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Nature. Author manuscript; available in PMC Jan 5, 2013.
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PMCID: PMC3398145
EMSID: UKMS47290

Butterfly genome reveals promiscuous exchange of mimicry adaptations among species

The Heliconius Genome Consortium, Kanchon K Dasmahapatra,1,* James R. Walters,2,* Adriana D. Briscoe,3 John W. Davey,4 Annabel Whibley,5 Nicola J. Nadeau,2 Aleksey V. Zimin,6 Daniel S. T. Hughes,7 Laura C. Ferguson,8 Simon H. Martin,2 Camilo Salazar,2,9 James J. Lewis,3 Sebastian Adler,10 Seung-Joon Ahn,11 Dean A. Baker,12 Simon W. Baxter,2 Nicola L. Chamberlain,13 Ritika Chauhan,14 Brian A. Counterman,15 Tamas Dalmay,16 Lawrence E. Gilbert,17 Karl Gordon,18 David G. Heckel,11 Heather M. Hines,19 Katharina J. Hoff,10 Peter W.H. Holland,8 Emmanuelle Jacquin-Joly,20 Francis M. Jiggins,21 Robert T. Jones,5 Durrell D. Kapan,22,23 Paul Kersey,7 Gerardo Lamas,24 Daniel Lawson,7 Daniel Mapleson,25 Luana S. Maroja,26 Arnaud Martin,3 Simon Moxon,27 William J. Palmer,21 Riccardo Papa,28 Alexie Papanicolaou,18 Yannick Pauchet,11 David A. Ray,29,30 Neil Rosser,1 Steven L. Salzberg,31 Megan A. Supple,32 Alison Surridge,2 Ayse Tenger-Trolander,13 Heiko Vogel,11 Paul A. Wilkinson,33 Derek Wilson,7 James A. Yorke,6 Furong Yuan,3 Alexi L. Balmuth,34 Cathlene Eland,34 Karim Gharbi,34 Marian Thomson,34 Richard A. Gibbs,35 Yi Han,35 Joy C. Jayaseelan,35 Christie Kovar,35 Tittu Mathew,35 Donna M. Muzny,35 Fiona Ongeri,35 Ling-Ling Pu,35 Jiaxin Qu,35 Rebecca L. Thornton,35 Kim C. Worley,35 Yuan-Qing Wu,35 Mauricio Linares,36 Mark L. Blaxter,4,34 Richard H. ffrench Constant,14 Mathieu Joron,5 Marcus R. Kronforst,13 Sean P. Mullen,37 Robert D. Reed,3 Steven E. Scherer,35 Stephen Richards,35 James Mallet,1,38,§ W. Owen McMillan,9 and Chris D. Jiggins2,9

Abstract

The evolutionary importance of hybridization and introgression has long been debated1. We used genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation2-5 . We sequenced the genome of Heliconius melpomene and compared it with other taxa to investigate chromosomal evolution in Lepidoptera and gene flow among multiple Heliconius species and races. Among 12,657 predicted genes for Heliconius, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organisation has remained broadly conserved since the Cretaceous, when butterflies split from the silkmoth lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, H. melpomene, H. timareta, and H. elevatus, especially at two genomic regions that control mimicry pattern. Closely related Heliconius species clearly exchange protective colour pattern genes promiscuously, implying a major role for hybridization in adaptive radiation.

The butterfly genus Heliconius (Nymphalidae: Heliconiinae) is associated with a suite of derived life-history and ecological traits, including pollen-feeding, extended life-span, augmented ultraviolet colour vision, ‘trap-lining’ foraging behavior, gregarious roosting and complex mating behaviours, and provides outstanding opportunities for genomic studies of adaptive radiation and speciation4, 6. The genus is best known for the hundreds of different colour pattern races seen among its 43 species, with repeated examples of both convergent evolution among distantly related species and divergent evolution between closely related taxa3. Geographic mosaics of multiple colour pattern races, such as in Heliconius melpomene (Fig. 1), converge to similar mosaics in other species, and this led to the hypothesis of mimicry2. Heliconius are unpalatable and Müllerian mimicry of warning colour patterns enables species to share the cost of educating predators3. Divergence in wing pattern is also associated with speciation and adaptive radiation due to a dual role in mimicry and mate selection3, 5. A particularly recent radiation is the melpomene-silvaniform clade, where mimetic patterns often appear polyphyletic (Fig. 1a). Most species in this clade occasionally hybridise in the wild with other clade members7. Gene genealogies at a small number of loci indicate introgression between species8, and one non-mimetic species, H. heurippa, has a hybrid origin9. Adaptive introgression of mimicry loci is therefore a plausible explanation for parallel evolution of multiple mimetic patterns in the melpomene-silvaniform clade.

Figure 1
Distribution, mimicry and phylogenetic relationships of sequenced taxa

A Heliconius melpomene melpomene stock from Darién, Panama (Fig. 1) was inbred via five generations of sib mating. A single male was sequenced to 38x coverage (after quality filtering) using combined 454 and Illumina technologies (Supplementary Information 1-8). The complete draft genome assembly of 269 Mb consists of 3,807 scaffolds with an N50 of 277 kb and contains 12,657 predicted protein-coding genes. RAD linkage mapping was used to assign and order 83% of the sequenced genome onto the 21 chromosomes (Supplementary Information 4). These data permit a considerably improved genome-wide chromosomal synteny comparison with the silkmoth Bombyx mori10, 11. Using 6,010 orthologues identified between H. melpomene and B. mori we found that 11 of 21 H. melpomene linkage groups show homology to single B. mori chromosomes and ten linkage groups have major contributions from two B. mori chromosomes (Fig. 2a and Supplementary Information 8), revealing several previously unidentified chromosomal fusions. These fusions on the Heliconius lineage most likely occurred after divergence from the sister genus Eueides4, which has the lepidopteran modal karyotype of n=3112. Three chromosomal fusions are evident in Bombyx (Fig. 2a, B. mori chromosomes 11, 23 and 24), as required for evolution of the Bombyx n=28 karyotype from the ancestral n=31 karyotype. Heliconius and Bombyx lineages diverged in the Cretaceous >100 MYA11, so the chromosomal structures of Lepidoptera genomes have remained highly conserved compared to those of flies or vertebrates13, 14. In contrast, small-scale rearrangements were frequent. In the comparison with Bombyx, we estimate 0.05-0.13 breaks/Mb/MY, and with the Monarch butterfly, Danaus plexippus, 0.04-0.29 breaks/Mb/MY. Although lower than previously suggested for Lepidoptera15, these rates are comparable to Drosophila (Supplementary Information 8).

Figure 2
Comparative analysis of synteny and expansion of the chemosensory genes

The origin of butterflies was associated with a switch from nocturnal to diurnal behaviour, and a corresponding increase in visual communication16. Heliconius have increased visual complexity through expression of a duplicate UV opsin6, in addition to the long wavelength, blue, and UV-sensitive opsins in Bombyx. We might therefore predict reduced complexity of olfactory genes, but in fact Heliconius and Danaus17 genomes have more chemosensory proteins (CSPs) than any other insect genome: 33 and 34 CSPs respectively (Supplementary Information 9), versus 24 in Bombyx and 3- 4 in Drosophila18. Lineage-specific CSP expansions were evident in both Danaus and Heliconius (Fig. 2b). In contrast, all three lepidopteran genomes possess similar numbers of odorant binding proteins and olfactory receptors (Supplementary Information 9). Hox genes are involved in body plan development and show strong conservation across animals. We identified four additional Hox genes located between the canonical Hox genes pb and zen, orthologous to shx genes in B. mori (Supplementary Information 10)19. These Hox gene duplications in the butterflies and Bombyx share a common origin, and are independent of the two tandem duplications known in dipterans (zen2, bcd). Immunity-related gene families are similar across all three lepidopterans (Supplementary Information 11), contrasting with extensive duplications and losses within dipterans20.

The Heliconius reference genome enabled rigorous tests for introgression among melpomene-silvaniform clade species. We used RAD resequencing to reconstruct a robust phylogenetic tree based on 84 individuals of H. melpomene and its relatives,sampling on average 12 Mb, or 4% of the genome (Fig 1a, Supplementary Information 12, 13, 18). We then tested for introgression between the sympatric co-mimetic postman races of H. melpomene aglaope and H. timareta ssp. nov. (Fig. 1) in Peru, employing ‘ABBA-BABA’ single nucleotide sites and Patterson’s D-statistics (Fig. 3a), originally developed to test for admixture between Neanderthals and modern humans21, 22 (Supplementary Information 12). Genome-wide we found an excess of ABBA sites, giving a significantly positive Patterson’s D = 0.037 ± 0.003 (two tailed Z-test for D = 0, P = 1 × 10−40), indicating greater genome-wide introgression between the sympatric mimetic taxa H. m. amaryllis and H. timareta ssp. nov., than between H. m. aglaope and H. timareta ssp. nov., which do not overlap spatially (Fig. 1b). These D-statistics yield an estimate of 2-5% of the genome exchanged21 between the two taxa (Supplementary Information 12). Eleven of the 21 chromosomes have significantly positive D-statistics (Fig. 3b,); interestingly, the strongest signals of introgressions were found on two chromosomes containing the known mimicry loci B/D and N/Yb (Fig. 3b, Supplementary Information 15).

Figure 3
Four-taxon ABBA-BABA test of introgression

Perhaps the best known case of Müllerian mimicry is the geographic mosaic of ~30 bold postman and rayed colour pattern races of H. melpomene (Fig. 1b, Supplementary Information 22), which mimic a near-identical colour pattern mosaic in H. erato (Fig. 1a), among other Heliconius. Mimicry variation is generally controlled by a few loci with major effects. Mimetic pattern differences between the postman H. melpomene amaryllis and rayed H. melpomene aglaope races studied here (Fig 1a) are controlled by the B/D (red pattern) and N/Yb (yellow pattern) loci23, 24. These loci are located on the same two chromosomes showing the strongest D- statistics in our RAD analysis (Fig. 3b). To test whether mimicry loci might be introgressed between co-mimetic H. timareta and H. melpomene (Fig. 1a)7, we resequenced the colour pattern regions B/D (0.7 Mb) and N/Yb (1.2 Mb), and 1.8 Mb of unlinked regions across the genome, from both postman and ray-patterned H. melpomene and H. timareta from Peru and Colombia, and six silvaniform outgroup taxa (Fig. 1a, Supplementary Information 12). To test for introgression at the B/D mimicry locus we compared rayed H. m. aglaope and postman H. m. amaryllis as the ingroup with postman H. timareta ssp. nov. (as in Fig. 3a) and found large, significant peaks of shared fixed ABBA nucleotide sites combined with an almost complete lack of BABA sites (Fig. 4b). This provides evidence that blocks of shared sequence variation in the B/D region were exchanged between postman H. timareta and postman H. melpomene, in the genomic region known to determine red mimicry patterns between races of H. melpomene23, 24 (Fig. 4a).

Figure 4
Evidence for adaptive introgression at the B/D mimicry locus

For a reciprocal test, we used the same H. melpomene races as the ingroup to compare with rayed H. timareta florencia at the B/D region. In this case, correspondingly large and significant peaks of BABA nucleotide sites are accompanied by virtual absence of ABBA sites (Fig. 4c) indicating that variation at the same mimicry locus was also shared between rayed H. timareta and rayed H. melpomene. Equivalent results in the N/Yb colour pattern region, controlling yellow colour pattern differences, are in the expected directions for introgression and highly significant for the test using postman H. timareta ssp. nov. (P = 6 × 10−34), although not significant with rayed H. timareta florencia (P = 0.13, Supplementary Information 17). In contrast hardly any ABBA or BABA sites are present in either comparison across 1.8 Mb in 55 genomic scaffolds unlinked to the colour pattern regions (Supplementary Information 21). These concordant, but reciprocal patterns, where fixed ABBA and BABA substitutions occur almost exclusively within large genomic blocks at two different colour pattern loci (449 and 99 sites for B/D and N/Yb respectively, Figs. 4b,c and Supplementary Information 17) would be very hard to explain via convergent functional site evolution or under coalescent fluctuations. Instead, our results imply that derived colour pattern elements have introgressed recently between both rayed and postman forms of H. timareta and H. melpomene.

To test whether colour pattern loci might be shared more broadly across the clade, we used sliding-window phylogenetic analyses along the colour pattern regions. For regions flanking and unlinked to colour pattern loci, tree topologies are similar to the overriding signal recovered from the genome as a whole (Supplementary Information 18). Races of H. melpomene and H. timareta each form separate monophyletic sister groups and both are separated from the more distantly related silvaniform species (Fig. 4d). By contrast, within the region of peak ABBA/BABA differences, the topologies switch dramatically. Races of H. melpomene and H. timareta group according to wing pattern, while the species themselves become polyphyletic (Figs. 4e,f, Supplementary Information 19, 20). Remarkably, the rayed H. elevatus, a member of the silvaniform clade according to genome average relationships (Fig. 1a, Supplementary Information 18), groups with rayed races of unrelated H. melpomene and H. timareta in small sections within both B/D and N/Yb colour pattern loci (Fig. 4e, Supplementary Information 19, 20). These results are again most readily explained by introgression and fixation of mimicry genes.

We have developed a de novo reference genome sequence that will facilitate evolutionary and ecological studies in this key group of butterflies. We have demonstrated repeated exchange of small (~100 kb) adaptive genome regions among multiple species in an adaptive radiation. Our genome-scale analysis provides considerably greater power than previous tests of introgression 8, 25, 26. As with H. heurippa9, our evidence suggests that H. elevatus was formed during a hybrid speciation event. The main genomic signal from this rayed species places it closest to H. pardalinus butleri (Fig. 1a), but colour pattern genomic regions resemble those of rayed races of H. melpomene (Fig. 4e and Supplementary Information 18-20). Colour pattern is important in mating behaviour in Heliconius5, and the transfer of mimetic pattern may have enabled the divergent sibling species H. elevatus to coexist with H. pardalinus across the Amazon. Although it was long suspected that introgression might be important in adaptive radiation1, our results from the most diverse terrestrial biome on the planet suggest that adaptive introgression is more pervasive than previously realized.

Methods summary

A full description of methods can be found in the Supplementary Information.

Supplementary Material

1

Supplementary Information is linked to the online version of the paper at www.nature.com/nature.

Acknowledgements

We thank the governments of Colombia, Peru and Panama for permission to collect and export butterflies. Sequencing was funded by contributions from consortium members. We thank Moisés Abanto for assistance in raising the inbred line. Individual laboratories were funded by the Leverhulme Trust (CDJ), John Fell Fund and Christ Church College, Oxford (LCF), The Royal Society (MJ, CDJ), NSF (WOM, MK, RR, SM, ADB), NIH (MK, SLS, JY), CNRS (MJ), ERC (MJ, PWHH), Banco de la República and COLCIENCAS (ML), and the BBSRC (JM, CDJ, MB, R ff-C).

Author contributions

Consortium leaders:

Chris D. Jiggins, W. Owen McMillan

Heliconius Genome Consortium PIs:

Richard ffrench-Constant, Marcus Kronforst, Mathieu Joron, James Mallet, Sean Mullen, Robert Reed, Mark Blaxter, Larry Gilbert, Mauricio Linares, Gerardo Lamas

Introgression study leader & corresponding author:

James Mallet

Lead investigators:

Kanchon Dasmahapatra, James Walters, Nicola Nadeau, Annabel Whibley, John Davey, Adriana Briscoe, Laura Ferguson, Daniel Hughes, Simon Martin, Camilo Salazar, James Lewis

Sequencing:

Stephen Richards, Steve Scherer, Alexi Balmuth, Marian Thomson, Karim Gharbi, Cathlene Eland, Mark Blaxter, Richard Gibbs, Yi Han, Joy Christina Jayaseelan, Christie Kovar, Tittu Mathew, Donna Marie Muzny, Fiona Ongeri, Ling-Ling Pu, Jiaxin Qu, Rebecca Lynn Thornton, Kim C. Worley, Yuan-Qing Wu

Assembly:

Aleksey Zimin, James Yorke, Steven Salzberg, Alexie Papanicolaou, Karl Gordon

RAD map and assembly verification:

John Davey, Simon Baxter, Mark Blaxter, Luana Maroja, Durrell D. Kapan, James Walters, Paul Wilkinson

Geographic distribution map:

Neil Rosser

Annotation:

James Walters, Daniel Hughes, Derek Wilson, Daniel Lawson, Katharina Hoff, Sebastian Adler, Paul Wilkinson

Genome browser and databases:

Daniel Hughes, James Lewis

Manual annotation and evolutionary analyses:

Olfactory proteins: Adriana Briscoe, Emmanuelle Jacquin-Joly, Furong Yuan

Hox genes: Laura Ferguson, Peter W. H. Holland, James Walters

Micro-RNAs: Alison Surridge, Tamas Dalmay, Daniel Mapleson, Simon Moxon

Immune genes: William Palmer, Francis Jiggins

P450 genes: Robert Jones and Ritika Chauhan

UGT genes: Heiko Vogel, Seung-Joon Ahn, David Heckel

Ribosomal Proteins: Yannick Pauchet

Manual annotation group: Simon Baxter, Mark Blaxter, Adriana Briscoe, Nicola Chamberlain, Brian Counterman, Laura Ferguson, Heather Hines, Chris Jiggins, Frank Jiggins, Mathieu Joron, Durrell Kapan, Marcus Kronforst, Jim Mallet, Arnaud Martin, Sean Mullen, Nicola Nadeau, William Palmer, Riccardo Papa, Megan Supple, Ayse Trolander, Annabel Whibley, Furong Yuan

Transposable elements: Brian Counterman, David Ray

Orthologue predictions: Dean Baker

Synteny: Annabel Whibley, John Davey, David Heckel, Karl Gordon

Introgression analysis: Kanchon Dasmahapatra, Nicola Nadeau, John Davey, Simon Martin, Camilo Salazar, Chris Jiggins, Mathieu Joron, James Mallet

Author Information The genome sequence has been submitted to European Nucleotide Archive with accession numbers HE667773-HE672081. The annotated genome is available on our genome browser at http://butterflygenome.org/ and will also be made available in the next release of ENSEMBL Genomes. Additional short read sequences have been submitted to the European Nucleotide Archive with accession numbers ERP000993 and ERP000991.

Reprints and permissions information is available at www.nature.com/ reprints.

The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature.

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