LETTER doi:10.1038/nature11041 Butterfly genome reveals promiscuous exchange of mimicry adaptations among species The Heliconius Genome Consortium* The evolutionary importance of hybridization and introgression has The butterfly genus Heliconius (Nymphalidae: Heliconiinae) is long been debated1. Hybrids are usually rare and unfit, but even associated with a suite of derived life-history and ecological traits, infrequent hybridization can aid adaptation by transferring bene- including pollen feeding, extended lifespan, augmented ultraviolet ficial traits between species. Here we use genomic tools to investigate colour vision, ‘trap-lining’ foraging behaviour, gregarious roosting and introgression in Heliconius, a rapidly radiating genus of neotropical complex mating behaviours, and provides outstanding opportunities for butterflies widely used in studies of ecology, behaviour, mimicry and genomic studies of adaptive radiation and speciation4,6. The genus is best speciation2–5. We sequenced the genome of Heliconius melpomene known for the hundreds of races with different colour patterns seen and compared it with other taxa to investigate chromosomal evolu- among its 43 species, with repeated examples of both convergent evolu- tion in Lepidoptera and gene flow among multiple Heliconius tion among distantly related species and divergent evolution between species and races. Among 12,669 predicted genes, biologically closely related taxa3. Geographic mosaics of multiple colour-pattern important expansions of families of chemosensory and Hox races, such as in Heliconius melpomene (Fig. 1), converge to similar genes are particularly noteworthy. Chromosomal organization mosaics in other species, and this led to the hypothesis of mimicry2. has remained broadly conserved since the Cretaceous period, when Heliconius are unpalatable to vertebrate predators and Mu¨llerian butterflies split from the Bombyx (silkmoth) lineage. Using mimicry of warning colour patterns enables species to share the cost genomic resequencing, we show hybrid exchange of genes between of educating predators3. As a result of its dual role in mimicry and mate three co-mimics, Heliconius melpomene, Heliconius timareta and selection, divergence in wing pattern is also associated with speciation Heliconius elevatus, especially at two genomic regions that control and adaptive radiation3,5. A particularly recent radiation is the mimicry pattern. We infer that closely related Heliconius species melpomene–silvaniform clade, in which mimetic patterns often seem exchange protective colour-pattern genes promiscuously, implying to be polyphyletic (Fig. 1a). Most species in this clade occasionally that hybridization has an important role in adaptive radiation. hybridize in the wild with other clade members7. Gene genealogies at a b H. melpomene H. numata rosina H. ethilla Silvaniform clade H. melpomene melpomene H. hecale 1,000 km H. pardalinus sergestus H. elevatus (rayed) H. pardalinus butleri H. melpomene H. timareta florencia aglaope/malleti (rayed) H. timareta ssp. nov. (postman) H. heurippa H. melpomene H. cydno amaryllis H. melpomene rosina (postman) H. melpomene H. melpomene melpomene (postman) p H. melpomene amaryllis (postman) H. melpomene aglaope/malleti (rayed) H. erato Figure 1 | Distribution, mimicry and phylogenetic relationships of H. melpomene races studied here (blue, yellow and purple), and the entire sequenced taxa. a, Phylogenetic relationship of sequenced species and distribution of H. melpomene (grey). The H. timareta races investigated have subspecies in the melpomene–silvaniform clade of Heliconius. Heliconius limited distributions (red) indicated by arrows and mimic sympatric races of elevatus falls in the silvaniform clade, but it mimics colour patterns of H. melpomene. Heliconius elevatus and the other silvaniform species are melpomene–timareta clade taxa. Most other silvaniforms mimic unrelated distributed widely across the Amazon basin (Supplementary Information, ithomiine butterflies24. b, Geographic distribution of postman and rayed section 22). *Lists of participants and their affiliations appear at the end of the paper. 00 MONTH 2012 | VOL 000 | NATURE | 1 ©2012 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER a small number of loci indicate introgression between species8, and The origin of butterflies was associated with a switch from nocturnal one non-mimetic species, Heliconius heurippa, has a hybrid origin9. to diurnal behaviour, and a corresponding increase in visual commun- Adaptive introgression of mimicry loci is therefore a plausible ication16. Heliconius have increased visual complexity through explanation for parallel evolution of multiple mimetic patterns in the expression of a duplicate ultraviolet opsin6, in addition to the long- melpomene–silvaniform clade. wavelength-, blue- and ultraviolet-sensitive opsins in Bombyx.We A Heliconius melpomene melpomene stock from Darie´n, Panama might therefore predict reduced complexity of olfactory genes, but (Fig. 1), was inbred through five generations of sib mating. We in fact Heliconius and Danaus17 genomes have more chemosensory sequenced a single male to 338 coverage (after quality filtering) using genes than any other insect genome: 33 and 34, respectively combined 454 and Illumina technologies (Supplementary Informa- (Supplementary Information, section 9). For comparison, there are tion, sections 1–8). The complete draft genome assembly, which is 24 in Bombyx and 3–4 in Drosophila18. Lineage-specific expansions 269 megabases (Mb) in size, consists of 3,807 scaffolds with an N50 of chemosensory genes were evident in both Danaus and Heliconius of 277 kb and contains 12,669 predicted protein-coding genes. (Fig. 2b). By contrast, all three lepidopteran genomes have similar Restriction-site-associated DNA (RAD) linkage mapping was used numbers of odorant binding proteins and olfactory receptors to assign and order 83% of the sequenced genome onto the 21 chro- (Supplementary Information, section 9). Hox genes are involved in mosomes (Supplementary Information, section 4). These data permit body plan development and show strong conservation across animals. a considerably improved genome-wide chromosomal synteny com- We identified four additional Hox genes located between the canonical parison with the silkmoth Bombyx mori10,11. Hox genes pb and zen, orthologous to shx genes in B. mori19 (Sup- Using 6,010 orthologues identified between H. melpomene and plementary Information, section 10). These Hox gene duplications B. mori, we found that 11 of 21 H. melpomene linkage groups show in the butterflies and Bombyx have a common origin and are inde- homology to single B. mori chromosomes and that ten linkage groups pendent of the two tandem duplications known in dipterans (zen2 have major contributions from two B. mori chromosomes (Fig. 2a and and bcd). Immunity-related gene families are similar across all three Supplementary Information, section 8), revealing several previously lepidopterans (Supplementary Information, section 11), whereas there unidentified chromosomal fusions. These fusions on the Heliconius are extensive duplications and losses within dipterans20. lineage most probably occurred after divergence from the sister genus The Heliconius reference genome allowed us to perform rigorous Eueides4, which has the lepidopteran modal karyotype of n 5 31 (ref. 12). tests for introgression among melpomene–silvaniform clade species. Three chromosomal fusions are evident in Bombyx (B. mori chromo- We used RAD resequencing to reconstruct a robust phylogenetic tree somes 11, 23 and 24; Fig. 2a), as required for evolution of the Bombyx based on 84 individuals of H. melpomene and its relatives, sampling on n 5 28 karyotype from the ancestral n 5 31 karyotype. Heliconius and average 12 Mb, or 4%, of the genome (Fig. 1a and Supplementary Bombyx lineages diverged in the Cretaceous, more than 100 million Information, sections 12–18). We then tested for introgression between years ago11, so the gross chromosomal structures of Lepidoptera the sympatric co-mimetic postman butterfly races of Heliconius genomes have remained highly conserved compared with those of flies melpomene amaryllis and H. timareta ssp. nov. (Fig. 1) in Peru, using or vertebrates13,14. By contrast, small-scale rearrangements were ‘ABBA/BABA’ single nucleotide sites and Patterson’s D-statistics frequent. In the comparison with Bombyx, we estimate there to be (Fig. 3a), originally developed to test for admixture between 0.05–0.13 breaks per megabase per million years, and in that with Neanderthals and modern humans21,22 (Supplementary Information, Danaus plexippus (Monarch butterfly), we estimate there to be section 12). Genome-wide, we found an excess of ABBA sites, giving a 0.04–0.29 breaks per megabase per million years. Although lower than significantly positive Patterson’s D of 0.037 6 0.003 (two-tailed Z-test previously suggested for Lepidoptera15, these rates are comparable to for D 5 0, P 5 1 3 10240), indicating greater genome-wide introgres- those in Drosophila (Supplementary Information, section 8). sion between the sympatric mimetic taxa H. melpomene amaryllis and 9 10 8 7 a 11 b Chemosensory proteins Heliconius 6 12 5 1 DpCSP5 10 Mb 4 HmCSP3 2 BmCSP3 Bm 13 3 Bootstrap support >80% H 24 DpCSP2mCSP5 CSP5 pCSP16 5 2 H CSP21 D HmCSP16 BmCSP16 mCSP2 BmCSP 14 BmCSP2 HmCSP21 Dp mCSP DpCSP20 B HmCSP20 BmCSP23 15 1 BmCSP25 B HmCSP2 mCSP16 DpCSP25 BmCSP7 16 DpCSP23 HmCSP7 2 HmCSP23 DpCSP7 DpCSP29 B 3 HmCSP22 u