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doublesex is a mimicry supergene

Abstract

One of the most striking examples of sexual dimorphism is sex-limited mimicry in butterflies, a phenomenon in which one sex—usually the female—mimics a toxic model species, whereas the other sex displays a different wing pattern1. Sex-limited mimicry is phylogenetically widespread in the swallowtail butterfly genus Papilio, in which it is often associated with female mimetic polymorphism1,2,3. In multiple polymorphic species, the entire wing pattern phenotype is controlled by a single Mendelian ‘supergene’4. Although theoretical work has explored the evolutionary dynamics of supergene mimicry5,6,7,8,9, there are almost no empirical data that address the critical issue of what a mimicry supergene actually is at a functional level. Using an integrative approach combining genetic and association mapping, transcriptome and genome sequencing, and gene expression analyses, we show that a single gene, doublesex, controls supergene mimicry in Papilio polytes. This is in contrast to the long-held view that supergenes are likely to be controlled by a tightly linked cluster of loci4. Analysis of gene expression and DNA sequence variation indicates that isoform expression differences contribute to the functional differences between dsx mimicry alleles, and protein sequence evolution may also have a role. Our results combine elements from different hypotheses for the identity of supergenes, showing that a single gene can switch the entire wing pattern among mimicry phenotypes but may require multiple, tightly linked mutations to do so.

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Figure 1: Polymorphic, sex-limited mimicry in Papilio polytes.
Figure 2: Mapping the mimicry supergene.
Figure 3: Expression of doublesex in P. polytes.

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Accessions

GenBank/EMBL/DDBJ

Sequence Read Archive

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Acknowledgements

We thank W. Wang for sharing genome sequence data, C. Robinett for providing the Dsx-DM monoclonal antibody, and E. Westerman, S. Nallu, M. Zhang, G. Garcia and N. Pierce for assistance and discussion. This project was funded by National Science Foundation grant DEB-1316037 to M.R.K.

Author information

Authors and Affiliations

Authors

Contributions

K.K. conceived the project and helped design the study, reared mapping families and samples for gene expression analysis and genome sequencing, performed bulk-segregant analysis and RAD mapping, and contributed to drafting the manuscript. W.Z. generated the reference genome sequences and transcriptome assemblies, performed association mapping, GWAS analysis, HKA tests, structural variant detection and linkage disequilibrium analyses, analysis of protein structure and synonymous/non-synonymous calculations, and contributed to drafting the manuscript. A.T.-T. assisted with butterfly husbandry, performed fine mapping, cDNA sequencing and qRT–PCR analyses. D.H.P. performed qRT–PCR analyses. A.M. and R.D.R. performed Dsx immunohistochemistry. S.P.M. helped design the project and contributed to drafting the manuscript. M.R.K. designed and directed the project, analysed data and wrote the manuscript.

Corresponding authors

Correspondence to K. Kunte or M. R. Kronforst.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Sequence data are available from NCBI SRA (SRP035394) and GenBank (KJ150616KJ150623).

Extended data figures and tables

Extended Data Figure 1 Amino acid substitutions in dsx.

The position of all amino acid substitutions fixed between cyrus and polytes alleles are shown relative to the DM and dimerization domains of dsx. doublesex is a transcription factor that binds DNA as a dimer. The DM domain is responsible for DNA binding and the dimerization domain is responsible for Dsx protein dimerization.

Extended Data Figure 2 Inferred effect of amino acid substitutions on Dsx protein structure.

a, Inferred secondary structure of the Doublesex protein from the cyrus allele of P. polytes, the polytes allele of P. polytes and Bombyx mori. Green helices represent alpha helix structure and blue arrows represent beta sheet structure. b, Inferred tertiary structure of the Doublesex protein from the cyrus allele of P. polytes, the polytes allele of P. polytes and Bombyx mori. Only female isoform 1 is pictured although all protein isoforms differ between cyrus and polytes in a similar way.

Extended Data Figure 3 Analyses of linkage disequilibrium around dsx.

af, Linkage disequilibrium, measured as r2, is elevated in the 1 Mb surrounding dsx in the analysis of all samples (first 1 Mb of the 4-Mb scaffold), compared to a 2-Mb region outside of dsx (last 2 Mb of the 4-Mb scaffold), but this pattern is not evident when analysing cyrus and polytes morph samples separately. g, Linkage disequilibrium heat map, measured as D′, across the 300-kb focal interval shows elevated linkage disequilibrium across dsx but not outside of the gene. Standard colour scheme: D′ < 1, LOD <2 (white); D′ = 1, LOD <2 (blue); D′ = 1, LOD ≥ 2 (pink and red).

Extended Data Figure 4 PCR assay for dsx inversion.

a, Analysis of genome sequence data indicated the presence of an inversion containing the gene dsx. PCR primers that span the breakpoints are expected to produce a product from samples that contain the ancestral, non-inverted sequence, but these should produce no PCR product from samples containing an inversion because the forward priming site is far away and in the opposite orientation. b, Using a series of partially overlapping PCR products, we located the likely 3′ breakpoint approximately 2 kb downstream of the dsx 3′ untranslated region. A second set of breakpoint and control primers produced identical results. All primers were located in regions of no polymorphism, based on genome re-sequencing data, and were tested with ten homozygous females of each phenotype.

Extended Data Table 1 Individuals used for genome re-sequencing
Extended Data Table 2 Candidate scaffolds identified from genome-wide association study
Extended Data Table 3 Results of HKA tests
Extended Data Table 4 De novo polytes scaffolds spanning possible inversion breakpoints

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Kunte, K., Zhang, W., Tenger-Trolander, A. et al. doublesex is a mimicry supergene. Nature 507, 229–232 (2014). https://doi.org/10.1038/nature13112

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