Balanced polymorphisms and their divergence in a Heliconius butterfly

The evolution of mimicry in similarly defended prey is well described by the Müllerian mimicry theory, which predicts the convergence of warning patterns in order to gain the most protection from predators. However, despite this prediction, we can find great diversity of color patterns among Müllerian mimics such as Heliconius butterflies in the neotropics. Furthermore, some species have evolved the ability to maintain multiple distinct warning patterns in single populations, a phenomenon known as polymorphic mimicry. The adaptive benefit of these polymorphisms is questionable since variation from the most common warning patterns is expected to be disadvantageous as novel signals are punished by predators naive to them. In this study, we use artificial butterfly models throughout Central and South America to characterize the selective pressures maintaining polymorphic mimicry in Heliconius doris. Our results highlight the complexity of positive frequency‐dependent selection, the principal selective pressure driving convergence among Müllerian mimics, and its impacts on interspecific variation of mimetic warning coloration. We further show how this selection regime can both limit and facilitate the diversification of mimetic traits.     

The population genetics of mimetic diversity in Heliconius butterflies

Theory predicts strong stabilizing selection on warning patterns within species and convergent evolution among species in Müllerian mimicry systems yet Heliconius butterflies exhibit extreme wing pattern diversity. One potential explanation for the evolution of this diversity is that genetic drift occasionally allows novel warning patterns to reach the frequency threshold at which they gain protection. This idea is controversial, however, because Heliconius butterflies are unlikely to experience pronounced population subdivision and local genetic drift. To examine the fine-scale population genetic structure of Heliconius butterflies we genotyped 316 individuals from eight Costa Rican Heliconius species with 1428 AFLP markers. Six species exhibited evidence of population subdivision and/or isolation by distance indicating genetic differentiation among populations. Across species, variation in the extent of local genetic drift correlated with the roles different species have played in generating pattern diversity: species that originally generated the diversity of warning patterns exhibited striking population subdivision while species that later radiated onto these patterns had intermediate levels of genetic diversity and less genetic differentiation among populations. These data reveal that Heliconius butterflies possess the coarse population genetic structure necessary for local populations to experience pronounced genetic drift which, in turn, could explain the origin of mimetic diversity.     

Condition dependence in biosynthesized chemical defenses of an aposematic and mimetic Heliconius butterfly

Aposematic animals advertise their toxicity or unpalatability with bright warning coloration. However, acquiring and maintaining chemical defenses can be energetically costly, and consequent associations with other important traits could shape chemical defense evolution. Here, we have tested whether chemical defenses are involved in energetic trade‐offs with other traits, or whether the levels of chemical defenses are condition dependent, by studying associations between biosynthesized cyanogenic toxicity and a suite of key life‐history and fitness traits in a Heliconius butterfly under a controlled laboratory setting. Heliconius butterflies are well known for the diversity of their warning color patterns and widespread mimicry and can both sequester the cyanogenic glucosides of their Passiflora host plants and biosynthesize these toxins de novo. We find energetically costly life‐history traits to be either unassociated or to show a general positive association with biosynthesized cyanogenic toxicity. More toxic individuals developed faster and had higher mass as adults and a tendency for increased lifespan and fecundity. These results thus indicate that toxicity level of adult butterflies may be dependent on individual condition, influenced by genetic background or earlier conditions, with maternal effects as one strong candidate mechanism. Additionally, toxicity was higher in older individuals, consistent with previous studies indicating accumulation of toxins with age. As toxicity level at death was independent of lifespan, cyanogenic glucoside compounds may have been recycled to release resources relevant for longevity in these long‐living butterflies. Understanding the origins and maintenance of variation in defenses is necessary in building a more complete picture of factors shaping the evolution of aposematic and mimetic systems.     

Variation of chemical compounds in wild Heliconiini reveals ecological factors involved in the evolution of chemical defenses in mimetic butterflies

Evolutionary convergence of color pattern in mimetic species is tightly linked with the evolution of chemical defenses. Yet, the evolutionary forces involved in natural variations of chemical defenses in aposematic species are still understudied. Herein, we focus on the evolution of chemical defenses in the butterfly tribe Heliconiini. These neotropical butterflies contain large concentrations of cyanogenic glucosides, cyanide‐releasing compounds acting as predator deterrent. These compounds are either de novo synthesized or sequestered from their Passiflora host plant, so that their concentrations may depend on host plant specialization and host plant availability. We sampled 375 wild Heliconiini butterflies across Central and South America, covering 43% species of this clade, and quantify individual variations in the different CGs using liquid chromatography coupled with tandem mass spectrometry. We detected new compounds and important variations in chemical defenses both within and among species. Based on the most recent and well‐studied phylogeny of Heliconiini, we show that ecological factors such as mimetic interactions and host plant specialization have a significant association with chemical profiles, but these effects are largely explained by phylogenetic relationships. Our results therefore suggest that shared ancestries largely contribute to chemical defense variation, pointing out at the interaction between historical and ecological factors in the evolution of Müllerian mimicry.     

A Narrow Heliconius cydno (Nymphalidae; Heliconiini) Hybrid Zone With Differences in Morph Sex Ratios1

In the western cordillera of the Cauca valley, Colombia, there is a narrow hybrid zone between two races of Heliconius cydno, one of which is polymorphic. Three large broods show that most of the phenotypic variation observed can be explained by four loci of major effect, named Sb, Yb, L, and K. Two further loci, G and Wo, were identified that control more minor phenotypic variation. Sb, Yb, and Wo are linked and the latter is differentially expressed between the sexes. The transition between H. c. cydnides in the north and H. c. weymeri to the south occurs over approximately 15 km. Collections from a single site near the center of the hybrid zone show that gene frequencies have been stable over the 10 yr from 1991 to 2001 and that color‐pattern genes are in Hardy–Weinberg equilibrium, with little evidence for linkage disequilibrium. This suggests that mating is random between color‐pattern forms. Nonetheless, there is evidence for variation in the sex ratio, with parental phenotypes and the most melanic hybrid phenotypes showing a fairly even sex ratio compared to a strong male bias observed in the remaining hybrids. We hypothesize that this might be explained by differential selection between sexes and phenotypes, phenomena that could have important implications for hybrid zone analysis and the evolution of Müllerian mimicry.     

Variation in wing pattern and palatability in a female‐limited polymorphic mimicry system

Checkerspot butterflies in the genera Euphydryas and Chlosyne exhibit phenotypic polymorphisms along a well‐defined latitudinal and elevational gradient in California. The patterns of phenotypic variation in Euphydryas chalcedona, Chlosyne palla, and Chlosyne hoffmanni suggest a mimetic relationship; in addition, the specific patterns of variation in C. palla suggest a female‐limited polymorphic mimicry system (FPM). However, the existence of polymorphic models runs counter to predictions of mimicry theory. Palatability trials were undertaken to assess whether or not the different color morphs of each species were distasteful or toxic to a generalized avian predator, the European starling (Sturnus vulgaris). Results indicate that the black morph of E. chalcedona is distasteful, but not toxic, to predators, while the red morph is palatable. C. hoffmanni and both color morphs of C. palla are palatable to predators. Predators that learn to reject black E. chalcedona also reject black C. palla, suggesting that the latter is a FPM of the former. C. hoffmanni does not appear to be involved in this mimetic relationship.     

Ghost mtDNA haplotypes generated by fortuitous NUMTs can deeply disturb infra‐specific genetic diversity and phylogeographic pattern

Nuclear copies of mitochondrial DNA (NUMTs or mitochondrial pseudogenes) are known to impede the detection of interspecific genetic diversity. But the effect of these artifacts on phylogeographic reconstruction remains under evaluated. In this study, we analysed a set of 115 sequences of a fragment of the cytochrome c oxidase subunit I gene (COI) of Monochamus galloprovincialis (Coleoptera, Cerambycidae) for which overlapping signals in sequencing electropherograms were observed. Comparison of full and corrected ‘ambiguities‐free’ data sets reveals the prevalence of numerous supernumerary haplotypes that deeply affect genetic diversity indices and phylogeographic patterns of this species. Slightly divergent pseudogenes were recovered in 49 of the 115 sequences. These results highlight the potential misdetection of NUMTs using current control methods and the consequences on phylogeographic structure. To test the frequency of unintended amplification of NUMTs, a cloning was performed on 15 individuals. An average of 3.72 and a maximum of six paralogous sequences with different levels of divergence were identified among individual cloned. Within individual pairwise distance between paralogs raised 1.4%. This work calls for awareness to the presence of undetected NUMTs within mitochondrial data sets, especially at infra‐specific level.     

The ecology and social behaviour of the Neotropical butterfly Heliconius xanthocles Bates in Colombia

The mimicry, population ecology¹, adult behaviour and life cycle of Heliconius xanthocles were studied at two sites in Colombian tropical forest. The results were compared with the known biology of other Heliconius species. H. xanthocles is probably unpalatable and is a Mullerian comimic of some other species. In the Amazon basin H. xanthocles belongs to the 'dennis ray' mimicry ring of the Heliconiini, which includes Eueides tales, H. bumeyi, H. elevatus, H. melpomene and H. erato. In the Rio Negro valley, where the latter species either do not exist or have different mimetic allegiances, the 'clennis-ray' pattern of H. xanthocles breaks down. The adults are pollen-feeders, like other Heliconius. but they also visit other food sources such as bird droppings. During a mark-recapture programme in the Rio Negro, adults were found to be almost entirely restricted to small areas near their foodplant Passiflorapraeacuta. Females were rarer than males, but this was probably due to a behavioural difference between the sexes. The males had a Fisher-Ford survival rate of 0.95 per day and a life expectancy of 19 days. Population size estimates of males were between 11 and 17 individuals. These estimates are lower than, but comparable to those for other Heliconius. Males as well as females visit the larval toodplant, and an adult male was observed hovering over a small group of larvae. The females lav batches of 12–41 eggs. The largest batch (41 eggs) was laid cooperatively by two females. The larvae are gregarious feeders on shoot-tips of Passiflora praeacuta. and all lile stages are described for the first time. Predation on the larvae by a bug is described. The pupa has a distinctive morphology unlike that of other Heliconius. The adult population ecology, mimicry and larval behaviour are discussed.     

Cryptic diversity,reproductive isolation and cytoplasmic incompatibility in a classic biological control success story

Molecular genetics and symbiont diagnostics have revolutionized our understanding of insect species diversity, and the transformative effects of bacterial symbionts on host life history. Encarsia inaron is a parasitoid wasp that has been shown to harbour two bacterial endosymbionts, Wolbachia and Cardinium. Known then as E. partenopea, it was introduced to the USA in the late 1980s from populations collected in Italy and Israel for the biological control of an ornamental tree pest, the ash whitefly, Siphoninus phillyreae. We studied natural populations from sites in the USA, the Mediterranean and the Middle East as well as from a Cardinium‐infected laboratory culture established from Italy, with the aims of characterizing these populations genetically, testing reproductive isolation, determining symbiont infection status in their native and introduced range, and determining symbiont role. The results showed that the two Encarsia populations introduced to the USA are genetically distinct, reproductively isolated, have different symbionts and different host–symbiont interactions, and can be considered different biological species. One (‘E. inaron’) is doubly infected by Wolbachia and Cardinium, while only Cardinium is present in the other (‘E. partenopea’). The Cardinium strains in the two species are distinct, although closely related, and crossing tests indicate that the Cardinium infecting ‘E. partenopea’ induces cytoplasmic incompatibility. The frequency of symbiont infection found in the native and introduced range of these wasps was similar, unlike the pattern seen in some other systems. These results also lead to a retelling of a successful biological control story, with several more characters than had been initially described.     

Variable Selection and the Coexistence of Multiple mimetic forms of the Butterfly Heliconius numata

Polymorphism in aposematic animals and coexistence of multiple mimicry rings within a habitat are not predicted by classical Müllerian mimicry. The butterfly Heliconius numata Cramer (Lepidoptera: Nymphalidae; Heliconiinae) is both polymorphic and aposematic. The polymorphism is due to variation at a single locus (or `supergene') which determines colour patterns involved in Müllerian mimicry. We sampled 11 sites in a small area (approx. 60×30km) of North-eastern Peru for H. numata and its co-mimics in the genus Melinaea and Athyrtis (Ithomiinae), and examined the role of temporal and spatial heterogeneity in the maintenance of polymorphism. Colour-patterns of Melinaea communities, which constitute the likely `mimetic environment' for H. numata, are differentiated on a more local scale than morphs of H. numata, but the latter do show a strong and significant response to local selection for colour-pattern. In contrast, analysis of enzyme polymorphism in H. numata across the region revealed no spatial structure, which is consistent with a high mobility of this species. Differences in spatial variability in the two taxa may have caused H. numata to become polymorphic, while temporal variability, not significant in this study, probably has a lesser effect. The mimetic polymorphism is therefore explained by means of multiple selection-migration clines at a single locus, a similar process to that which explains narrow hybrid zones between geographic races of other Heliconius butterflies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Defining the colour pattern phenotype in bumble bees (Bombus): a new model for evo devo

Few insects exhibit the striking colour pattern radiation found in bumble bees (Bombus), which have diversified globally into a wide range of colours and patterns. Their potent sting is often advertised by conspicuous bands of contrasting colour commonly mimicked by scores of harmless (Batesian mimics) and noxious species (Müllerian co‐mimics). Despite extensive documentation of colour pattern diversification, next to nothing is known about the genetic regulation of pattern formation in bumble bees, hindering progress toward a more general model of the evolution of colour pattern mimicry. A critical first step in understanding the colour pattern genotype is an unambiguous understanding of the phenotype under selection, which has not been objectively defined in bumble bees. Here, we quantitatively define the principal colour pattern elements that comprise the phenotype array across all species. Matrix analysis of meticulously scored colour patterns of ～95% of described species indicates there are 12 discrete primary ‘ground plan’ elements in common among all species, many of which correspond to segmentation patterning. Additional secondary elements characterize individual species and geographical variants. The boundaries of these elements appear to correspond to expression patterns of Hox genes in Drosophila and Apis but also suggest novel post‐Hox specialization of abdominal patterning. Our findings provide the first foundation for exploring candidate genes regulating adaptive pattern variation in bumble bees and broaden the framework for understanding common genetic mechanisms of pattern evolution in insects. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 384–404.     

Cryptic habitats and cryptic diversity: unexpected patterns of connectivity and phylogeographical breaks in a Mediterranean endemic marine cave mysid

The marine cave‐dwelling mysid Hemimysis margalefi is distributed over the whole Mediterranean Sea, which contrasts with the poor dispersal capabilities of this brooding species. In addition, underwater marine caves are a highly fragmented habitat which further promotes strong genetic structuring, therefore providing highly informative data on the levels of marine population connectivity across biogeographical regions. This study investigates how habitat and geography have shaped the connectivity network of this poor disperser over the entire Mediterranean Sea through the use of several mitochondrial and nuclear markers. Five deeply divergent lineages were observed among H. margalefi populations resulting from deep phylogeographical breaks, some dating back to the Oligo‐Miocene. Whether looking at the intralineage or interlineage levels, H. margalefi populations present a high genetic diversity and population structuring. This study suggests that the five distinct lineages observed in H. margalefi actually correspond to as many separate cryptic taxa. The nominal species, H. margalefi sensu stricto, corresponds to the westernmost lineage here surveyed from the Alboran Sea to southeastern Italy. Typical genetic breaks such as the Almeria‐Oran Front or the Siculo‐Tunisian Strait do not appear to be influential on the studied loci in H. margalefi sensu stricto. Instead, population structuring appears more complex and subtle than usually found for model species with a pelagic dispersal phase. The remaining four cryptic taxa are all found in the eastern basin, but incomplete lineage sorting is suspected and speciation might still be in process. Present‐day population structure of the different H. margalefi cryptic species appears to result from past vicariance events started in the Oligo‐Miocene and maintained by present‐day coastal topography, water circulation and habitat fragmentation.     

Geographic variation in mimetic precision among different species of coral snake mimics

Batesian mimicry is widespread, but whether and why different species of mimics vary geographically in resemblance to their model is unclear. We characterized geographic variation in mimetic precision among four Batesian mimics of coral snakes. Each mimic occurs where its model is abundant (i.e. in ‘deep sympatry’), rare (i.e. at the sympatry/allopatry boundary or ‘edge sympatry’) and absent (i.e. in allopatry). Geographic variation in mimetic precision was qualitatively different among these mimics. In one mimic, the most precise individuals occurred in edge sympatry; in another, they occurred in deep sympatry; in the third, they occurred in allopatry; and in the fourth, precise mimics were not concentrated anywhere throughout their range. Mimicry was less precise in allopatry than in sympatry in only two mimics. We present several nonmutually exclusive hypotheses for these patterns. Generally, examining geographic variation in mimetic precision – within and among different mimics – offers novel insights into the causes and consequences of mimicry.     

Hybridization and transgressive exploration of colour pattern and wing morphology in Heliconius butterflies

Hybridization can generate novel phenotypes distinct from those of parental lineages, a phenomenon known as transgressive trait variation. Transgressive phenotypes might negatively or positively affect hybrid fitness, and increase available variation. Closely related species of Heliconius butterflies regularly produce hybrids in nature, and hybridization is thought to play a role in the diversification of novel wing colour patterns despite strong stabilizing selection due to interspecific mimicry. Here, we studied wing phenotypes in first‐ and second‐generation hybrids produced by controlled crosses between either two co‐mimetic species of Heliconius or between two nonmimetic species. We quantified wing size, shape and colour pattern variation and asked whether hybrids displayed transgressive wing phenotypes. Discrete traits underlain by major‐effect loci, such as the presence or absence of colour patches, generate novel phenotypes. For quantitative traits, such as wing shape or subtle colour pattern characters, hybrids only exceed the parental range in specific dimensions of the morphological space. Overall, our study addresses some of the challenges in defining and measuring phenotypic transgression for multivariate traits and our data suggest that the extent to which transgressive trait variation in hybrids contributes to phenotypic diversity depends on the complexity and the genetic architecture of the traits.     

Phylogeography of Heliconius cydno and its closest relatives: disentangling their origin and diversification

The origins of the extraordinary diversity within the Neotropics have long fascinated biologists and naturalists. Yet, the underlying factors that have given rise to this diversity remain controversial. To test the relative importance of Quaternary climatic change and Neogene tectonic and paleogeographic reorganizations in the generation of biodiversity, we examine intraspecific variation across the Heliconius cydno radiation and compare this variation to that within the closely related Heliconius melpomene and Heliconius timareta radiations. Our data, which consist of both mtDNA and genome‐scan data from nearly 2250 amplified fragment length polymorphism (AFLP) loci, reveal a complex history of differentiation and admixture at different geographic scales. Both mtDNA and AFLP phylogenies suggest that H. timareta and H. cydno are probably geographic extremes of the same radiation that probably diverged from H. melpomene prior to the Pliocene–Pleistocene boundary, consistent with hypotheses of diversification that rely on geological events in the Pliocene. The mtDNA suggests that this radiation originated in Central America or the northwestern region of South America, with a subsequent colonization of the eastern and western slopes of the Andes. Our genome‐scan data indicate significant admixture among sympatric H. cydno/H. timareta and H. melpomene populations across the extensive geographic ranges of the two radiations. Within H. cydno, both mtDNA and AFLP data indicate significant population structure at local scales, with strong genetic differences even among adjacent H. cydno colour pattern races. These genetic patterns highlight the importance of past geoclimatic events, intraspecific gene flow, and local population differentiation in the origin and establishment of new adaptive forms.     

Interplay between Developmental Flexibility and Determinism in the Evolution of Mimetic Heliconius Wing Patterns

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Sharp genetic discontinuity across a unimodal Heliconius hybrid zone

Hybrid zones are powerful natural systems to study evolutionary processes to gain an understanding of adaptation and speciation. In the Cauca Valley (Colombia), two butterfly races, Heliconius cydno cydnides and Heliconius cydno weymeri, meet and hybridize. We characterized this hybrid zone using a combination of mitochondrial DNA (mtDNA) sequences, amplified fragment length polymorphisms (AFLPs), microsatellites and sequences for nuclear loci within and outside of the genomic regions that cause differences in wing colour pattern. The hybrid zone is largely composed of individuals of mixed ancestry. However, there is strong genetic discontinuity between the hybridizing races in mtDNA and, to a lesser extent, in all nuclear markers surveyed. The mtDNA clustering of H. c. cydnides with the H. cydno race from the Magdalena Valley and H. c. weymeri with the H. cydno race from the pacific coast suggests that H. c. cydnides colonized the Cauca Valley from the north, whereas H. c. weymeri did so by crossing the Andes in the southern part, implying a secondary contact origin. Colonization of the valley by H. cydno was accompanied by mimicry shift. Strong ecological isolation, driven by locally adaptive differences in mimetic wing patterns, is playing an important role in maintaining the hybrid zone. However, selection on wing pattern alone is not sufficient to explain the genetic discontinuity observed. There is evidence for differences in male mating preference, but the contribution of additional barriers needs further investigation. Overall, our results support the idea that speciation is a cumulative process, where the combination of multiple isolation barriers, combined with major phenotypic differences, facilitates population divergence in face of gene flow.     

Paleoclimatic evolution as the main driver of current genomic diversity in the widespread and polymorphic Neotropical songbird Arremon taciturnus

Several factors have been proposed as drivers of species diversification in the Neotropics, including environmental heterogeneity, the development of drainage systems and historical changes in forest distribution due to climatic oscillations. Here, we investigate which drivers contributed to the evolutionary history and current patterns of diversity of a polymorphic songbird (Arremon taciturnus) that is widely distributed in Amazonian and Atlantic forests as well as in Cerrado gallery and seasonally‐dry forests. We use genomic, phenotypic and habitat heterogeneity data coupled with climatic niche modelling. Results suggest the evolutionary history of the species is mainly related to paleoclimatic changes, although changes in the strength of the Amazon river as a barrier to dispersal, current habitat heterogeneity and geographic distance were also relevant. We propose an ancestral distribution in the Guyana Shield, and recent colonization of areas south of the Amazon river at ~380 to 166 kya, and expansion of the distribution to southern Amazonia, Cerrado and the Atlantic Forest. Since then, populations south of the Amazon River have been subjected to cycles of isolation and possibly secondary contact due to climatic changes that affected habitat heterogeneity and population connectivity. Most Amazonian rivers are not associated with long lasting isolation of populations, but some might act as secondary barriers, susceptible to crossing under specific climatic conditions. Morphological variation, while stable in some parts of the distribution, is not a reliable indicator of genetic structure or phylogenetic relationships.