Evolutionary dynamics of genome size and content during the adaptive radiation of Heliconiini butterflies.

Heliconius butterflies, a speciose genus of Müllerian mimics, represent a classic example of an adaptive radiation that includes a range of derived dietary, life history, physiological and neural traits. However, key lineages within the genus, and across the broader Heliconiini tribe, lack genomic resources, limiting our understanding of how adaptive and neutral processes shaped genome evolution during their radiation. Here, we generate highly contiguous genome assemblies for nine Heliconiini, 29 additional reference-assembled genomes, and improve 10 existing assemblies. Altogether, we provide a dataset of annotated genomes for a total of 63 species, including 58 species within the Heliconiini tribe. We use this extensive dataset to generate a robust and dated heliconiine phylogeny, describe major patterns of introgression, explore the evolution of genome architecture, and the genomic basis of key innovations in this enigmatic group, including an assessment of the evolution of putative regulatory regions at the Heliconius stem. Our work illustrates how the increased resolution provided by such dense genomic sampling improves our power to generate and test gene-phenotype hypotheses, and precisely characterize how genomes evolve.

Evolutionary and ecological processes influencing chemical defense variation in an aposematic and mimetic Heliconius butterfly.

Chemical defences against predators underlie the evolution of aposematic coloration and mimicry, which are classic examples of adaptive evolution. Surprisingly little is known about the roles of ecological and evolutionary processes maintaining defence variation, and how they may feedback to shape the evolutionary dynamics of species. Cyanogenic Heliconius butterflies exhibit diverse warning color patterns and mimicry, thus providing a useful framework for investigating these questions. We studied intraspecific variation in de novo biosynthesized cyanogenic toxicity and its potential ecological and evolutionary sources in wild populations of Heliconius erato along environmental gradients, in common-garden broods and with feeding treatments. Our results demonstrate substantial intraspecific variation, including detectable variation among broods reared in a common garden. The latter estimate suggests considerable evolutionary potential in this trait, although predicting the response to selection is likely complicated due to the observed skewed distribution of toxicity values and the signatures of maternal contributions to the inheritance of toxicity. Larval diet contributed little to toxicity variation. Furthermore, toxicity profiles were similar along steep rainfall and altitudinal gradients, providing little evidence for these factors explaining variation in biosynthesized toxicity in natural populations. In contrast, there were striking differences in the chemical profiles of H. erato from geographically distant populations, implying potential local adaptation in the acquisition mechanisms and levels of defensive compounds. The results highlight the extensive variation and potential for adaptive evolution in defense traits for aposematic and mimetic species, which may contribute to the high diversity often found in these systems.

The dynamics of cyanide defences in the life cycle of an aposematic butterfly: Biosynthesis versus sequestration.

Heliconius butterflies are highly specialized in Passiflora plants, laying eggs and feeding as larvae only on them. Interestingly, both Heliconius butterflies and Passiflora plants contain cyanogenic glucosides (CNglcs). While feeding on specific Passiflora species, Heliconius melpomene larvae are able to sequester simple cyclopentenyl CNglcs, the most common CNglcs in this plant genus. Yet, aromatic, aliphatic, and modified CNglcs have been reported in Passiflora species and they were never tested for sequestration by heliconiine larvae. As other cyanogenic lepidopterans, H. melpomene also biosynthesize the aliphatic CNglcs linamarin and lotaustralin, and their toxicity does not rely exclusively on sequestration. Although the genes encoding the enzymes in the CNglc biosynthesis have not yet been biochemically characterized in butterflies, the cytochromes P450 CYP405A4, CYP405A5, CYP405A6 and CYP332A1 have been hypothesized to be involved in this pathway in H. melpomene. In this study, we determine how the CNglc composition and expression of the putative P450s involved in the biosynthesis of these compounds vary at different developmental stages of Heliconius butterflies. We also establish which kind of CNglcs H. melpomene larvae can sequester from Passiflora. By analysing the chemical composition of the haemolymph from larvae fed with different Passiflora diets, we show that H. melpomene is able to sequestered prunasin, an aromatic CNglcs, from P. platyloba. They are also able to sequester amygdalin, gynocardin, [C13/C14]linamarin and [C13/C14]lotaustralin painted on the plant leaves. The CNglc tetraphyllin B-sulphate from P. caerulea is not detected in the larval haemolymph, suggesting that such modified CNglcs cannot be sequestered by Heliconius. Although pupae and virgin adults contain dihydrogynocardin resulting from larval sequestration, this compound was metabolized during adulthood, and not used as nuptial gift or transferred to the offspring. Thus, we speculate that dihydrogynocardin is catabolized to recycle nitrogen and glucose, and/or to produce fitness signals during courtship. Mature adults have a higher concentration of CNglcs than any other developmental stages due to increased de novo biosynthesis of linamarin and lotaustralin. Accordingly, all CYP405As are expressed in adults, whereas larvae mostly express CYP405A4. Our results shed light on the importance of CNglcs for Heliconius biology and their coevolution with Passiflora.

Sequestration and biosynthesis of cyanogenic glucosides in passion vine butterflies and consequences for the diversification of their host plants.

The colorful heliconiine butterflies are distasteful to predators due to their content of defense compounds called cyanogenic glucosides (CNglcs), which they biosynthesize from aliphatic amino acids. Heliconiine larvae feed exclusively on Passiflora plants where ~30 kinds of CNglcs have been reported. Among them, some CNglcs derived from cyclopentenyl glycine can be sequestered by some Heliconius species. In order to understand the evolution of biosynthesis and sequestration of CNglcs in these butterflies and its consequences for their arms race with Passiflora plants, we analyzed the CNglc distribution in selected heliconiine and Passiflora species. Sequestration of cyclopentenyl CNglcs is not an exclusive trait of Heliconius, since these compounds were present in other heliconiines such as Philaethria, Dryas and Agraulis, and in more distantly related genera Cethosia and Euptoieta. Thus, it is likely that the ability to sequester cyclopentenyl CNglcs arose in an ancestor of the Heliconiinae subfamily. Biosynthesis of aliphatic CNglcs is widespread in these butterflies, although some species from the sara-sapho group seem to have lost this ability. The CNglc distribution within Passiflora suggests that they might have diversified their cyanogenic profile to escape heliconiine herbivory. This systematic analysis improves our understanding on the evolution of cyanogenesis in the heliconiine-Passiflora system.