Riccardo Papa

Butterfly genome reveals promiscuous exchange of mimicry adaptations among species

The evolutionary importance of hybridization and introgression has long been debated. Hybrids are usually rare and unfit, but even infrequent hybridization can aid adaptation by transferring beneficial traits between species. Here we use genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation. 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 12,669 predicted genes, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organization has remained broadly conserved since the Cretaceous period, when butterflies split from the Bombyx (silkmoth) lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, Heliconius melpomene, Heliconius timareta and Heliconius elevatus, especially at two genomic regions that control mimicry pattern. We infer that closely related Heliconius species exchange protective colour-pattern genes promiscuously, implying that hybridization has an important role in adaptive radiation.

optix Drives the Repeated Convergent Evolution of Butterfly Wing Pattern Mimicry

Heliconius butterfly wing pattern mimicry is driven by cis-regulatory variation of the optix gene. Mimicry—whereby warning signals in different species evolve to look similar—has long served as a paradigm of convergent evolution. Little is known, however, about the genes that underlie the evolution of mimetic phenotypes or to what extent the same or different genes drive such convergence. Here, we characterize one of the major genes responsible for mimetic wing pattern evolution in Heliconius butterflies. Mapping, gene expression, and population genetic work all identify a single gene, optix, that controls extreme red wing pattern variation across multiple species of Heliconius. Our results show that the cis-regulatory evolution of a single transcription factor can repeatedly drive the convergent evolution of complex color patterns in distantly related species, thus blurring the distinction between convergence and homology.

A Conserved Supergene Locus Controls Colour Pattern Diversity in Heliconius Butterflies

We studied whether similar developmental genetic mechanisms are involved in both convergent and divergent evolution. Mimetic insects are known for their diversity of patterns as well as their remarkable evolutionary convergence, and they have played an important role in controversies over the respective roles of selection and constraints in adaptive evolution. Here we contrast three butterfly species, all classic examples of Müllerian mimicry. We used a genetic linkage map to show that a locus, Yb, which controls the presence of a yellow band in geographic races of Heliconius melpomene, maps precisely to the same location as the locus Cr, which has very similar phenotypic effects in its co-mimic H. erato. Furthermore, the same genomic location acts as a “supergene”, determining multiple sympatric morphs in a third species, H. numata. H. numata is a species with a very different phenotypic appearance, whose many forms mimic different unrelated ithomiine butterflies in the genus Melinaea. Other unlinked colour pattern loci map to a homologous linkage group in the co-mimics H. melpomene and H. erato, but they are not involved in mimetic polymorphism in H. numata. Hence, a single region from the multilocus colour pattern architecture of H. melpomene and H. erato appears to have gained control of the entire wing-pattern variability in H. numata, presumably as a result of selection for mimetic “supergene” polymorphism without intermediates. Although we cannot at this stage confirm the homology of the loci segregating in the three species, our results imply that a conserved yet relatively unconstrained mechanism underlying pattern switching can affect mimicry in radically different ways. We also show that adaptive evolution, both convergent and diversifying, can occur by the repeated involvement of the same genomic regions.

Diversification of complex butterfly wing patterns by repeated regulatory evolution of a Wnt ligand

Although animals display a rich variety of shapes and patterns, the genetic changes that explain how complex forms arise are still unclear. Here we take advantage of the extensive diversity of Heliconius butterflies to identify a gene that causes adaptive variation of black wing patterns within and between species. Linkage mapping in two species groups, gene-expression analysis in seven species, and pharmacological treatments all indicate that cis-regulatory evolution of the WntA ligand underpins discrete changes in color pattern features across the Heliconius genus. These results illustrate how the direct modulation of morphogen sources can generate a wide array of unique morphologies, thus providing a link between natural genetic variation, pattern formation, and adaptation.

Genomic Hotspots for Adaptation: The Population Genetics of Mullerian Mimicry in Heliconius erato

Wing pattern evolution in Heliconius butterflies provides some of the most striking examples of adaptation by natural selection. The genes controlling pattern variation are classic examples of Mendelian loci of large effect, where allelic variation causes large and discrete phenotypic changes and is responsible for both convergent and highly divergent wing pattern evolution across the genus. We characterize nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium (LD), and candidate gene expression patterns across two unlinked genomic intervals that control yellow and red wing pattern variation among mimetic forms of Heliconius erato. Despite very strong natural selection on color pattern, we see neither a strong reduction in genetic diversity nor evidence for extended LD across either patterning interval. This observation highlights the extent that recombination can erase the signature of selection in natural populations and is consistent with the hypothesis that either the adaptive radiation or the alleles controlling it are quite old. However, across both patterning intervals we identified SNPs clustered in several coding regions that were strongly associated with color pattern phenotype. Interestingly, coding regions with associated SNPs were widely separated, suggesting that color pattern alleles may be composed of multiple functional sites, conforming to previous descriptions of these loci as “supergenes.” Examination of gene expression levels of genes flanking these regions in both H. erato and its co-mimic, H. melpomene, implicate a gene with high sequence similarity to a kinesin as playing a key role in modulating pattern and provides convincing evidence for parallel changes in gene regulation across co-mimetic lineages. The complex genetic architecture at these color pattern loci stands in marked contrast to the single casual mutations often identified in genetic studies of adaptation, but may be more indicative of the type of genetic changes responsible for much of the adaptive variation found in natural populations.

Wing patterning gene redefines the mimetic history of Heliconius butterflies

The mimetic butterflies Heliconius erato and Heliconius melpomene have undergone parallel radiations to form a near-identical patchwork of over 20 different wing-pattern races across the Neotropics. Previous molecular phylogenetic work on these radiations has suggested that similar but geographically disjunct color patterns arose multiple times independently in each species. The neutral markers used in these studies, however, can move freely across color pattern boundaries, and therefore might not represent the history of the adaptive traits as accurately as markers linked to color pattern genes. To assess the evolutionary histories across different loci, we compared relationships among races within H. erato and within H. melpomene using a series of unlinked genes, genes linked to color pattern loci, and optix, a gene recently shown to control red color-pattern variation. We found that although unlinked genes partition populations by geographic region, optix had a different history, structuring lineages by red color patterns and supporting a single origin of red-rayed patterns within each species. Genes closely linked (80–250 kb) to optix exhibited only weak associations with color pattern. This study empirically demonstrates the necessity of examining phenotype-determining genomic regions to understand the history of adaptive change in rapidly radiating lineages. With these refined relationships, we resolve a long-standing debate about the origins of the races within each species, supporting the hypothesis that the red-rayed Amazonian pattern evolved recently and expanded, causing disjunctions of more ancestral patterns.

Convergent Evolution in the Genetic Basis of Müllerian Mimicry in Heliconius Butterflies

The neotropical butterflies Heliconius melpomene and H. erato are Müllerian mimics that display the same warningly colored wing patterns in local populations, yet pattern diversity between geographic regions. Linkage mapping has previously shown convergent red wing phenotypes in these species are controlled by loci on homologous chromosomes. Here, AFLP bulk segregant analysis using H. melpomene crosses identified genetic markers tightly linked to two red wing-patterning loci. These markers were used to screen a H. melpomene BAC library and a tile path was assembled spanning one locus completely and part of the second. Concurrently, a similar strategy was used to identify a BAC clone tightly linked to the locus controlling the mimetic red wing phenotypes in H. erato. A methionine rich storage protein (MRSP) gene was identified within this BAC clone, and comparative genetic mapping shows red wing color loci are in homologous regions of the genome of H. erato and H. melpomene. Subtle differences in these convergent phenotypes imply they evolved independently using somewhat different developmental routes, but are nonetheless regulated by the same switch locus. Genetic mapping of MRSP in a third related species, the “tiger” patterned H. numata, has no association with wing patterning and shows no evidence for genomic translocation of wing-patterning loci.

Localization of Müllerian Mimicry Genes on a Dense Linkage Map of Heliconius erato

We report a dense genetic linkage map of Heliconius erato, a neotropical butterfly that has undergone a remarkable adaptive radiation in warningly colored mimetic wing patterns. Our study exploited natural variation segregating in a cross between H. erato etylus and H. himera to localize wing color pattern loci on a dense linkage map containing amplified fragment length polymorphisms (AFLP), microsatellites, and single-copy nuclear loci. We unambiguously identified all 20 autosomal linkage groups and the sex chromosome (Z). The map spanned a total of 1430 Haldane cM and linkage groups varied in size from 26.3 to 97.8 cM. The average distance between markers was 5.1 cM. Within this framework, we localized two major color pattern loci to narrow regions of the genome. The first gene, D, responsible for red/orange elements, had a most likely placement in a 6.7-cM region flanked by two AFLP markers on the end of a large 87.5-cM linkage group. The second locus, Sd, affects the melanic pattern on the forewing and was found within a 6.3-cM interval between flanking AFLP loci. This study complements recent linkage analysis of H. eratos comimic, H. melpomene, and forms the basis for marker-assisted physical mapping and for studies into the comparative genetic architecture of wing-pattern mimicry in Heliconius.

Population genomics of parallel hybrid zones in the mimetic butterflies, H. melpomene and H. erato

Hybrid zones can be valuable tools for studying evolution and identifying genomic regions responsible for adaptive divergence and underlying phenotypic variation. Hybrid zones between subspecies of Heliconius butterflies can be very narrow and are maintained by strong selection acting on color pattern. The comimetic species, H. erato and H. melpomene, have parallel hybrid zones in which both species undergo a change from one color pattern form to another. We use restriction-associated DNA sequencing to obtain several thousand genome-wide sequence markers and use these to analyze patterns of population divergence across two pairs of parallel hybrid zones in Peru and Ecuador. We compare two approaches for analysis of this type of data-alignment to a reference genome and de novo assembly-and find that alignment gives the best results for species both closely (H. melpomene) and distantly (H. erato, ∼15% divergent) related to the reference sequence. Our results confirm that the color pattern controlling loci account for the majority of divergent regions across the genome, but we also detect other divergent regions apparently unlinked to color pattern differences. We also use association mapping to identify previously unmapped color pattern loci, in particular the Ro locus. Finally, we identify a new cryptic population of H. timareta in Ecuador, which occurs at relatively low altitude and is mimetic with H. melpomene malleti.

The Functional Basis of Wing Patterning in Heliconius Butterflies: The Molecules Behind Mimicry

Wing-pattern mimicry in butterflies has provided an important example of adaptation since Charles Darwin and Alfred Russell Wallace proposed evolution by natural selection >150 years ago. The neotropical butterfly genus Heliconius played a central role in the development of mimicry theory and has since been studied extensively in the context of ecology and population biology, behavior, and mimicry genetics. Heliconius species are notable for their diverse color patterns, and previous crossing experiments revealed that much of this variation is controlled by a small number of large-effect, Mendelian switch loci. Recent comparative analyses have shown that the same switch loci control wing-pattern diversity throughout the genus, and a number of these have now been positionally cloned. Using a combination of comparative genetic mapping, association tests, and gene expression analyses, variation in red wing patterning throughout Heliconius has been traced back to the action of the transcription factor optix. Similarly, the signaling ligand WntA has been shown to control variation in melanin patterning across Heliconius and other butterflies. Our understanding of the molecular basis of Heliconius mimicry is now providing important insights into a variety of additional evolutionary phenomena, including the origin of supergenes, the interplay between constraint and evolvability, the genetic basis of convergence, the potential for introgression to facilitate adaptation, the mechanisms of hybrid speciation in animals, and the process of ecological speciation.