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Evidence for the evolutionary nascence of a novel sex determination pathway in honeybees
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"LETTERS Evidence for the evolutionary nascence of a novel sex determination pathway in honeybees Martin Hasselmann 1 *, Tanja Gempe 1 *, Morten Schi�tt 1,2 , Carlos Gustavo Nunes-Silva 3 , Marianne Otte 1 & Martin Beye 1 Sex determination in honeybees (Apis mellifera) is governed by heterozygosity ata single locus harbouring the complementary sex determiner (csd) gene 1 , in contrast to the well-studied sex chro- mosomesystemofDrosophilamelanogaster 2 .Beesheterozygousat csd are females, whereas homozygotes and hemizygotes (haploid individuals)aremales.Althoughatleast15different csdallelesare known among natural bee populations 3 , the mechanisms linking allelic interactions to switching of the sexual development pro- gramme are still obscure. Here we report a new component of the sex-determining pathway in honeybees, encoded 12kilobases upstream of csd. The gene feminizer (fem) is the ancestrally conserved progenitor gene from which csd arose and encodes an SR-type protein, harbouring an Arg/Ser-rich domain. Fem shares the same arrangement of Arg/Ser- and proline-rich-domain with the Drosophila principal sex-determining gene transformer (tra), but lacks conserved motifs except for a 30-amino-acid motif that Fem shares only with Tra of another fly, Ceratitis capitata 4 . Like tra, the fem transcript is alternatively spliced. The male-specific splicevariantcontainsaprematurestopcodonandyieldsnofunc- tional product, whereas the female-specific splice variant encodes the functional protein. We show that RNA interference (RNAi)- induced knockdowns of the female-specific fem splice variant result in male bees, indicating that the fem product is required for entire female development. Furthermore, RNAi-induced knockdowns of female allelic csd transcripts result in the male- specific fem splice variant, suggesting that the fem gene imple- mentstheswitchofdevelopmentalpathwayscontrolledbyhetero- zygosity at csd. Comparative analysis of fem and csd coding sequences from five bee species indicates a recent origin of csd in the honeybee lineage from the fem progenitor and provides evidence for positive selection at csd accompanied by purifying selection at fem. The fem locus in bees uncovers gene duplication and positive selection as evolutionary mechanisms underlying the origin of a novel sex determination pathway. Wehaveidentifiedasecondswitchgene,fem,inthehoneybeethat, besides csd, also localizes to the sex determination locus (SDL; Fig. 1a). The SDL defines the genomic region that is always hetero- zygousinfemales 5 andthuspossiblyharboursextragenesinvolvedin sex determination. We isolated a new part of the SDL (26kilobases (kb)) by assembling sequences of a previously analysed region (21kb) 1 ,fragmentsfromshotguncloning,contigsfromthehoneybee genome project 6 and site-specific amplicons. In our previous study 1 we failed to isolate more parts of the SDL because SDL sequences are AT-rich and are under-represented in our various cloning and shot- gun sequencing strategies 1,6 . We now report three extra genes within the SDL. We tested all five genes located within the SDL (Fig. 1a) for sex-determining function by RNAi knockdown experiments. Only csd and the new fem gene, located 12kb upstream of csd, have sex determinationfunction(Fig.1b).RNAi-inducedknockdownsoffem in females result in a developmental switch to entire male head differentiation (Fig. 1b), whereas knockdowns in males do not affect head development. Repressing the function of csd by RNAi results in apparently the same male-like development in females, but again does not affect head differentiation in males (bottom right panel of Fig. 1b). These findings indicate that fem is the second binary switch gene of the sex determination pathway that, when active, regulates the entire developmental programme of females but not that of males. The fem gene encodes a protein that has a carboxy-terminal Arg/Ser-rich and Pro-rich domain with a high degree of sequence identity to the Csd protein (.70% identical amino acid residues, Fig. 1c), but no similarity to other proteins in the database by apply- ingBLASTprogramsearches.FemhasanextraArg/Ser-domaininits amino terminus, but lacks the hypervariable region of Csd (Fig. 1c) 3 . Thus, both genes are evidently paralogues coding for SR-type proteins,whicharethoughttobeinvolvedgenerallyintheregulation of RNA splicing 7 . We characterized sex-specific transcripts of fem and identified maleandfemalefemtranscriptswiththesame59untranslatedregion (UTR)sequence,butdifferencesintheirdownstreamexoncomposi- tion(Fig.2a).Male-specificfemtranscriptsretainafullexon3,which contains astopcodon, sothattranslationterminatesprematurely. In females,thispartofexon3plusexons4and5aresplicedout,leading toacompleteopenreadingframethattranslatesintoaproteinof403 amino acids. Consistent with the allelic mode of csd activation 1 ,we isolated csd transcripts that differ in their sequence composition, but not in their combination of exons (Fig. 1c). To test whether fem in femalesisregulatedbytheactivityofthecsdgene,werepressedcsdin early embryogenesis by RNAi and studied fem transcripts in fourth instar larvae. These females have a predominant transcript of the male composition (Fig. 2b), establishing that splicing of fem is regu- lated in response to the function of csd. Next, we examined whether transcriptionallevelsof csdareregulatedinresponsetofemfunction. We repressed fem in early embryogenesis and measured csd messen- gerRNAlevelsatthemiddlestageofembryogenesis.Weobservedno differences in csd mRNA levels between fem knockdown and mock short-interfering-RNA-treated (siRNA-treated) embryos (P.0.1, t-test; Fig. 2c). Taken together, these results indicate that the binary switch of the sex determination pathway is implemented by alterna- tivesplicingofthefemtranscriptinresponsetoheterozygosityatcsd. Wecomparedourfindings withthe D. melanogaster pathway.The genes fem and tra seem to have equivalent functions in sex deter- mination, belong to the same family of SR-type proteins, share the *These authors contributed equally to this work. 1 DepartmentofGenetics,HeinrichHeineUniversityDuesseldorf,Universitaetsstrasse1,40225Duesseldorf,Germany. 2 CentreforSocialEvolution,DepartmentofBiology,University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark. 3 Grupo de Pesquisas em Abelhas (GPA), Instituto Nacional de Pesquisas da Amazo?nia (INPA) Avenida Andre� Arau�jo 2936, 69060-001 Manaus, AM, Brazil. Vol 454|24 July 2008|doi:10.1038/nature07052 519 �2008 Macmillan Publishers Limited. All rights reserved samearrangementofregionsenrichedwitharginineandserine(Arg/ Ser domain) and proline (Pro-rich region), but harbour no signifi- cant identity in sequence motifs (Fig. 1c). Such identify may not be expected given the rapid divergence of this sequence between differ- ent dipteran species 4,8 . The tra gene of D. melanogaster functions as a switch gene, isregulated sex-specifically byalternative splicing and is necessary for the entire development of females 9,10 . It is also part of the sex determination cascade that communicates the upstream X:A (ratioofXchromosomestoautosomesets)andSexlethal(Sxl)signal to the downstream gene doublesex (dsx), which controls the activity of the final target genes necessary for sexual differentiation 11?13 . When we compared Fem with the orthologue of Tra from the mediterranean fly C. capitata 4 , in addition to the same arrangement ofdomains(Fig.1c),weidentifieda30-amino-acidmotifinwhich15 residuesareidentical(yellowandframedboxinFig.1c).Onthebasis ofequivalent function andregulationofsex determination, thesame arrangement of Arg/Ser- and Pro-enriched domains and the con- served sequence motif, weconcludethat fem and tra have acommon evolutionary origin. Thesehomologies establish acommon ancestral pathway of sexual regulation at the level of the tra gene across insect orders and,300million years (Myr) of independent evolution. We compared the coding sequences of the honeybee paralogues fem and csd with their orthologues from two related Asian honeybee species (Apis cerana, Apis dorsata), as well as fem sequences from the stingless bee (Melipona compressipes) and the bumble bee (Bombus terrestris)?both of which are members of the major sister branches of honeybees (corbiculate bees)?and the jewel wasp (Nasonia vitri- pennis), to obtain information on the evolutionary relationship and functional divergence of these genes. Significantly, the csd gene is unique to the honeybee lineage. The geneduplicationeventcanbeplacedonthephylogenybycomparing the gene family tree with the phylogeny of the bee species (Fig. 3a). The fem and csd sequences of honeybees form a single clade in the gene tree irrespective of whether we analyse synonymous (Fig. 3a) or amino acid substitutions (Supplementary Fig. 1). We next addressed whether anaccelerated substitution rate inthestingless bee sequence offers an alternative explanation to the clustering of honeybee fem and csd sequences. The stingless bee sequence had a lower relative substitution rate than honeybees (relative rate test, Supplementary Table 1), suggesting that the fem and csd clade in the gene tree is the consequence of a duplication event within the honeybee lineage. Given some uncertainty in the current phylogenetic relationships among the major sister branches of corbiculate bees, we included a b Females Diploid males Control fem RNAi csd RNAi GB11211 csdfem GF3FMHiMKFHi-52RB 5 kb 0.0 0.0 0.40.5 c Fem 77% 72% C. capitata Tra 403 aa 429 aa 419 aa 197 aa D. melanogaster Tra A. mellifera 407 aa 423 aa 50 amino acids GB13727 GB30480 cM Genetic markers Csd alleles FEM 152 INPEDVMLKRRTGEGSKPIFEREEI 176 +NP +V++KRR GEGSKP+F+R++I CcTRA 37 VNPSEVVIKRRFGEGSKPLFQRDDI 61 Figure 1 | Sex-determining genes within the SDL genomic region. a,DiagramoftheidentifiedgeneswithintheSDLgenomicregion 5 .Genesare orientated 59 to 39 according to the direction of the arrows; the names of previously analysed genes 1 are underlined. b, Head development of males andfemalestreatedwithfemandcsdsiRNAsinearlyembryogenesis.Frontal viewoffemale(workers,controlontheleft,n58)anddiploidmale(control on the left, n518) heads are shown. Seventy-eight per cent (n517) of females treated with fem siRNAs and 75% (n527) of females treated with csd siRNAs develop the entire head structures of males. Diploid males treated with fem (n59) or csd (n519) siRNAs have normally developed male heads. Scale bars, 1mm. c, Domain diagrams of Csd, Fem and Tra proteins. Arg/Ser domains are indicated by red, the hypervariable region by green, and the common Pro-rich region by blue boxes. The sequence motif shared across insect orders (Fem and C. capitata Tra (Cc-Tra 4 )) and its schematic location (yellow boxes) are shown. The percentage of amino acid identities of Fem and Csd domains are indicated. aa, amino acids. b csd siRNA 1234 350 bp- 1.6 kb- c 0 5 10 15 20 Control fem siRNA ATG 1 kb TGA AAA ATG TAA AAA a Relative csd transcription (%) Figure 2 | Structureoffemsplicevariantsandthefunctionalrelationshipof fem and csd genes. a, Female and male splicing diagram of the fem gene. Common exons are marked in white, and male-specific exons and exon extensionareingrey.Translationalstartandstopsitesaswellasthepoly(A) addition sites are indicated. b, The processing of fem transcripts in response to the repression of csd function by RNAi. Fragments corresponding to transcripts of female (,350bp) and male (,1.6kb) composition were amplified by RT?PCR reactions. c, csd mRNA amounts in response to repression of fem by RNAi in early embryogenesis. Relative transcription amountsofcsdinfemknockdownembryos(n56)andmocksiRNA-treated control embryos (n59). Error bars represent s.d. LETTERS NATURE|Vol 454|24 July 2008 520 �2008 Macmillan Publishers Limited. All rights reserved the N. vitripennis protein sequence as a further distant out-group (,120Myr of divergence) in our gene tree analysis (Fig. 3b) and again found a clustering of honeybee Csd and Fem proteins and no evidence for relative differences in substitution rates (relative rate test,SupplementaryTable2).Insupportofarecentduplicationevent in the honeybee lineage, we detected only a single copy of this gene family in the bumble bee (B. terrestris) and the jewel wasp (N. vitri- pennis). Our Southern blot hybridization of fem DNA to B. terrestris genomic DNA showed single bands in four different restrictions, supporting the presence of a single genomic locus (Supplementary Fig. 2). By searching the sequenced genome of N. vitripennis,we identifiedonlyasinglegenewithhomologiestofem.Thecloseevolu- tionary relationship of honeybee csd and fem genes thus strongly suggests that gene duplication occurred after the split of the stingless bee,bumblebeeandhoneybee(,70Myrago) 14 butbefore honeybee divergence (,10Myr ago). Consequently, the csd gene is not the universal molecular basis of complementary sex determination in a variety of hymenopteran insects (bees, ants and wasps) 15,16 , suggest- ing that other unknown molecular signals are the primary sex deter- miners in these species. Further analysis suggests that the csd-based sex determination was shaped by positive selection. An excess of non-synonymous to syn- onymous substitutions in a branch of a phylogeny indicates that positive darwinian selection has operated in enhancing the fixation of amino acid changes. A significant excess of non-synonymous over synonymous substitutions is observed in the branch immediately after gene duplication, indicating the action of positive selection in theriseofcsd(Fig.3c;branchpointi?ii,P510 24 ,one-tailedFisher?s exact test). This finding is consistent whether we used different honeybee divergence scenarios or included varying numbers of csd alleles in the analysis. Six substitutions that are fixed in the csd gene are components of a coiled-coil motif which encodes protein-bind- ing properties 17 (Fig. 3d)?a prerequisite for the function of csd- based sex determination 18 . In addition to branch i?ii, an excess of non-synonymous over synonymous changes is also found in the branch ii?iii (P50.007, one-tailed Fisher?s exact test) leading to the western honeybee (A. mellifera). This latter excess is, however, significantly lower than the former (P50.016, one-tailed Fisher?s exacttest)suggestingthatstrongerpositiveselectionoperatedduring theearlyformationofthecsdgenethanduringlineage-specificdiver- gence. The fem gene?the progenitor of csd?is under purifying selection, showing an excess of synonymous to non-synonymous substitutions in branches of fem (P,0.05, one-tailed Fisher?s exact test; Fig. 3c). This low evolutionary divergence of Fem proteins is consistent with our previous result indicating an ancestral sex-deter- mining function for fem. Evidently,femhasretaineditssexdetermination functionwhereas its recent duplicate, csd, evolved an allelic mode of SR-type protein activation through positive selection. The csd-based sex determina- tion system controls sex-specific splicing of its progenitor transcript, thus implementing the switch of male and female pathways. These findings suggest that gene duplication of an existing major sex Phylogenetic tree Approximate divergence time (millions of years ago) A. mellifera M. compressipes B. terrestris N. vitripennis 120100 6080 40 20 0 Bees Wasp a Gene tree fem a n a r e c a r e f i l l e m a t a s r o d A. mellifera A. dorsata A. cerana csd fem M. compressipes fem B. terrestris Apis 100 100 100 100 100 0.05 Synonymous substitutions per site b A. mellifera A. cerana A. dorsata Fem B. terrestris 91 98 97 95 Fem M. compressipes 0.05 A. cerana Csd Apis Fem Apis Fem N. vitripennis A. dorsata A. mellifera c M. compressipes A. mellifera A. dorsata A. mellifera A. dorsata fem csd A. cerana A. cerana i ii iii 6/42 2/30 7/42 0/8 10/27 39/0 7/1 29/36 29/27 75/38 49/11 * 25/0 ** 5/0 19/10 20/7 6/8 * 5/12 ** 0/2 4/12 ** 1/8 ** d CSRDRNREYKEK-DRRYEKLH--NEKEKLLEERTSRKRYSR CSRDRNREHRKK-DRQYEKLH--NEKEKLLEERTSRKRYSH CSRDRNREYKKK-DRQYEKLY--NEKEKLLQEKTSRKRYSR CS--KNREYNKKKDSQYEKLY--NEKEKLLQEKTSRKRYSR CSRDRNKEYKKK-ARQYEKLRTDNEKEKLSQEKTSRKRYSR CSGDRNKEYKKK-DRQYEKLRTDNEKEKLSQEKTSRKRYSR CSRDRSREYKKK-DRRYDQLH--NVEEKHLRERTSRRRYSR CSRDRSREYKKK-DRRYDQLH--NVEEKHLRERTNRRRYSR CSRDRSREYKKK-DRRYDQLH--NVEEKHLRERTSRRRYSR Coiled-coil Csd Fem A. mellifera A. dorsata A. cerana A. mellifera A. dorsata A. cerana Amino acid substitutions per site Figure 3 | Nucleotide and amino acid substitutions in the evolution of csd and fem genes. a, Comparison of the phylogenetic 14,30 (top panel) and gene (bottom panel) tree. Branches derived from csd and fem sequences areindicated by red and blue boxes, respectively. Species sources are marked by coloured frames. Numbers represent bootstrap values.80%. b, Gene tree of Fem and Csd protein sequences including the wasp N. vitripennis. Numbers denote bootstrap values.80%. c, Numbers of non- synonymous (N) and synonymous (S) substitutions along branches of the gene tree. Non-synonymous to synonymous substitution values per branch (a N /a S ) and per branch and site (b N /b S ) 31000 are shown belowandon thelines,respectively. Level of significance of Fisher?s exact tests for positive (bold numbers) and for purifying (underlined numbers) selection 29 . Single asterisk, P,0.05; double asterisk, P,0.01. i, ii and iii (coloured in blue) define branches between nodes of the csd genealogy. d, Comparison of fixed amino acid substitutions between Csd and Fem proteins involved in coiled-coil formation. Fixed differences are marked by boxes; the ones in Csd that are components of the coiled- coil motif (red frame) are coloured in green. NATURE|Vol 454|24 July 2008 LETTERS 521 �2008 Macmillan Publishers Limited. All rights reserved determining gene, followed by positive selection in one of the dupli- cates,favouredtheoriginationofanewupstreamsignaland,thereby, the creation of a novel sex determination pathway. The upwards growth of this pathway is consistent with theory 19,20 predictingthatnewsignalsareco-optedupstreamofacascadeduring the course of evolution. Furthermore, it has been proposed that the origin of alternative sex determination signals involve a selective advantage,suchasthepossibilitytomodifysexratios 21 ortoimprove the quality of signals 21,22 . Our findings provide direct evidence for a role of strong positive selection in the formation of a new sex deter- mination system and are thus consistent with previous suggestions. Weproposethatinourcasethereductionofrecombinationatthesex determination locus 5 (Fig. 1a) results in a gradual loss of the levels of adaptation of the gene 23,24 , which would facilitate the evolution of alternative initial signals of complementary sex determination. This process may thus relate to the evolution of chromosomal systems in which the cessation of recombination 25,26 results in a degradation of genes 27 and the extinction of the sex chromosomes 28 . In either case, our findings provide strong support for the role of positive selection in shaping the growth of a developmental pathway. METHODS SUMMARY Queens producing 50% female and 50% diploid males were derived from brother?sister crosses (inbred crosses). Male-producing queens laying exclu- sively unfertilized, haploid eggs were obtained from non-mated, CO 2 -treated queens. Female-producing queens were obtained from queens inseminated by semenofasinglemale.ThesequencereadswereassembledusingStadenPackage software. Potential exons and genes within the SDL were detected by gene pre- diction programs as described previously 1 . Transcriptionally active genes in embryogenesis were identified by reverse transcription PCR (RT?PCR) of embryonic complementary DNA. The full sequences of transcripts were obtained by 59 and 39 RACE experiments. siRNA (50?100pg per embryo) and double-stranded RNA 1 were injected into embryos at the syncytial stage. Fragments corresponding to sex-specific fem transcripts were amplified by RT?PCR reactions from individual fourth instar larvae and resolved by agarose gel electrophoresis. Freshly hatched larvae were reared in vitro at 34.5uC and saturated humidity. Relative transcriptional levels of csd were calculated by comparing cycle thresholds to the reference gene, elongation factor 1-alpha. Fifty-one csd (n515, A. mellifera; n517, A. cerana; n519, A. dorsata) and ten fem codingsequences (n54, A. mellifera; n52, A. cerana; n52, A. dorsata; n51, M. compressipes; n51, B. terrestris) were isolated by high-fidelity PCR amplificationsofcDNAsderivedfromRNApreparationsofembryos.Genetrees wereobtainedbytheminimumevolutionmethodbyapplyingtheNei?Gojobori distance with Jukes?Cantor correction for nucleotide and Poisson-corrected distances for amino acid differences. The non-synonymous and synonymous substitution values for each branch of the gene tree were obtained by least- squares method. Fisher?s exact test for selection was performed on the numbers of changed and unchanged non-synonymous and synonymous sites 29 . Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Received 5 November 2007; accepted 1 May 2008. Published online 25 June 2008. 1. Beye,M.,Hasselmann,M.,Fondrk,M.K.,Page,R.E.&Omholt,S.W.Thegenecsd is the primary signal for sexual development in the honeybee and encodes an SR- type protein. Cell 114, 419?429 (2003). 2. Cline, T. W. & Meyer, B. J. Vive la diffe�rence: males vs females in flies vs worms. Annu. Rev. Genet. 30, 637?702 (1996). 3. Hasselmann, M. & Beye, M. Signatures of selection among sex-determining alleles of the honey bee. Proc. Natl Acad. Sci. USA 101, 4888?4893 (2004). 4. Pane,A.,Salvemini,M.,Bovi,P.D.,Polito,C.&Saccone,G.Thetransformergenein Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate. Development 129, 3715?3725 (2002). 5. Hasselmann, M. & Beye, M. Pronounced differences of recombination activity at the sex determination locus (SDL) of the honey bee, a locus under strong balancing selection. Genetics 174, 1469?1480 (2006). 6. Honeybee Genome Sequencing Consortium.. Insights into social insects fromthe genome of the honeybee Apis mellifera. Nature 443, 931?949 (2006). 7. Blencowe, B. J., Bowman, J. A., McCracken, S. & Rosonina, E. SR-related proteins and theprocessingof messenger RNAprecursors. Biochem. Cell Biol. 77,277?291 (1999). 8. Kulathinal,R.J.,Skwarek,L.,Morton,R.A.&Singh,R.S.Rapidevolutionofthesex- determining gene, transformer: structural diversity and rate heterogeneity among sibling species of Drosophila. Mol. Biol. Evol. 20, 441?452 (2003). 9. Butler,B., Pirrotta,V., Irminger-Finger, I.&Nothiger, R.Thesex-determining gene tra of Drosophila: molecular cloning and transformation studies. EMBO J. 5, 3607?3613 (1986). 10. Boggs,R.T.,Gregor,P.,Idriss,S.,Belote,J.M.&McKeown,M.Regulationofsexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. Cell 50, 739?747 (1987). 11. Bell, L. R., Maine, E. M., Schedl, P. & Cline, T. W. Sex-lethal,aDrosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins. Cell 55, 1037?1046 (1988). 12. Keyes, L. N., Cline, T. W. & Schedl, P. The primary sex determination signal of Drosophila acts at the level of transcription. Cell 68, 933?943 (1992). 13. Hoshijima, K., Inoue, K., Higuchi, I., Sakamoto, H. & Shimura, Y. Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila. Science 252, 833?836 (1991). 14. Grimaldi, D. & Engel, M. S. Evolution of the Insects 454?467 (Cambridge Univ. Press, UK, 2005). 15. Kerr, W. E. Sex determination in bees. XXI. Number of XO-heteroalleles in a natural population of Melipona compressipes fasciculata (Apidae). Insectes Soc. 34, 274?279 (1987). 16. Cook, J. M. Sex determination in the Hymenoptera: a review of models and evidence. Heredity 71, 421?435 (1993). 17. Burkhard, P., Stetefeld, J. & Strelkov, S. V. Coiled coils: a highly versatile protein folding motif. Trends Cell Biol. 11, 82?88 (2001). 18. Beye, M. The dice of fate: the csd gene and how its allelic composition regulates sexual development in the honey bee, Apis mellifera. Bioessays 26, 1131?1139 (2004). 19. Wilkins, A.S.Moving upthehierarchy: Ahypothesis ontheevolution ofagenetic sex determination pathway. Bioessays 17, 71?77 (1995). 20. No�thiger, R. & Steinemann-Zwicky, M. A single principle for sex determination in insects. Cold Spring Harb. Symp. Quant. Biol. 50, 615?621 (1985). 21. Bull, J. J. Evolution of Sex Determining Mechanisms (Benjamin/Cummings Publishing Company, Menlo Park, California, 1983). 22. Pomiankowski, A., Nothiger, R. & Wilkins, A. The evolution of the Drosophila sex- determination pathway. Genetics 166, 1761?1773 (2004). 23. Hill, W. G. & Robertson, A. The effect of linkage on limits to artificial selection. Genet. Res. 8, 269?294 (1966). 24. Otto, S. P. & Lenormand, T. Resolving the paradox of sex and recombination. Nature Rev. Genet. 3, 252?261 (2002). 25. Lewis, D. The evolution of sex in flowering plants. Biol. Rev. 17, 46?67 (1942). 26. Charlesworth, B. & Charlesworth, D. A model for the evolution of dioecy and gynodioecy. Am. Nat. 112, 975?997 (1978). 27. Charlesworth, D., Charlesworth, B. & Marais, G. Steps in the evolution of heteromorphic sex chromosomes. Heredity 95, 118?128 (2005). 28. Graves, J. A. Sex chromosome specialization and degeneration in mammals. Cell 124, 901?914 (2006). 29. Nei, M. & Kumar, S. Molecular Evolution and Phylogenetics 216?221 (Oxford Univ. Press, New York, 2000). 30. Engel, M. S. Monophyly and extensive extinction of advanced eusocial bees: insights from an unexpected Eocene diversity. Proc. Natl Acad. Sci. USA 98, 1661?1664 (2001). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We thank W. Martin, A. Wilkins, T. Eltz and J. Baines for comments on the manuscript; E.-M. Theilenberg, M. Mueller-Borg and C. Schulte fortechnicalsupport;D.Titeraforprovidingbeecrosses;J.Pflugfelder,N.Koeniger, G. Koeniger, J. Bozic and S. Tingek for collecting honeybee samples; K. Lunau for providing bumble bee samples; and M. Griese for bee-keeping support. This work was supported by grants from the Deutsche Forschungsgemeinschaft DFG. Author Contributions M.H. performed the evolutionary nucleotide analysis, isolatedgenesequencesfromdifferentspeciesandsupervisedsome experiments; T.G. performed the gene studies; M.S. assembled SDL sequences and identified genes; C.G.N.-S. characterized M. compressipes sequences; M.O. analysed domain structures; and M.B. performed the experimental design, supervised the research project and wrote the manuscript. Author Information The sequences generated in this study are available from GenBank under the accession numbers EU101387 (GB11211), EU101388 (fem F ), EU101389 (fem M ), EU101390 (csd), EU101391 (GB30480), EU101392 (GB13727), EU139305 (M. compressipes fem), EU288185 (B. terrestris fem), and EU100885?EU100941(Apisfemandcsdsequencesfrompopulationsanddifferent species). SDL assembly and sequence annotation data are available in the Third Party Annotation Section of the DDBJ/EMBL/GenBank databases under the accession number TPA: BK006346. Reprints and permissions information is available at www.nature.com/reprints. Correspondence and requests for materials should be addressed to M.B. (martin.beye@uni-duesseldorf.de). LETTERS NATURE|Vol 454|24 July 2008 522 �2008 Macmillan Publishers Limited. All rights reserved METHODS Bee material. Queens producing 50% female and 50% diploid males were derived from brother?sister crosses (inbred crosses). Male-producing queens were obtained from non-mated, CO 2 treated queens. Female-producing queens were obtained from queens that were inseminated with the semen of a single male. Samples of honeybee species for evolutionary nucleotide analyses were collected in Borneo (Tenom, A. cerana and A. dorsata), Thailand (Samut Songkram, A. cerana; Wanmanaow, A. dorsata), Slovenia (Litija, A. mellifera) and South Africa (Pretoria, A. mellifera). Samples of stingless bees (Melipona compressipes) were collected in Brazil (Manaus). Samples of bumble bees (B. terrestris) were obtained from the Koppert Company. Sequence analysis. Sequences of the SDL (AADG05006532, AADG05006533, DQ681226, AY352277 (GenBank accession numbers), and 250636813, 165754571, 566358507, 566314258, 173514040, 566864637, 240540892 and 160908628 (NCBI trace archive numbers)) were assembled using the Staden Package software. Ordering of genomic sequences was verified by cDNA sequences. Potential exons were predicted, and transcriptionally active genes were identified by RT?PCR of embryonic cDNA. The first-strand cDNA was generated by reverse transcription with oligo(dT) primer or random hexamers. RACE experiments were performed to isolate the 59 and 39 ends of genes. Sequences were obtained from high-fidelity PCR amplifications of embryonic cDNA and at least three independent clones. We used BLAST programs and a low complexity filter option to compare genes with the database. Domains were analysed by PROSITE database comparisons (http://www.expasy.org/prosite/). The coiled-coil region was predicted by COILS program (http://www.ch. embnet.org/software/COILS_form.html) with a probability of one. Sequences ofthecoiled-coilmotifcomparison(Fig.3d)werederivedfromGenBankacces- sion numbers: EU100885, EU100893, EU100921, EU100928, EU100904, EU100902, EU100940, EU100938, EU100936. Fem and Csd and Tra proteins (N. vitripennis Fem, XM_001604744 and EU780924; C. capitata Tra, AF434936; andD.melanogasterTra,P11596)werecomparedbyBLAST2sequenceprogram (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi). Functionalanalysisofgenes.RNAiknockdownswereinducedinearlyembryo- genesis at the syncytial stage (0?4h after egg deposition) in haploid and diploid males and females. dsRNAs were generated from cloned cDNAs of genes GB11211, GB13727 and GB30480, and from a DNA marker sequence which we used as an RNAi control. The fem and csd siRNAs were synthesized (MWG BioTech) and injected at a concentration of 50?100pg per embryo. Sequences for the nonsense siRNAs used in the control experiments were obtained by scrambling the nucleotide composition of fem and csd siRNA sequences. Injection of these mock siRNAs did not affect sexual differentiation (data not shown). Individuals derived from inbred crosses (producing diploid males and females)weresubjectto csdgenotypingafterphenotypeanalysisattheadultand pupal stage. Head development differs substantially between females (workers) and males (Fig. 1b). Females (workers) have a triangular shaped head, narrow oval-shaped compound eyes, a long proboscis and 12 antennal segments, whereashaploidanddiploidmaleshavearound-shapedhead,largeoval-shaped compound eyes that nearly join in a medio?dorsal position, a short proboscis and 13 antennal segments. Sex-specific splicing of fem transcripts were analysed by RT?PCR reactions with RNA prepared from the fourth instar larvae. Amplifiedfragmentswerecomposedofexons3?6?7?8(size,350bp)andexons 3?4?5?6?7?8 (size,1.6 kb) corresponding to the female and male transcripts, respectively. Hatched larvae were reared in vitro. To quantify the mRNA levels with a BioRad Chromo4, aliquots of first stranded cDNA were amplified, and real-time fluorimetric intensity of SYBR green was monitored. Each sample was runtwiceintriplereplicates.Todeterminerelativetranscriptionallevelsofcsd,2 was raised to the power of DCts values that were obtained by comparing cycle thresholds (Cts) to those of the reference gene, elongation factor 1-alpha (DCts5Cts control 2Cts target ). Individual fem knockdown embryos were iden- tified by testing individual transcription levels against the distribution of mock- siRNA-treated control samples using t-test statistics. Multiple comparisons against the distribution were adjusted using the Bonferroni procedure. Statistical analysis was carried out using the SPSS 15.0 software. Sequences of oligonucleotide primers and siRNAs are listed in the Supplementary Information. Phylogenetic and molecular evolutionary analysis of nucleotide and amino acid substitutions. Theproposedphylogenetic treeshowstheevolutionary rela- tionshipanddivergencetimesofspeciesunderstudyandisbasedonamberfossils andmorphologicaldata.Sequenceswereobtainedfromclonedhigh-fidelityPCR fragments of cDNAs prepared from embryonic mRNA. Extra primer sets to amplify csd from A. cerana and A. dorsata samples were: csd_forCer2/ csd_rev4CIII/; csd_IIIfor/ csd_IIIrev3 (Supplementary Information). The csd (A. mellifera, n515; A. cerana, n517; A. dorsata, n519) and fem sequences (lacking the Asn/Tyr repeat (hypervariable region); A. mellifera, n54; A. cerana, n52;A.dorsata,n52;M.compressipes,n51;B.terrestris,n51)werealignedby the BioEdit program and edited manually to improve the conformity with the open reading frame.Gaps in thesequencealignments were completely deleted in the evolutionary analyses. Trees of coding sequences were obtained by the min- imum evolution method. We applied the Nei?Gojobori distance with Jukes? Cantor correction for synonymous differences and Poisson-corrected distances for amino acid differences which are implemented in the MEGA Version 3.1 program. Gene tree analysis including the wasp N. vitripennis was confined to theC-andN-terminalpartsoftheFemprotein,theregionforwhichwedetected sufficient homology (amino acid positions 1?54 and 301?404, XP_001604794). RelativeratetestsforsubstitutionratedifferenceswereanalysedusingtheRRTree version 1.1 program onsynonymous and amino acid differences using either the B.terrestrisortheN.vitripennisfemsequencesasanout-group.Non-synonymous andsynonymoussubstitution analysisofbranchesofthegenetreewereobtained by least square method implemented in the b N ?b S program. The analysis includeduptosixcsdallelesperhoneybeespecies.Fisher?sexacttestforselection wasperformedonthenumbersofchangedandunchangednon-synonymousand synonymous sites. The number of potential non-synonymous and synonymous sites in the branch analysis are 647 and 277, respectively. For Southern blotting onto Hybond N1membranes standard procedures were used. doi:10.1038/nature07052 �2008 Macmillan Publishers Limited. All rights reserved "
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