Mimicry among Heliconius butterflies provides a classic example of coevolution but unresolved relationships among mimetic subspecies have prevented examination of codiversification between species. We present amplified fragment length polymorphism and mtDNA datasets for the major comimetic races of Heliconius erato and H. melpomene. The AFLP data reveal unprecedented resolution, clustering samples by geography and race in both species. Our results show that, although H. erato and H. melpomene co-occur, mimic each other, and exhibit parallel shifts in color pattern, they experienced very different modes of diversification and geographic histories. Our results suggest that H. erato originated on the western side of South America whereas H. melpomene originated in the east. H. erato underwent rapid diversification and expansion with continued gene-flow following diversification, resulting in widely dispersed sister taxa. In contrast, H. melpomene underwent a slower pace of diversification with lower levels of gene flow, producing a stepwise directional expansion from west to east. Our results also suggest that each of the three main wing pattern phenotypes originated and/or was lost multiple times in each species. The rayed pattern is likely to be the ancestral phenotype in H. erato whereas postman or red patch is likely to be ancestral in H. melpomene. Finally, H. cydno and H. himera are monophyletic entities clearly nested within H. melpomene and H. erato, rather than being their respective sister species. Estimates of mtDNA divergence suggest a minimum age of 2.8 and 2.1 My for H. erato and H. melpomene, respectively, placing their origins in the late Pliocene.

Heliconiines are called passion vine butterflies because they feed exclusively on Passiflora plants during the larval stage. Many features of Passiflora and heliconiines indicate that they have radiated and speciated in association with each other, and therefore this model system was one of the first examples used to exemplify coevolution theory. Three major adaptations of Passiflora plants supported arguments in favour of their coevolution with heliconiines: unusual variation of leaf shape within the genus; the occurrence of yellow structures mimicking heliconiine eggs; and their extensive diversity of defence compounds called cyanogenic glucosides. However, the protection systems of Passiflora plants go beyond these three features. Trichomes, mimicry of pathogen infection through variegation, and production of extrafloral nectar to attract ants and other predators of their herbivores, are morphological defences reported in this plant genus. Moreover, Passiflora plants are well protected chemically, not only by cyanogenic glucosides, but also by other compounds such as alkaloids, flavonoids, saponins, tannins and phenolics. Heliconiines can synthesize cyanogenic glucosides themselves, and their ability to handle these compounds was probably one of the most crucial adaptations that allowed the ancestor of these butterflies to feed on Passiflora plants. Indeed, it has been shown that Heliconius larvae can sequester cyanogenic glucosides and alkaloids from their host plants and utilize them for their own benefit. Recently, it was discovered that Heliconius adults have highly accurate visual and chemosensory systems, and the expansion of brain structures that can process such information allows them to memorize shapes and display elaborate pre-oviposition behaviour in order to defeat visual barriers evolved by Passiflora species. Even though the heliconiine-Passiflora model system has been intensively studied, the forces driving host-plant preference in these butterflies remain unclear. New studies have shown that host-plant preference seems to be genetically controlled, but in many species there is some plasticity in this choice and preferences can even be induced. Although much knowledge regarding the coevolution of Passiflora plants and heliconiine butterflies has accumulated in recent decades, there remain many exciting unanswered questions concerning this model system.

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.

Neotropical Entomology 37(3): 247-252 (2008) Interação Herbívoro-Tricoma: o Caso de Heliconius charithonia (L.) (Lepidoptera: Nymphalidae) e Passifl ora lobata (Killip) Hutch. (Passifl oraceae) RESUMO -Apesar de as evidências mostrarem que herbívoros são negativamente afetados pelos tricomas, há também relatos de contra-adaptações que sobrepujam as defesas das plantas. Este estudo busca os prováveis mecanismos usados pelas larvas da borboleta ninfalídea Heliconius charithonia (L.) que permitem que elas se alimentem de uma planta hospedeira que é, presumivelmente, protegida por tricomas uncinados (curvados) (Passifl ora lobata (Killip) Hutch.). Para isso realizou-se observação direta de movimento e comportamento da larva, análise de fezes, microscopia eletrônica de varredura da superfície foliar e análise experimental do movimento de larvas em plantas com e sem tricomas (removidos manualmente). O experimento foi feito comparando o comportamento dessas larvas com o de larvas de um não-especialista, Heliconius pachinus Salvin. As larvas de H. charithonia são capazes de se desvencilhar do aprisionamento pelos tricomas usando força física. Além disso, ao movimentar-se, a larva espalha fi os de seda sobre os tricomas e retira suas pontas com as mandíbulas. De fato, pontas de tricoma foram encontradas nas fezes das larvas. A remoção experimental dos tricomas auxiliou o movimento da larva não-especialista, mas não teve efeitos notáveis sobre a larva especialista. Os resultados confi rmam que os tricomas são capazes de deter um herbívoro não especializado (H. pachinus). Os exatos mecanismos responsáveis pelo sucesso de H. charithonia ainda são desconhecidos, mas sugere-se que a combinação de mecanismos comportamentais e de resistência física estejam envolvidos e estudos futuros necessitam verifi car a possibilidade de resistência física no tegumento das larvas. PALAVRAS-CHAVE: Herbivoria, defesa mecânica, interação inseto-plantaABSTRACT -Trichomes reduce herbivore attack on plants by physically and/or chemically inhibiting movement or other activities. Despite evidence that herbivores are negatively affected by trichomes there also reports of insect counter-adaptations that circumvent the plant's defense. This paper reports on a study that investigated the likely mechanisms employed by larvae of the nymphalid butterfl y, Heliconius charithonia (L.), that allow it to feed on a host that is presumably protected by hooked trichomes (Passifl ora lobata (Killip) Hutch). Evidence were gathered using data from direct observations of larval movement and behavior, faeces analysis, scanning electron microscopy of plant surface and experimental analysis of larval movement on plants with and without trichomes (manually removed). The latter involved a comparison with a non specialist congener, Heliconius pachinus Salvin. Observations showed that H. charithonia larvae are capable of freeing themselves from entrapment on trichome tips by physical force. Moreover, wandering larvae lay silk mats on the trichomes and remove their tips ...

Evolution of pollen feeding in Heliconius has allowed exploitation of rich amino acid sources and dramatically reorganized life-history traits. In Heliconius, eggs are produced mainly from adult-acquired resources, leaving somatic development and maintenance to larva effort. This innovation may also have spurred evolution of chemical defence via amino acid-derived cyanogenic glycosides. In contrast, nonpollen-feeding heliconiines must rely almost exclusively on larval-acquired resources for both reproduction and defence. We tested whether adult amino acid intake has an immediate influence on cyanogenesis in Heliconius. Because Heliconius are more distasteful to bird predators than close relatives that do not utilize pollen, we also compared cyanogenesis due to larval input across Heliconius species and nonpollen-feeding relatives. Except for one species, we found that varying the amino acid diet of an adult Heliconius has negligible effect on its cyanide concentration. Adults denied amino acids showed no decrease in cyanide and no adults showed cyanide increase when fed amino acids. Yet, pollenfeeding butterflies were capable of producing more defence than nonpollenfeeding relatives and differences were detectable in freshly emerged adults, before input of adult resources. Our data points to a larger role of larval input in adult chemical defence. This coupled with the compartmentalization of adult nutrition to reproduction and longevity suggests that one evolutionary consequence of pollen feeding, shifting the burden of reproduction to adults, is to allow the evolution of greater allocation of host plant amino acids to defensive compounds by larvae.