ody size is one of the fctors tht contriutes to symmetric ehviourl isoltion. However, even in the sence of ody size divergence, Pcific Ocen femles still hve 93.5% proility (95% confidence intervl ) of choosing conspecific mle (Fig. 3), suggesting tht other fctors re involved in Pcific Ocen femle choice. We found tht difference in mle dorsl ehviour lso contriutes to symmetric ehviourl isoltion. Dorsl is component of mle mting ehviour specific to threespine sticklecks in which the mle rises his dorsl spines nd pricks the femle. This ehviour might help the mle ssess the femle, provide tctile stimultion to induce femle spwning, or ct s wy for the mle to disply his dorsl spines to the femle 23,24. In Jpn Se mles, the Femle meiosis LG19 (X 1 ) LG19 FISH proe Stn33 Stn6 Stn7 Stn274 Stn284 Stn256 Stn Mp LG9 (X 2 ) Stn15 Stn17 X 1 X 1 X 2 X 2 X 1 X 2 LG19 FISH proe Mle meiosis LG9-LG19 Stn6 Stn8 Stn225 Stn113 Stn114 Stn115 neo-y Jpn Se femle (2n = 42) Jpn Se mle (2n = 41) LG9 FISH proe Stn256 Stn284 Cyp Mp Sex Stn12 (mle) Stn15 Stn17 Stn 1º E N 4º N 3º N N Akkeshi By 13º E Se of Jpn 14º E Pcific Ocen Jpnese rchipelgo Mid2 Upstrem Mid1 Bekneushi River Lke Lke Akkeshi Akkeshi 6 dults 6 juveniles 7 dults 7 juveniles Pro of ssignment Jpn Se form Pro of ssignment Pro of ssignment Lke () Pro of ssignment Lke (84) Mid1 (1) Pcific Ocen form Mid2 (122) Mid2 (7) (46) (37) (22) (138) Upstrem (13) Upstrem (77) (11) (24) LG9 FISH proe Stn113 Stn225 Stn116 1 cm Figure 1 Genetic nd cytogenetic evidence of fusion etween one copy of LG9 nd the Y chromosome in Jpn Se mles., A cross etween Jpn Se individuls ws used to crete linkge mp. The femle meiotic mps (X X recomintion) of LG19 (X 1 ) nd LG9 (X 2 ) re to the left, nd the mle meiotic mp (X Y recomintion) of LG9/19 (neo-y) is to the right. LG19 (X 1 ) is in green nd LG9 (X 2 ) is in mgent. Genetic distnces etween the mrkers re drwn to scle. Scle r, 1 centimorgns (cm). Severl LG9 nd LG19 mrkers do not recomine with ech other or with the sexdetermintion locus (Sex) in mles. The coloured rs to the left of the mp indicte the reltive position of the FISH proes., The X 1 nd X 2 chromosomes from representtive Akkeshi Jpn Se femle metphse spred nd the X 1,X 2 nd neo-y chromosomes from representtive Akkeshi Jpn Se mle metphse spred re shown. A LG9 BAC (mgent) nd LG19 BAC (green) were used s proes for FISH. 9 Mcmilln Pulishers Limited. All rights reserved Figure 2 Distriution of the Jpnese threespine stickleck species pir in region of symptry., The top pnel shows mp of the Jpnese rchipelgo, indicting the loction of Akkeshi in estern Hokkido, Jpn. The ottom pnel shows mp of our four collection sites (Lke, Mid1, Mid2 nd Upstrem) in Lke Akkeshi nd the Bekneushi mrsh. Scle r, 2 km., The top pnels show representtive photos of Jpn Se mle nd Pcific Ocen mle. Scle r, 1 mm. Genetic nlysis of fish collected in 6 (n 5 61) nd 7 (n 5 368) ws performed using STRUCTURE. There re two genetic clusters in Akkeshi (Supplementry Fig. 2), with the Jpn Se cluster shown in red, nd the Pcific Ocen cluster shown in green. In the STRUCTURE plots, ech individul is indicted y single verticl line. The proility of ssignment to the Jpn Se cluster or the Pcific Ocen cluster is indicted y the extent of the coloured r. Fish collected from the sme loction re grouped together, with loctions seprted y thick lck lines. The numer of individuls per loction is indicted in prenthesis. The Jpn Se form is mostly found in the lke, wheres the Pcific Ocen form is mostly found in the upstrem region of the Bekneushi River. The midstrem region (Mid2) is hyrid zone tht contins oth prentl forms nd hyrids.

3 NATURE Vol Octoer 9 dorsl disply is gretly exggerted, nd the mle pushes the femle upwrds during dorsl (Fig. 3) 19. Furthermore, dorsl spine length is sexully dimorphic (mles hve longer spines thn femles) in the Jpn Se form 25. In contrst, in the Pcific Ocen form, the dorsl disply is wek (Fig. 3), nd dorsl spine length is not sexully dimorphic 19,25. We found tht the Pcific Ocen femles frequently escped nd did not resume mting fter they encountered the ggressive dorsl of Jpn Se mles, wheres the Jpn Se femles did not escpe from mles fter dorsl (Fig. 3). Genetic mpping of isolting rriers To investigte the chromosoml loctions of the isolting rriers etween the two forms, we ckcrossed F 1 hyrid (Jpn Se femle 3 Pcific Ocen mle) femles to Pcific Ocen mles nd conducted quntittive trit locus (QTL) mpping of mle dorsl, mle dorsl spine length, mle ody size, nd hyrid mle sterility. Individuls were genotyped with 9 single nucleotide polymorphism (SNP) mrkers nd 13 other microstellite mrkers tht together spn the stickleck genome. Hyrid mle sterility nd mle ody size mpped to LG19 (Fig. 4, Supplementry Fig. 4 nd Supplementry Tle 1), which is the ncestrl X chromosome shred y the Jpn Se form nd Pcific Ocen form. Dorsl nd first dorsl spine length mpped to distinct loctions on LG9 (Fig. 4, Supplementry Fig. 4 nd Supplementry Tle 1), which is the neo-x chromosome in the Jpn Se form. A genome-wide scn for episttic interctions identified significnt conspecific interction etween loci on LG19 for hyrid mle sterility (log likelihood rtio of linkge (LOD) compring the full model with interction to the dditive model 5 5.8; genome-wide significnce threshold ( 5.5); Supplementry Fig. 5). No significnt episttic interctions were found for ny other trits or loci exmined. Our dt demonstrte tht loci importnt for oth prezygotic nd postzygotic isoltion mp to the X chromosomes in this nturl verterte system. This lrge X-effect is unlikely to result from n overrepresenttion of these chromosomes in the stickleck genome, s LG9 nd LG19 comprise just 9.% of the stickleck genome (.2 megses (M) ech of M totl 4.5% for ech linkge group in the stickleck genome ssemly; BROAD S1, Fe 6). Becuse mpping in ckcross proly overestimtes the effects of the hemizygous X chromosome 26, we lso performed QTL mpping of the sme trits in n independent F 2 intercross to ensure tht the oserved lrge X-effect ws not simply the result of our ckcross mpping strtegy. We still detect QTL for dorsl nd first dorsl spine length on LG9 nd QTL for mle sterility nd ody size on LG19, with the ddition of single QTL for testis size on LG1 (Supplementry Fig. 6 nd Supplementry Tle 2). Proility of choosing conspecific mle Proility of choosing conspecific mle Pcific Ocen femle choice 1 1 Mle ody size difference (mm) (Pcific Ocen mle Jpn Se mle) Jpn Se femle choice Mle swim-up distnce during dorsl (mm) Percentge of femle escpe 1 1 Mle ody size difference (mm) (Jpn Se mle Pcific Ocen mle) PO mle PO mle 8 JS mle Pcific Ocen femle 8 JS mle Pcific Ocen femle Grvid femle Dorsl disply (swim-up) Courting mle 16 PO mle 16 PO mle JS mle Jpn Se femle JS mle Jpn Se femle Figure 3 Behviourl isoltion results from divergence in mle ody size nd mle dorsl ehviour., The top pnel indictes the preferences of Pcific Ocen femles (n 5 3), nd the ottom pnel indictes the preferences of Jpn Se femles (n 5 29) s function of mle ody size divergence. The horizontl xes indicte the ody size difference in mm etween mles (conspecific mle stndrd length minus heterospecific mle stndrd length). Ech symol indictes mte choice tril, in which 1 indictes the femle chose conspecific mle, nd indictes the femle chose heterospecific mle. Trils were conducted with lortory-rered Pcific Ocen mles (solid circles), resident freshwter Pcific mles (solid tringles), or without size mnipultion (open circles). The logistic regression curves indicte the proility of femle choosing conspecific mle for given difference in mle ody size., The top pnel shows the distnce Pcific Ocen (PO) or Jpn Se (JS) mle moved upwrds during dorsl with Pcific Ocen or Jpn Se femles. The ottom pnel shows the percentge of trils in which femle escped fter dorsl y either Pcific Ocen or Jpn Se mle. The smple size for ech mting pir is shown ove ech column. Lower cse letters ove the rs represent pirs tht re significntly different from ech other (pirwise Mnn Whitney U-test, P,.5 fter Bonferroni correction). FISH proe FISH proe 9 Mcmilln Pulishers Limited. All rights reserved LG19 (X 1 ) Sperm numer Body length c ss ss Stn7 LG19 LG19 37 Stn274.5 Stn235* 31 Idh P X P Y J X1 P Y Stn256 ss ss Stn192 Cyp19* Mp 8 8 P X P Y J X1 P Y LG9 (X 2 ) Dorsl Dorsl spine length 15 ss ss Stn1 LG9 LG Stn99 P 9 P 9 J X2 P 9 ss ss Stn* 1.5 ss Stn Stn11.5 ss Stn113 ss * P Stn P 9 J X2 P 9 1 cm Dorsl Body length Sperm numer Adjusted dorsl spine length Figure 4 Genetic mpping of isolting rriers., Genetic linkge mps of LG19 (X 1 ; green) nd LG9 (X 2 ; mgent) in the ckcross. The loctions of FISH proes re indicted to the left of the mp, wheres the nmes of genetic mrkers re indicted to the right. The sterisk indictes the mrker closest to the QTL pek., For ech QTL, the LOD score is indicted cross the top nd is plotted reltive to the positions of the mrkers indicted in, with distnce in cm indicted. The hyrid mle sterility QTL is represented y sperm numer, the ody size QTL is represented y ody length, nd the dorsl QTL is represented y men dorsl. The dorsl spine length QTL is represented y first dorsl spine length, which ws nlysed with ody length s n intercting covrite. Dshed lines indicte the genome-wide significnce thresholds determined y permuttion tests ( 5.5). c, For ech trit, the phenotypic vlues (men 6 s.e.m.) re indicted for genotypes t the mrker closest to the QTL pek; tht is, Cyp19 for sperm numer, Stn235 for ody length, SNP ss for men dorsl, nd Stn for dorsl spine length. Smple sizes for ech genotypic clss re shown in the grph. These were the only genomic regions with significnt phenotype-genotype ssocitions fter Bonferroni correction (P,.1), detected either y Kruskl Wllis test (sperm numer, ody length, nd dorsl ) or y nlysis of covrince (ANCOVA) (dorsl spine length). 1

4 NATURE Vol Octoer 9 Discussion Mpping of hyrid mle sterility to the ncestrl X chromosome (LG19) is consistent with previous studies on hyrid mle sterility in other systems 5,6, suggesting tht the lrge X-effect on hyrid mle sterility is common cross diverse tx. Although the resons for the lrge X-effect on hyrid sterility re deted 5, in some cses it results from genetic conflict in the form of sex rtio meiotic drive 27,28. However, we find no evidence for sex rtio distortion in the wekly fertile F 1 hyrid mles resulting from cross etween Jpn Se femles nd Pcific Ocen mles (Supplementry Tle 3). We do, however, find evidence for conspecific epistsis etween X-linked loci, which is commonly oserved for hyrid mle sterility in Drosophil 29,3. Despite this finding of common lrge X-effect cross widely seprted tx, we found no evidence for ny effect of the neo-x chromosome on hyrid mle sterility. It my e tht the reltive ge nd/or levels of degenertion of sex chromosomes re importnt fctors in determining whether the X chromosome contriutes to the evolution of hyrid mle sterility. Unlike mle sterility, mle courtship disply trits conferring ehviourl isoltion etween the Jpn Se nd Pcific Ocen forms mp to oth the ncestrl X chromosome nd the neo-x chromosome. Interestingly, mle ody size is sexully dimorphic in oth the Jpn Se nd Pcific Ocen forms 25 nd mps to the ncestrl X chromosome. In contrst, first dorsl spine length is only sexully dimorphic in the Jpn Se form 25, nd the dorsl disply is exggerted in the Jpn Se mles; these trits oth mp to the neo-x chromosome. Thus, these disply trits might hve evolved s result of differentil fitness effects etween mles nd femles specificlly in the Jpn Se linege. Although our cross design did not llow us to directly test whether these trits mpped to the neo-y chromosome s predicted y theory 7,8, it is possile tht selection for linkge etween mle eneficil trits, such s dorsl nd dorsl spine length, nd the sex-determintion locus ctully promoted the spred of the fusion etween LG9 nd the Y chromosome in the Jpn Se popultion 7. Alterntively, these mle eneficil trits my hve ccumulted on the neo-x chromosome 3 fter the formtion of the neo-y chromosome in the lst million yers. In either cse, our dt provide direct empiricl evidence linking sex chromosome turnover nd reproductive isoltion etween closely relted species. The turnover of sex chromosomes etween species is common in mny groups of nimls, where closely relted species often differ in sex-determintion nd sex-chromosome systems 9,1. Although it hs een suggested tht sex-chromosome turnover might drive rpid specition in cichlid fishes in which femle colour trit is linked to n invding ZW sex-chromosome system 15 17, this hypothesis hs not een directly tested. Given the potentil role of sexul ntgonism in driving oth sex-chromosome evolution 1,3,7,8 nd specition 31,32, we suggest tht sex-chromosome divergence etween closely relted species should e given further considertion s n importnt mechnism contriuting to the evolution of reproductive isoltion. METHODS SUMMARY Threespine sticklecks of the Pcific Ocen nd the Jpn Se form were collected with seine nets nd minnow trps in Lke Akkeshi nd the Beukneushi River in My July of 3 8. Jpn Se fish were lso collected from n lloptric site (Cpe of Benkei) on the west cost of Hokkido in 8. For cytogenetic nd ehviourl studies, live fish were trnsported to the Fred Hutchinson Cncer Reserch Center; ll experimentl procedures were pproved y the Institutionl Animl Cre nd Use Committee (IACUC numer 1575). Cytogenetics nd FISH were performed on Jpn Se mles (n 5 2) nd femle (n 5 1) from Akkeshi, s well s Jpn Se mles (n 5 2) nd femles (n 5 3) from the lloptric site, using fluorescently lelled cteril rtificil chromosome (BAC) clones s previously descried 22. For popultion genetic nlysis, 969 fish were genotyped with 12 neutrl microstellite mrkers (Supplementry Tle 4). Mte-choice experiments were conducted s previously descried 19. For QTL mpping, Jpn Se femle nd Pcific Ocen 2 mle were crossed to otin n F 1 hyrid fmily (J 1 3 P 1 ). These F 1 femles were crossed with mles resulting from cross etween Pcific Ocen femle nd nother Pcific Ocen mle (P 2 3 P 3 ) to generte ckcross progeny. At mturity, 76 ckcross mles were phenotyped for trits relted to ody size, fertility nd dorsl ehviour. These mles were genotyped with LG9 nd LG19 microstellites, s well s pnel of SNP mrkers distriuted cross the stickleck genome (Supplementry Tle 5). All DNA isoltion nd microstellite genotyping were conducted s previously descried 19 ; SNP genotyping ws performed using Illumin Golden Gte rrys. The genotypes of 9 SNPs nd 14 microstellites were used to crete linkge mp in JoinMp 3. (ref. 33), nd QTL nlyses were performed in MpQTL 4. (ref. 34) nd R/qtl 35. Received 4 My; ccepted August 9. Pulished online 27 Septemer 9. 9 Mcmilln Pulishers Limited. All rights reserved 1. Rice, W. R. The ccumultion of sexully ntgonistic genes s selective gent promoting the evolution of reduced recomintion etween primitive sex chromosomes. Evolution 41, (1987). 2. Rice, W. R. Genetic hitchhiking nd the evolution of reduced genetic ctivity of the Y sex chromosome. Genetics 116, (1987). 3. Rice, W. R. Sex chromosomes nd the evolution of sexul dimorphism. Evolution 38, (1984). 4. Chrlesworth, B., Coyne, J. A. & Brton, N. H. The reltive rtes of evolution of sex chromosomes nd utosomes. Am. Nt. 13, (1987). 5. Presgrves, D. C. Sex chromosomes nd specition in Drosophil. Trends Genet. 24, (8). 6. Qvrnström, A. & Biley, R. I. Specition through evolution of sex-linked genes. Heredity 12, 4 15 (9). 7. Chrlesworth, D. & Chrlesworth, B. Sex differences in fitness nd selection for centric fusions etween sex-chromosomes nd utosomes. Genet. Res. 35, (198). 8. vn Doorn, G. S. & Kirkptrick, M. Turnover of sex chromosomes induced y sexul conflict. Nture 449, (7). 9. Ezz, T., Stiglec, R., Veyrunes, F. & Grves, J. A. M. Reltionships etween verterte ZW nd XY sex chromosome systems. Curr. Biol. 16, R736 R743 (6). 1. Mnk, J. E., Promislow, D. E. L. & Avise, J. C. Evolution of lterntive sexdetermining mechnisms in teleost fishes. Biol. J. Linn. Soc. 87, (6). 11. Worm, R. A. et l. Comprtive genome nlysis of the primry sex-determining locus in slmonid fishes. Genome Res. 13, (3). 12. Tnk, K., Tkehn, Y., Nruse, K., Hmguchi, S. & Skizumi, M. Evidence for different origins of sex chromosomes in closely relted Oryzis fishes: sustitution of the mster sex-determining gene. Genetics 177, (7). 13. White, M. J. D. Animl Cytology nd Evolution (Univ. Press, 1973). 14. Ross, J. A. et l. Turnover of sex chromosomes in the stickleck fishes (Gsterosteide). PLoS Genet. 5, e1391 (9). 15. Seehusen, O., vn Alphen, J. J. M. & Lnde, R. Color polymorphism nd sex rtio distortion in cichlid fish s n incipient stge in symptric specition y sexul selection. Ecol. Lett. 2, (1999). 16. Lnde, R., Seehusen, O. & vn Alphen, J. J. M. Mechnisms of rpid symptric specition y sex reversl nd sexul selection in cichlid fish. Genetic , (1). 17. Kocher, T. D. Adptive evolution nd explosive specition: the cichlid fish model. Nture Rev. Genet. 5, (4).. Higuchi, M. & Goto, A. Genetic evidence supporting the existence of two distinct species in the genus Gsterosteus round Jpn. Environ. Biol. Fishes 47, 1 16 (1996). 19. Kitno, J., Mori, S. & Peichel, C. L. Phenotypic divergence nd reproductive isoltion etween symptric popultions of ndromous threespine sticklecks. Biol. J. Linn. Soc. 91, (7).. Kingsley, D. M. & Peichel, C. L. in Biology of the Three-Spined Stickleck (eds Östlund-Nilsson, S., Myer, I. & Huntingford, F.) (CRC Press, 7). 21. Peichel, C. L. et l. The mster sex-determintion locus in threespine sticklecks is on nscent Y chromosome. Curr. Biol. 14, (4). 22. Ross, J. A. & Peichel, C. L. Moleculr cytogenetic evidence of rerrngements on the Y chromosome of the threespine stickleck fish. Genetics 179, (8). 23. Foster, S. A. in The Evolutionry Biology of the Threespine Stickleck (eds Bell, M. A. & Foster, S. A.) (Oxford Univ. Press, 1994). 24. Wootton, R. J. A Functionl Biology of Sticklecks (Croom Helm, 1984). 25. Kitno, J., Mori, S. & Peichel, C. L. Sexul dimorphism in the externl morphology of the threespine stickleck (Gsterosteus culetus). Copei 7, (7). 26. Wu, C.-I. & Dvis, A. W. Evolution of postmting reproductive isoltion: the composite nture of Hldne s rule nd its genetic ses. Am. Nt. 142, (1993). 27. To, Y., Hrtl, D. L. & Lurie, C. C. Sex-rtio segregtion distortion ssocited with reproductive isoltion in Drosophil. Proc. Ntl Acd. Sci. USA 98, (1). 28. Phdnis, N. & Orr, H. A. A single gene cuses oth mle sterility nd segregtion distortion in Drosophil hyrids. Science 323, (9).

5 NATURE Vol Octoer Orr, H. A. & Irving, S. Complex epistsis nd the genetic sis of hyrid sterility in the Drosophil pseudooscur Bogot-USA hyridiztion. Genetics 158, 9 11 (1). 3. Swmur, K., Roote, J., Wu, C.-I. & Ymmoto, M. T. Genetic complexity underlying hyrid mle sterility in Drosophil. Genetics 166, (4). 31. Gvrilets, S. Fitness Lndscpesnd theoriginof Species (Princeton Univ. Press, 4). 32. Arnqvist, G. & Rowe, L. Sexul Conflict (Princeton Univ. Press, 5). 33. Vn Ooijen, J. W. & Voorrips, R. E. JoinMp 3., Softwre for the Clcultion of Genetic Linkge Mps (Plnt Reserch Interntionl, 1). 34. Vn Ooijen, J. W., Boer, M. P., Jnsen, R. C. & Mlieprd, C. MpQTL 4., Softwre for the Clcultion of QTL Positions on Genetic Mps (Plnt Reserch Interntionl, 2). 35. Bromn, K. W., Wu, H., Sen, S. & Churchill, G. A. R/qtl: QTL mpping in experimentl crosses. Bioinformtics 19, (3). Supplementry Informtion is linked to the online version of the pper t Acknowledgements We thnk M. Nishitni, J. Kitjim, M. Nishid, S. Tkeym, T. Andoh, T. Kuwhr, C. Torii, Akkeshi Fisheries Coopertive Assocition, Akkeshi Wterfowl Center, S. Brdy, A. Southwick, ll memers of the Peichel lortory, nd mny field ssistnts for technicl help nd discussion. We thnk J. Boughmn, T. Brdshw, H. Mlik, J. McKinnon, N. Phdnis nd D. Schluter for comments on the mnuscript. We lso thnk the Brod Institute for the pulic relese of n initil stickleck genome ssemly. This reserch ws supported y the Uehr Memoril Foundtion (J.K.), Grnt-in-Aid for Scientific Reserch from the Ministry of Eduction, Culture, Sports, Science, nd Technology of Jpn, nd Wter nd People Project of Reserch Institute for Humnity nd Nture (S.M.), Akkeshi Town Grnts-in-Aid for Scientific Reserch in the Lke Akkeshi-Bekneushi Wetlnd (M.K.), Burroughs Wellcome Fund Creer Awrd in the Biomedicl Sciences (C.L.P.), nd Ntionl Institutes of Helth grnts T32 GM727 (J.A.R.), R1 GM754 (C.L.P.) nd P5 HG2568 (R.M.M., D.M.K. nd C.L.P.). Author Contriutions J.K., S.M. nd C.L.P. conceived nd designed the study. F.C.J., Y.F.C., D.M.A., J.G., J.S., R.M.M. nd D.M.K. contriuted new regents nd crried out the SNP genotyping experiments for genome-wide linkge mpping. J.K., J.A.R., S.M., M.K. nd C.L.P. performed ll other experiments nd nlysed the dt. J.K. nd C.L.P. wrote the mnuscript. Author Informtion All SNP informtion hs een deposited t Reprints nd permissions informtion is ville t Correspondence nd requests for mterils should e ddressed to C.L.P. (cpeichel@fhcrc.org). 9 Mcmilln Pulishers Limited. All rights reserved 3