Abstract
Sex determination in honeybees (Apis mellifera) is governed by heterozygosity at a single locus harbouring the complementary sex determiner (csd) gene1, in contrast to the well-studied sex chromosome system of Drosophila melanogaster2. Bees heterozygous at csd are females, whereas homozygotes and hemizygotes (haploid individuals) are males. Although at least 15 different csd alleles are known among natural bee populations3, the mechanisms linking allelic interactions to switching of the sexual development programme are still obscure. Here we report a new component of the sex-determining pathway in honeybees, encoded 12 kilobases 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 capitata4. Like tra, the fem transcript is alternatively spliced. The male-specific splice variant contains a premature stop codon and yields no functional 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 implements the switch of developmental pathways controlled by heterozygosity 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.
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Primary accessions
GenBank/EMBL/DDBJ
Data deposits
The sequences generated in this study are available from GenBank under the accession numbers EU101387 (GB11211), EU101388 (femF), EU101389 (femM), EU101390 (csd), EU101391 (GB30480), EU101392 (GB13727), EU139305 (M. compressipes fem), EU288185 (B. terrestris fem), and EU100885–EU100941 (Apis fem and csd sequences from populations and different 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.
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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 for technical support; D. Titera for providing bee crosses; 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, isolated gene sequences from different species and supervised some 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.
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Hasselmann, M., Gempe, T., Schiøtt, M. et al. Evidence for the evolutionary nascence of a novel sex determination pathway in honeybees. Nature 454, 519–522 (2008). https://doi.org/10.1038/nature07052
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DOI: https://doi.org/10.1038/nature07052
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