The evolution and ecology of multiple antipredator defences.

Prey seldom rely on a single type of antipredator defence, often using multiple defences to avoid predation. In many cases, selection in different contexts may favour the evolution of multiple defences in a prey. However, a prey may use multiple defences to protect itself during a single predator encounter. Such "defence portfolios" that defend prey against a single instance of predation are distributed across and within successive stages of the predation sequence (encounter, detection, identification, approach (attack), subjugation and consumption). We contend that at present, our understanding of defence portfolio evolution is incomplete, and seen from the fragmentary perspective of specific sensory systems (e.g., visual) or specific types of defences (especially aposematism). In this review, we aim to build a comprehensive framework for conceptualizing the evolution of multiple prey defences, beginning with hypotheses for the evolution of multiple defences in general, and defence portfolios in particular. We then examine idealized models of resource trade-offs and functional interactions between traits, along with evidence supporting them. We find that defence portfolios are constrained by resource allocation to other aspects of life history, as well as functional incompatibilities between different defences. We also find that selection is likely to favour combinations of defences that have synergistic effects on predator behaviour and prey survival. Next, we examine specific aspects of prey ecology, genetics and development, and predator cognition that modify the predictions of current hypotheses or introduce competing hypotheses. We outline schema for gathering data on the distribution of prey defences across species and geography, determining how multiple defences are produced, and testing the proximate mechanisms by which multiple prey defences impact predator behaviour. Adopting these approaches will strengthen our understanding of multiple defensive strategies.

First chromosome scale genomes of ithomiine butterflies (Nymphalidae: Ithomiini): Comparative models for mimicry genetic studies.

The ithomiine butterflies (Nymphalidae: Danainae) represent the largest known radiation of Müllerian mimetic butterflies. They dominate by number the mimetic butterfly communities, which include species such as the iconic neotropical Heliconius genus. Recent studies on the ecology and genetics of speciation in Ithomiini have suggested that sexual pheromones, colour pattern and perhaps hostplant could drive reproductive isolation. However, no reference genome was available for Ithomiini, which has hindered further exploration on the genetic architecture of these candidate traits, and more generally on the genomic patterns of divergence. Here, we generated high-quality, chromosome-scale genome assemblies for two Melinaea species, M. marsaeus and M. menophilus, and a draft genome of the species Ithomia salapia. We obtained genomes with a size ranging from 396 to 503 Mb across the three species and scaffold N50 of 40.5 and 23.2 Mb for the two chromosome-scale assemblies. Using collinearity analyses we identified massive rearrangements between the two closely related Melinaea species. An annotation of transposable elements and gene content was performed, as well as a specialist annotation to target chemosensory genes, which is crucial for host plant detection and mate recognition in mimetic species. A comparative genomic approach revealed independent gene expansions in ithomiines and particularly in gustatory receptor genes. These first three genomes of ithomiine mimetic butterflies constitute a valuable addition and a welcome comparison to existing biological models such as Heliconius, and will enable further understanding of the mechanisms of adaptation in butterflies.

Improved Basic Cytogenetics Challenges Holocentricity of Butterfly Chromosomes.

Mitotic chromosomes of butterflies, which look like dots or short filaments in most published data, are generally considered to lack localised centromeres and thus to be holokinetic. This particularity, observed in a number of other invertebrates, is associated with meiotic particularities known as "inverted meiosis," in which the first division is equational, i.e., centromere splitting-up and segregation of sister chromatids instead of homologous chromosomes. However, the accurate analysis of butterfly chromosomes is difficult because (1) their size is very small, equivalent to 2 bands of a mammalian metaphase chromosome, and (2) they lack satellite DNA/heterochromatin in putative centromere regions and therefore marked primary constrictions. Our improved conditions for basic chromosome preparations, here applied to 6 butterfly species belonging to families Nymphalidae and Pieridae challenges the holocentricity of their chromosomes: in spite of the absence of primary constrictions, sister chromatids are recurrently held together at definite positions during mitotic metaphase, which makes possible to establish karyotypes composed of acrocentric and submetacentric chromosomes. The total number of chromosomes per karyotype is roughly inversely proportional to that of non-acrocentric chromosomes, which suggests the occurrence of frequent robertsonian-like fusions or fissions during evolution. Furthermore, the behaviour and morphological changes of chromosomes along the various phases of meiosis do not seem to differ much from those of canonical meiosis. In particular, at metaphase II chromosomes clearly have 2 sister chromatids, which refutes that anaphase I was equational. Thus, we propose an alternative mechanism to holocentricity for explaining the large variations in chromosome numbers in butterflies: (1) in the ancestral karyotype, composed of about 62 mostly acrocentric chromosomes, the centromeres, devoid of centromeric heterochromatin/satellite DNA, were located at contact with telomeric heterochromatin; (2) the instability of telomeric heterochromatin largely contributed to drive the multiple rearrangements, principally chromosome fusions, which occurred during butterfly evolution.

Comparative transcriptome analysis at the onset of speciation in a mimetic butterfly-The Ithomiini Melinaea marsaeus.

Ecological speciation entails divergent selection on specific traits and ultimately on the developmental pathways responsible for these traits. Selection can act on gene sequences but also on regulatory regions responsible for gene expression. Mimetic butterflies are a relevant system for speciation studies because wing colour pattern (WCP) often diverges between closely related taxa and is thought to drive speciation through assortative mating and increased predation on hybrids. Here, we generate the first transcriptomic resources for a mimetic butterfly of the tribe Ithomiini, Melinaea marsaeus, to examine patterns of differential expression between two subspecies and between tissues that express traits that likely drive reproductive isolation; WCP and chemosensory genes. We sequenced whole transcriptomes of three life stages to cover a large catalogue of transcripts, and we investigated differential expression between subspecies in pupal wing discs and antennae. Eighteen known WCP genes were expressed in wing discs and 115 chemosensory genes were expressed in antennae, with a remarkable diversity of chemosensory protein genes. Many transcripts were differentially expressed between subspecies, including two WCP genes and one odorant receptor. Our results suggest that in M. marsaeus the same genes as in other mimetic butterflies are involved in traits causing reproductive isolation, and point at possible candidates for the differences in those traits between subspecies. Differential expression analyses of other developmental stages and body organs and functional studies are needed to confirm and expand these results. Our work provides key resources for comparative genomics in mimetic butterflies, and more generally in Lepidoptera.

The genomics of mimicry: Gene expression throughout development provides insights into convergent and divergent phenotypes in a Müllerian mimicry system.

A common goal in evolutionary biology is to discern the mechanisms that produce the astounding diversity of morphologies seen across the tree of life. Aposematic species, those with a conspicuous phenotype coupled with some form of defence, are excellent models to understand the link between vivid colour pattern variations, the natural selection shaping it, and the underlying genetic mechanisms underpinning this variation. Mimicry systems in which multiple species share the same conspicuous phenotype can provide an even better model for understanding the mechanisms of colour production in aposematic species, especially if comimics have divergent evolutionary histories. Here we investigate the genetic mechanisms by which vivid colour and pattern are produced in a Müllerian mimicry complex of poison frogs. We did this by first assembling a high-quality de novo genome assembly for the mimic poison frog Ranitomeya imitator. This assembled genome is 6.8 Gbp in size, with a contig N50 of 300 Kbp R. imitator and two colour morphs from both Ranitomeya fantastica and R. variabilis which R. imitator mimics. We identified a large number of pigmentation and patterning genes that are differentially expressed throughout development, many of them related to melanocyte development, melanin synthesis, iridophore development and guanine synthesis. Polytypic differences within species may be the result of differences in expression and/or timing of expression, whereas convergence for colour pattern between species does not appear to be due to the same changes in gene expression. In addition, we identify the pteridine synthesis pathway (including genes such as qdpr and xdh) as a key driver of the variation in colour between morphs of these species. Finally, we hypothesize that genes in the keratin family are important for producing different structural colours within these frogs.

Assessing the Role of Developmental and Environmental Factors in Chemical Defence Variation in Heliconiini Butterflies.

Chemical defences in animals are both incredibly widespread and highly diverse. Yet despite the important role they play in mediating interactions between predators and prey, extensive differences in the amounts and types of chemical compounds can exist between individuals, even within species and populations. Here we investigate the potential role of environment and development on the chemical defences of warningly coloured butterfly species from the tribe Heliconiini, which can both synthesize and sequester cyanogenic glycosides (CGs). We reared 5 Heliconiini species in captivity, each on a single species-specific host plant as larvae, and compared them to individuals collected in the wild to ascertain whether the variation in CG content observed in the field might be the result of differences in host plant availability. Three of these species were reared as larvae on the same host plant, Passiflora riparia, to further test how species, sex, and age affected the type and amount of different defensive CGs, and how they affected the ratio of synthesized to sequestered compounds. Then, focusing on the generalist species Heliconius numata, we specifically explored variation in chemical profiles as a result of the host plant consumed by caterpillars and their brood line, using rearing experiments carried out on two naturally co-occurring host plants with differing CG profiles. Our results show significant differences in both the amount of synthesized and sequestered compounds between butterflies reared in captivity and those collected in the field. We also found a significant effect of species and an effect of sex in some, but not all, species. We show that chemical defences in H. numata continue to increase throughout their life, likely because of continued biosynthesis, and we suggest that variation in the amount of synthesized CGs in this species does not appear to stem from larval host plants, although this warrants further study. Interestingly, we detected a significant effect of brood lines, consistent with heritability influencing CG concentrations in H. numata. Altogether, our results point to multiple factors resulting in chemical defence variation in Heliconiini butterflies and highlight the overlooked effect of synthesis capabilities, which may be genetically determined to some extent.

3-Acetoxy-fatty acid isoprenyl esters from androconia of the ithomiine butterfly Ithomia salapia.

Male ithomiine butterflies (Nymphalidae: Danainae) have hairpencils on the forewings (i.e., androconia) that disseminate semiochemicals during courtship. While most ithomiines are known to contain derivatives of pyrrolizidine alkaloids, dihydropyrrolizines, or γ-lactones in these androconia, here we report on a new class of fatty acid esters identified in two subspecies, Ithomia salapia aquinia and I. s. derasa. The major components were identified as isoprenyl (3-methyl-3-butenyl) (Z)-3-acetoxy-11-octadecenoate, isoprenyl (Z)-3-acetoxy-13-octadecenoate (12) and isoprenyl 3-acetoxyoctadecanoate (11) by GC/MS and GC/IR analyses, microderivatizations, and synthesis of representative compounds. The absolute configuration of 12 was determined to be R. The two subspecies differed not only in the composition of the ester bouquet, but also in the composition of more volatile androconial constituents. While some individuals of I. s. aquinia contained ithomiolide A (3), a pyrrolizidine alkaloid derived γ-lactone, I. s. derasa carried the sesquiterpene α-elemol (8) in the androconia. These differences might be important for the reproductive isolation of the two subspecies, in line with previously reported low gene exchange between the two species in regions where they co-occur. Furthermore, the occurrence of positional isomers of unsaturated fatty acid derivatives indicates activity of two different desaturases within these butterflies, Δ9 and Δ11, which has not been reported before in male Lepidoptera.

Contrasting genomic and phenotypic outcomes of hybridization between pairs of mimetic butterfly taxa across a suture zone.

Hybrid zones, whereby divergent lineages come into contact and eventually hybridize, can provide insights on the mechanisms involved in population differentiation and reproductive isolation, and ultimately speciation. Suture zones offer the opportunity to compare these processes across multiple species. In this paper we use reduced-complexity genomic data to compare the genetic and phenotypic structure and hybridization patterns of two mimetic butterfly species, Ithomia salapia and Oleria onega (Nymphalidae: Ithomiini), each consisting of a pair of lineages differentiated for their wing colour pattern and that come into contact in the Andean foothills of Peru. Despite similarities in their life history, we highlight major differences, both at the genomic and phenotypic level, between the two species. These differences include the presence of hybrids, variations in wing phenotype, and genomic patterns of introgression and differentiation. In I. salapia, the two lineages appear to hybridize only rarely, whereas in O. onega the hybrids are not only more common, but also genetically and phenotypically more variable. We also detected loci statistically associated with wing colour pattern variation, but in both species these loci were not over-represented among the candidate barrier loci, suggesting that traits other than wing colour pattern may be important for reproductive isolation. Our results contrast with the genomic patterns observed between hybridizing lineages in the mimetic Heliconius butterflies, and call for a broader investigation into the genomics of speciation in Ithomiini - the largest radiation of mimetic butterflies.

Chemistry of the Androconial Secretion of the Ithomiine Butterfly Oleria onega.

Ithomiine butterflies use pyrrolizidine alkaloids (PAs) as precursors for male pheromones, such as dihydropyrrolizines or lactones. In contrast to most other ithomiine genera, none of these compounds have ever been detected in Oleria species. The absence of these compounds is thought to be the result of limited access to PA-containing plants. Here we investigate the contents of the androconia of Oleria onega caught in the wild when PA containing plants were abundant. Although the PA lycopsamine was detected in the hairpencils, none of the other known PA-derived compounds were present. Instead, the unsubstituted core of the PA necine base, 1-methylene-1H-pyrrolizine (13), a very unstable compound, was found. The identity of this compound was proven by synthesis. Although its formation in nature appears very likely, 13 is also formed during GC analysis of PAs, making its natural occurrence uncertain. Nevertheless, its reactivity makes it a good candidate for a signaling compound, because its rapid degradation can be used to convey spatial and temporal information. In addition, several other compounds, likely used in intraspecific communication, were identified. All of these compounds are reported for the first time as natural products or from insects. These include 9-hydroxynonanoic acid (21) and (Z)-9-hydroxy-6-enoic acid (18), as well as their condensation products with 11-hexadecenoic- and octadecenoic acids. Furthermore, self-condensation products, such as (Z)-9-[(9-hydroxynon-6-enoyl)oxy]- and 9-[(9-hydroxynonanoyl)oxy]nonanoic acid and non-6-enoic acids (35, 36, 38, 40) were identified, together with the known compounds 2-heptadecanol (39) and 6,10,14-trimethylpentadecan-2-ol (37). In summary, O. onega appears to lack enzymes to produce dihydropyrrolizines. In stark contrast to other ithomiine genera, a unique blend of oxidized fatty acids seems to be used instead.

Why has transparency evolved in aposematic butterflies? Insights from the largest radiation of aposematic butterflies, the Ithomiini.

Defended species are often conspicuous and this is thought to be an honest signal of defences, i.e. more toxic prey are more conspicuous. Neotropical butterflies of the large Ithomiini tribe numerically dominate communities of chemically defended butterflies and may thus drive the evolution of mimetic warning patterns. Although many species are brightly coloured, most are transparent to some degree. The evolution of transparency from a warning-coloured ancestor is puzzling as it is generally assumed to be involved in concealment. Here, we show that transparent Ithomiini species are indeed less detectable by avian predators (i.e. concealment). Surprisingly, transparent species are not any less unpalatable, and may in fact be more unpalatable than opaque species, the latter spanning a larger range of unpalatability. We put forth various hypotheses to explain the evolution of weak aposematic signals in these butterflies and other cryptic defended prey. Our study is an important step in determining the selective pressures and constraints that regulate the interaction between conspicuousness and unpalatability.

Does divergent selection predict the evolution of mate preference and reproductive isolation in the tropical butterfly genus Melinaea (Nymphalidae: Ithomiini)?

Many studies have shown that speciation can be facilitated when a trait under divergent selection also causes assortative mating. In Müllerian mimetic butterflies, a change in wing colour pattern can cause reproductive isolation. However, colour pattern divergence does not always lead to reproductive isolation. Understanding how divergent selection affects speciation requires identifying the mechanisms that promote mate preference and/or choosiness. This study addresses whether shifts in wing colour pattern drives mate preference and reproductive isolation in the tropical butterfly genus Melinaea (Nymphalidae: Ithomiini), and focuses on five taxa that form a speciation continuum, from subspecies to fully recognized species. Using genetic markers, wing colour pattern quantification, male pheromone characterization and behavioural assays of mating preference, we characterize the extent of genetic and phenotypic differentiation between taxa and compare it to the level of reproductive isolation. We show strong premating isolation between the closely related species M. satevis and M. marsaeus, in addition to genetic and phenotypic (colour pattern and pheromones) differentiation. By contrast, M. menophilus and M. marsaeus consist of pairs of subspecies that differ for colour pattern but that cannot be differentiated genetically. Pheromonal differentiation of subspecies was significant only for M. marsaeus, although most individuals were indistinguishable. Melinaea menophilus and M. marsaeus also differ in the strength of assortative mating, suggesting that mate preference has evolved only in M. marsaeus, consistent with selection against maladaptive offspring, as subspecific 'hybrids' of M. marsaeus have intermediate, non-mimetic colour patterns, unlike those of M. menophilus which display either parental phenotypes. We conclude that a shift in colour pattern per se is not sufficient for reproductive isolation, but rather, the evolution of assortative mating may be caused by selection against maladaptive intermediate phenotypes. This study suggests that mate preference and assortative mating evolve when adaptive, and that even in the early stages of divergence, reproductive isolation can be nearly complete due to mating preferences.

Heterozygosity and Chain Multivalents during Meiosis Illustrate Ongoing Evolution as a Result of Multiple Holokinetic Chromosome Fusions in the Genus Melinaea (Lepidoptera, Nymphalidae).

Mitotic and meiotic chromosomes from 2 taxa of the genus Melinaea, M. satevis cydon and M. "satevis" tarapotensis (Lepidoptera: Nymphalidae), and from hybrids produced in captivity were obtained using an improved spreading technique and were subsequently analyzed. In one of the taxa, the presence of trivalents and tetravalents at diakinesis/metaphase I is indicative of heterozygosity for multiple chromosome fusions or fissions, which might explain the highly variable number of chromosomes previously reported in this genus. Two large and complex multivalents were observed in the meiotic cells of the hybrid males (32 chromosomes) obtained from a cross between M. "s." tarapotensis (28 chromosomes) and M. s. cydon (40-43 chromosomes). The contribution of the 2 different haploid karyotypes to these complex figures during meiosis is discussed, and a taxonomic revision is proposed. We conclude that chromosome evolution is active and ongoing, that the karyotype of the common ancestor consisted of at least 48 chromosomes, and that evolution by chromosome fusion rather than fission is responsible for this pattern. Complex chromosome evolution in this genus may drive reproductive isolation and speciation, and highlights the difficulties inherent to the systematics of this group. We also show that Melinaea chromosomes, classically considered as holocentric, are attached to unique, rather than multiple, spindle fibers.

Unravelling the role of host plant expansion in the diversification of a Neotropical butterfly genus.

BACKGROUND: Understanding the processes underlying diversification is a central question in evolutionary biology. For butterflies, access to new host plants provides opportunities for adaptive speciation. On the one hand, locally abundant host species can generate ecologically significant selection pressure. But a diversity of host plant species within the geographic range of each population and/or species might also eliminate any advantage conferred by specialization. This paper focuses on four Melinaea species, which are oligophagous on the family Solanaceae: M. menophilus, M. satevis, M. marsaeus, and finally, M. mothone. We examined both female preference and larval performance on two host plant species that commonly occur in this butterfly's native range, Juanulloa parasitica and Trianaea speciosa, to determine whether the different Melinaea species show evidence of local adaptation. RESULT: In choice experiments, M. mothone females used both host plants for oviposition, whereas all other species used J. parasitica almost exclusively. In no choice experiment, M. mothone was the only species that readily accepted T. speciosa as a larval host plant. Larval survival was highest on J. parasitica (82.0 % vs. 60.9 %) and development took longer on T. speciosa (14.12 days vs. 13.35 days), except for M. mothone, which did equally well on both host plants. For all species, average pupal weight was highest on J. parasitica (450.66 mg vs. 420.01 mg), although this difference was least apparent in M. mothone. CONCLUSION: We did not find that coexisting species of Melinaea partition host plant resources as expected if speciation is primarily driven by host plant divergence. Although M. mothone shows evidence of local adaptation to a novel host plant, T. speciosa, which co-occurs, it does not preferentially lay more eggs on or perform better on this host plant than on host plants used by other Melinaea species and not present in its distributional range. It is likely that diversification in this genus is driven by co-occurring Müllerian mimics and the resulting predation pressure, although this is also likely made possible by greater niche diversity as a consequence of plasticity for potential hosts.

Defensive responses by a social caterpillar are tailored to different predators and change with larval instar and group size.

Gregariousness in animals is widely accepted as a behavioral adaptation for protection from predation. However, predation risk and the effectiveness of a prey's defense can be a function of several other factors, including predator species and prey size or age. The objective of this study was to determine if the gregarious habit of Malacosoma disstria caterpillars is advantageous against invertebrate natural enemies, and whether it is through dilution or cooperative defenses. We also examined the effects of larval growth and group size on the rate and success of attacks. Caterpillars of M. disstria responded with predator-specific behaviors, which led to increased survival. Evasive behaviors were used against stinkbugs, while thrashing by fourth instar caterpillars and holding on to the silk mat by second instar caterpillars was most efficient against spider attacks. Collective head flicking and biting by groups of both second and fourth instar caterpillars were observed when attacked by parasitoids. Increased larval size decreased the average number of attacks by spiders but increased the number of attacks by both stinkbugs and parasitoids. However, increased body size decreased the success rate of attacks by all three natural enemies and increased handling time for both predators. Larger group sizes did not influence the number of attacks from predators but increased the number of attacks and the number of successful attacks from parasitoids. In all cases, individual risk was lower in larger groups. Caterpillars showed collective defenses against parasitoids but not against the walking predators. These results show that caterpillars use different tactics against different natural enemies. Overall, these tactics are both more diverse and more effective in fourth instar than in second instar caterpillars, confirming that growth reduces predation risk. We also show that grouping benefits caterpillars through dilution of risk, and, in the case of parasitoids, through group defenses. The decreased tendency to aggregate in the last larval instar may therefore be linked to decreasing predation risk.

Territorial aggressiveness on the arboreal ant Azteca alfari by Camponotus blandus in French Guiana due to behavioural constraints.

This study reports new information on interactions between two sympatric ant species, the plant-ant Azteca alfari (Dolichoderinae) living in association with the myrmecophyte Cecropia obtusa (Cecropiaceae) and Camponotus blandus (Formicinae), a ground-nesting, arboreal-foraging species. Workers of A. alfari forage only on the foliage and the upper parts of the trunk of their host Cecropia, while C. blandus nests in the ground but frequently forages and patrols pioneer tree foliage, including Cecropia. The activity pattern of A. alfari and the number of C. blandus on Cecropia obtusa was monitored hourly during a two-day period in a disturbed area in French Guiana. The maximum activity of C. blandus occurred between 8:30 and 12:30, at which time A. alfari had retreated within the domatia and were least present on the trunks. Even though aggressive confrontations were observed, C. blandus workers often initiate confrontations but do not prey on A. alfari nor exploit food bodies produced by Cecropia, the principal food source of A. alfari. Hence hostility appears to be the result of territoriality. Differences in their foraging rhythms are proposed as promoting resource and territory partitioning in this ant assemblage.

Courtship behavior in relation to the female sex pheromone in the parasitoid, Aphidius ervi (Hymenoptera: Braconidae).

Mating in the aphid parasitoid, Aphidius ervi, is mediated by sex pheromones. Virgin females produce pheromones that stimulate both upwind flight and elicit close-range courtship behavior by males. Field studies and laboratory bioassays demonstrated that time of day and adult age affect both the emission of, and receptivity to, the sex pheromones. In contrast, mating affected female pheromone production, but not male responsiveness.

Silicon and tin-directed Tiffeneau-Demjanov reaction.

Silicon and tin substituents surprisingly have only a moderate directing effect on the Tiffeneau-Demjanov reaction. The low selectivity is rationalised as being due to the reactive nature of the diazonium ion leaving group, the weaker oxydiazene leaving group was found to give better yields of the silicon-directed ring expanded product.