Warning signals are seductive: relative contributions of color and pattern to predator avoidance and mate attraction in Heliconius butterflies.

Visual signaling in animals can serve many uses, including predator deterrence and mate attraction. In many cases, signals used to advertise unprofitability to predators are also used for intraspecific communication. Although aposematism and mate choice are significant forces driving the evolution of many animal phenotypes, the interplay between relevant visual signals remains little explored. Here, we address this question in the aposematic passion-vine butterfly Heliconius erato by using color- and pattern-manipulated models to test the contributions of different visual features to both mate choice and warning coloration. We found that the relative effectiveness of a model at escaping predation was correlated with its effectiveness at inducing mating behavior, and in both cases wing color was more predictive of presumptive fitness benefits than wing pattern. Overall, however, a combination of the natural (local) color and pattern was most successful for both predator deterrence and mate attraction. By exploring the relative contributions of color versus pattern composition in predation and mate preference studies, we have shown how both natural and sexual selection may work in parallel to drive the evolution of specific animal color patterns.

Multi-allelic major effect genes interact with minor effect QTLs to control adaptive color pattern variation in Heliconius erato.

Recent studies indicate that relatively few genomic regions are repeatedly involved in the evolution of Heliconius butterfly wing patterns. Although this work demonstrates a number of cases where homologous loci underlie both convergent and divergent wing pattern change among different Heliconius species, it is still unclear exactly how many loci underlie pattern variation across the genus. To address this question for Heliconius erato, we created fifteen independent crosses utilizing the four most distinct color pattern races and analyzed color pattern segregation across a total of 1271 F2 and backcross offspring. Additionally, we used the most variable brood, an F2 cross between H. himera and the east Ecuadorian H. erato notabilis, to perform a quantitative genetic analysis of color pattern variation and produce a detailed map of the loci likely involved in the H. erato color pattern radiation. Using AFLP and gene based markers, we show that fewer major genes than previously envisioned control the color pattern variation in H. erato. We describe for the first time the genetic architecture of H. erato wing color pattern by assessing quantitative variation in addition to traditional linkage mapping. In particular, our data suggest three genomic intervals modulate the bulk of the observed variation in color. Furthermore, we also identify several modifier loci of moderate effect size that contribute to the quantitative wing pattern variation. Our results are consistent with the two-step model for the evolution of mimetic wing patterns in Heliconius and support a growing body of empirical data demonstrating the importance of major effect loci in adaptive change.

The benefit of being a social butterfly: communal roosting deters predation.

Aposematic passion-vine butterflies from the genus Heliconius form communal roosts on a nightly basis. This behaviour has been hypothesized to be beneficial in terms of information sharing and/or anti-predator defence. To better understand the adaptive value of communal roosting, we tested these two hypotheses in field studies. The information-sharing hypothesis was addressed by examining following behaviour of butterflies departing from natural roosts. We found no evidence of roost mates following one another to resources, thus providing no support for this hypothesis. The anti-predator defence hypothesis was tested using avian-indiscriminable Heliconius erato models placed singly and in aggregations at field sites. A significantly higher number of predation attempts were observed on solitary models versus aggregations of models. This relationship between aggregation size and attack rate suggests that communally roosting butterflies enjoy the benefits of both overall decreased attack frequency as well as a prey dilution effect. Communal roosts probably deter predators through collective aposematism in which aggregations of conspicuous, unpalatable prey communicate a more effective repel signal to predators. On the basis of our results, we propose that predation by birds is a key selective pressure maintaining Heliconius communal roosting behaviour.

UV photoreceptors and UV-yellow wing pigments in Heliconius butterflies allow a color signal to serve both mimicry and intraspecific communication.

Mimetic wing coloration evolves in butterflies in the context of predator confusion. Unless butterfly eyes have adaptations for discriminating mimetic color variation, mimicry also carries a risk of confusion for the butterflies themselves. Heliconius butterfly eyes, which express recently duplicated ultraviolet (UV) opsins, have such an adaptation. To examine bird and butterfly color vision as sources of selection on butterfly coloration, we studied yellow wing pigmentation in the tribe Heliconiini. We confirmed, using reflectance and mass spectrometry, that only Heliconius use 3-hydroxy-DL-kynurenine (3-OHK), which looks yellow to humans but reflects both UV- and long-wavelength light, whereas butterflies in related genera have chemically unknown yellow pigments mostly lacking UV reflectance. Modeling of these color signals reveals that the two UV photoreceptors of Heliconius are better suited to separating 3-OHK from non-3-OHK spectra compared with the photoreceptors of related genera or birds. The co-occurrence of potentially enhanced UV vision and a UV-reflecting yellow wing pigment could allow unpalatable Heliconius private intraspecific communication in the presence of mimics. Our results are the best available evidence for the correlated evolution of a color signal and color vision. They also suggest that predator visual systems are error prone in the context of mimicry.

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.

Genomic hotspots for adaptation: the population genetics of Müllerian 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.

Highly conserved gene order and numerous novel repetitive elements in genomic regions linked to wing pattern variation in Heliconius butterflies.

BACKGROUND: With over 20 parapatric races differing in their warningly colored wing patterns, the butterfly Heliconius erato provides a fascinating example of an adaptive radiation. Together with matching races of its co-mimic Heliconius melpomene, H. erato also represents a textbook case of Müllerian mimicry, a phenomenon where common warning signals are shared amongst noxious organisms. It is of great interest to identify the specific genes that control the mimetic wing patterns of H. erato and H. melpomene. To this end we have undertaken comparative mapping and targeted genomic sequencing in both species. This paper reports on a comparative analysis of genomic sequences linked to color pattern mimicry genes in Heliconius. RESULTS: Scoring AFLP polymorphisms in H. erato broods allowed us to survey loci at approximately 362 kb intervals across the genome. With this strategy we were able to identify markers tightly linked to two color pattern genes: D and Cr, which were then used to screen H. erato BAC libraries in order to identify clones for sequencing. Gene density across 600 kb of BAC sequences appeared relatively low, although the number of predicted open reading frames was typical for an insect. We focused analyses on the D- and Cr-linked H. erato BAC sequences and on the Yb-linked H. melpomene BAC sequence. A comparative analysis between homologous regions of H. erato (Cr-linked BAC) and H. melpomene (Yb-linked BAC) revealed high levels of sequence conservation and microsynteny between the two species. We found that repeated elements constitute 26% and 20% of BAC sequences from H. erato and H. melpomene respectively. The majority of these repetitive sequences appear to be novel, as they showed no significant similarity to any other available insect sequences. We also observed signs of fine scale conservation of gene order between Heliconius and the moth Bombyx mori, suggesting that lepidopteran genome architecture may be conserved over very long evolutionary time scales. CONCLUSION: Here we have demonstrated the tractability of progressing from a genetic linkage map to genomic sequence data in Heliconius butterflies. We have also shown that fine-scale gene order is highly conserved between distantly related Heliconius species, and also between Heliconius and B. mori. Together, these findings suggest that genome structure in macrolepidoptera might be very conserved, and show that mapping and positional cloning efforts in different lepidopteran species can be reciprocally informative.