Evidence of attack deflection suggests adaptive evolution of wing tails in butterflies.

Predation is a powerful selective force shaping many behavioural and morphological traits in prey species. The deflection of predator attacks from vital parts of the prey usually involves the coordinated evolution of prey body shape and colour. Here, we test the deflection effect of hindwing (HW) tails in the swallowtail butterfly Iphiclides podalirius. In this species, HWs display long tails associated with a conspicuous colour pattern. By surveying the wings within a wild population of I. podalirius, we observed that wing damage was much more frequent on the tails. We then used a standardized behavioural assay employing dummy butterflies with real I. podalirius wings to study the location of attacks by great tits Parus major. Wing tails and conspicuous coloration of the HWs were struck more often than the rest of the body by birds. Finally, we characterized the mechanical properties of fresh wings and found that the tail vein was more fragile than the others, suggesting facilitated escape ability of butterflies attacked at this location. Our results clearly support the deflective effect of HW tails and suggest that predation is an important selective driver of the evolution of wing tails and colour pattern in butterflies.

The effect of captivity on craniomandibular and calcaneal ontogenetic trajectories in wild boar.

Deciphering the plastic (i.e., nonheritable) changes induced by human control over wild animals in the archeological record is challenging. Previous studies detected morphological markers associated with captivity in the cranium, mandible, and calcaneus of adult wild boar (Sus scrofa) but the developmental trajectories leading up to these changes during ontogeny remain unknown. To assess the impact of growth in a captive environment on morphological structures during postnatal ontogeny, we used an experimental approach focusing on the same three structures and taxon. We investigated the form and size differences of captive-reared and wild-caught wild boar during growth using three-dimensional landmark-based geometric morphometrics. Our results provide evidence of an influence of captivity on the morphology of craniomandibular structures, as wild specimens are smaller than captive individuals at similar ages. The food resources inherent to anthropogenic environments may explain some of the observed differences between captive-reared and wild specimens. The calcaneus presents a different contrasted pattern of plasticity as captive and wild individuals differ in terms of form but not in terms of size. The physically more constrained nature of the calcaneus and the direct influence of mobility reduction on this bone may explain these discrepancies. These results provide new methodological perspectives for bioarchaeological approaches as they imply that the plastic mark of captivity can be observed in juvenile specimens in the same way it has been previously described in adults.

Long-term evolution of quantitative traits in the Drosophila melanogaster species subgroup.

Quantitative genetics aims at untangling the genetic and environmental effects on phenotypic variation. Trait heritability, which summarizes the relative importance of genetic effects, is estimated at the intraspecific level, but theory predicts that heritability could influence long-term evolution of quantitative traits. The phylogenetic signal concept bears resemblance to heritability and it has often been called species-level heritability. Under certain conditions, such as trait neutrality or contribution to phylogenesis, within-species heritability and between-species phylogenetic signal should be correlated. Here, we investigate the potential relationship between these two concepts by examining the evolution of multiple morphological traits for which heritability has been estimated in Drosophila melanogaster. Specifically, we analysed 42 morphological traits in both sexes on a phylogeny inferred from 22 nuclear genes for nine species of the melanogaster subgroup. We used Pagel's λ as a measurement of phylogenetic signal because it is the least influenced by the number of analysed taxa. Pigmentation traits showed the strongest concordance with the phylogeny, but no correlation was found between phylogenetic signal and heritability estimates mined from the literature. We obtained data for multiple climatic variables inferred from the geographical distribution of each species. Phylogenetic regression of quantitative traits on climatic variables showed a significantly positive correlation with heritability. Convergent selection, the response to which depends on the trait heritability, may have led to the null association between phylogenetic signal and heritability for morphological traits in Drosophila. We discuss the possible causes of discrepancy between both statistics and caution against their confusion in evolutionary biology.

Divergence of climbing escape flight performance in Morpho butterflies living in different microhabitats.

Habitat specialization can influence the evolution of animal movement in promoting divergent locomotor abilities adapted to contrasting environmental conditions, differences in vegetation clutter or predatory communities. While the effect of habitat on the evolution of locomotion and particularly escape performance has been well investigated in terrestrial animals, it remains understudied in flying animals. Here, we investigated whether specialization of Morpho butterfly species into different vertical strata of the Amazonian forest affects the performance of upward escape flight manoeuvres. Using stereoscopic high-speed videography, we compared the climbing flight kinematics of seven Morpho species living either in the forest canopy or in the understory. We show that butterflies from canopy species display strikingly higher climbing speed and steeper ascent angle compared with understory species. Although climbing speed increased with wing speed and angle of attack, the higher climb angle observed in canopy species was best explained by their higher body pitch angle, resulting in more upward-directed aerodynamic thrust forces. Climb angle also scales positively with weight-normalized wing area, and this weight-normalized wing area was higher in canopy species. This shows that a combined divergence in flight behaviour and morphology contributes to the evolution of increased climbing flight abilities in canopy species.

Constraints associated with captivity alter craniomandibular integration in wild boar.

The domestication process is associated with substantial phenotypic changes through time. However, although morphological integration between biological structures is purported to have a major influence on the evolution of new morphologies, little attention has been paid to the influence of domestication on the magnitude of integration. Here, we assessed the influence of constraints associated with captivity, considered as one of the crucial first steps in the domestication process, on the integration of cranial and mandibular structures. We investigated the craniomandibular integration in Western European Sus scrofa using three-dimensional (3D) landmark-based geometric morphometrics. Our results suggest that captivity is associated with a lower level of integration between the cranium and the mandible. Plastic responses to captivity can thus affect the magnitude of integration of key functional structures. These findings underline the critical need to develop integration studies in the context of animal domestication to better understand the processes accountable for the set-up of domestic phenotypes through time.

Punctuational ecological changes rather than global factors drive species diversification and the evolution of wing phenotypes in Morpho butterflies.

Assessing the relative importance of geographical and ecological drivers of evolution is paramount to understand the diversification of species and traits at the macroevolutionary scale. Here, we use an integrative approach, combining phylogenetics, biogeography, ecology and quantified phenotypes to investigate the drivers of both species and phenotypic diversification of the iconic Neotropical butterfly genus Morpho. We generated a time-calibrated phylogeny for all known species and inferred historical biogeography. We fitted models of time-dependent (accounting for rate heterogeneity across the phylogeny) and paleoenvironment-dependent diversification (accounting for global effect on the phylogeny). We used geometric morphometrics to assess variation of wing size and shape across the tree and investigated their dynamics of evolution. We found that the diversification of Morpho is best explained when considering variable diversification rates across the tree, possibly associated with lineages occupying different microhabitat conditions. First, a shift from understory to canopy was characterized by an increased speciation rate partially coupled with an increasing rate of wing shape evolution. Second, the occupation of dense bamboo thickets accompanying a major host-plant shift from dicotyledons towards monocotyledons was associated with a simultaneous diversification rate shift and an evolutionary 'jump' of wing size. Our study points to a diversification pattern driven by punctuational ecological changes instead of a global driver or biogeographic history.

Convergence in sympatry: Evolution of blue-banded wing pattern in Morpho butterflies.

Species interactions such as mimicry can promote trait convergence but disentangling this effect from those of shared ecology, evolutionary history, and niche conservatism is often challenging. Here by focusing on wing colour pattern variation within and between three butterfly species living in sympatry in a large proportion of their range, we tested the effect of species interactions on trait diversification. These butterflies display a conspicuous iridescent blue coloration on the dorsal side of their wings and a cryptic brownish colour on the ventral side. Combined with an erratic and fast flight, these colour patterns increase the difficulty of capture by predators and contribute to the high escape abilities of these butterflies. We hypothesize that, beyond their direct contribution to predator escape, these wing patterns can be used as signals of escape abilities by predators, resulting in positive frequency-dependent selection favouring convergence in wing pattern in sympatry. To test this hypothesis, we quantified dorsal wing pattern variations of 723 butterflies from the three species sampled throughout their distribution, including sympatric and allopatric situations and compared the phenotypic distances between species, sex and localities. We detected a significant effect of localities on colour pattern, and higher inter-specific resemblance in sympatry as compared to allopatry, consistent with the hypothesis of local convergence of wing patterns. Our results provide support to the existence of escape mimicry in the wild and stress the importance of estimating trait variation within species to understand trait variation between species, and to a larger extent, trait diversification at the macro-evolutionary scale.

Cyclin G and the Polycomb Repressive complexes PRC1 and PR-DUB cooperate for developmental stability.

In Drosophila, ubiquitous expression of a short Cyclin G isoform generates extreme developmental noise estimated by fluctuating asymmetry (FA), providing a model to tackle developmental stability. This transcriptional cyclin interacts with chromatin regulators of the Enhancer of Trithorax and Polycomb (ETP) and Polycomb families. This led us to investigate the importance of these interactions in developmental stability. Deregulation of Cyclin G highlights an organ intrinsic control of developmental noise, linked to the ETP-interacting domain, and enhanced by mutations in genes encoding members of the Polycomb Repressive complexes PRC1 and PR-DUB. Deep-sequencing of wing imaginal discs deregulating CycG reveals that high developmental noise correlates with up-regulation of genes involved in translation and down-regulation of genes involved in energy production. Most Cyclin G direct transcriptional targets are also direct targets of PRC1 and RNAPolII in the developing wing. Altogether, our results suggest that Cyclin G, PRC1 and PR-DUB cooperate for developmental stability.

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.

Effects of natural wing damage on flight performance in Morpho butterflies: what can it tell us about wing shape evolution?

Flying insects frequently experience wing damage during their life. Such irreversible alterations of wing shape affect flight performance and ultimately fitness. Insects have been shown to compensate for wing damage through various behavioural adjustments, but the importance of damage location over the wings has scarcely been studied. Using natural variation in wing damage, we tested how the loss of different wing parts affects flight performance. We quantified flight performance in two species of large butterflies, Morpho helenor and Morpho achilles, caught in the wild and displaying large variation in the extent and location of wing damage. We artificially generated more severe wing damage in our sample to contrast natural versus higher magnitude wing loss. Wing shape alteration across our sample was quantified using geometric morphometrics to test the effect of different damage distributions on flight performance. Our results show that impaired flight performance clearly depends on damage location over the wings, pointing to a relative importance of different wing parts for flight. A deteriorated forewing leading edge most critically affected flight performance, specifically decreasing flight speed and the proportion of gliding flight. In contrast, the most frequent natural damage, deteriorated wing margin, had no detectable effect on flight behaviour. Damage located on the hindwings - although having a limited effect on flight - was associated with reduced flight height, suggesting that the forewings and hindwings play different roles in butterfly flight. By contrasting harmless and deleterious consequences of various types of wing damage, our study highlights different selective regimes acting on morphological variations of butterfly wings.

Recurrent specialization on a toxic fruit in an island Drosophila population.

Recurrent specialization on similar host plants offers a unique opportunity to unravel the evolutionary and genetic mechanisms underlying dietary shifts. Recent studies have focused on ecological races belonging to the same species, but it is hard in many cases to untangle the role of adaptive introgression versus distinct mutations in facilitating recurrent evolution. We discovered on the island of Mayotte a population of the generalist fly Drosophila yakuba that is strictly associated with noni (Morinda citrifolia). This case strongly resembles Drosophila sechellia, a genetically isolated insular relative of D. yakuba whose intensely studied specialization on toxic noni fruits has always been considered a unique event in insect evolution. Experiments revealed that unlike mainland D. yakuba strains, Mayotte flies showed strong olfactory attraction and significant toxin tolerance to noni. Island females strongly discriminated against mainland males, suggesting that dietary adaptation has been accompanied by partial reproductive isolation. Population genomic analysis indicated a recent colonization (∼29 kya), at a time when year-round noni fruits may have presented a predictable resource on the small island, with ongoing migration after colonization. This relatively recent time scale allowed us to search for putatively adaptive loci based on genetic variation. Strong signals of genetic differentiation were found for several detoxification genes, including a major toxin tolerance locus in D. sechellia Our results suggest that recurrent evolution on a toxic resource can involve similar historical events and common genetic bases, and they establish an important genetic system for the study of early stages of ecological specialization and speciation.

Deciphering the Routes of invasion of Drosophila suzukii by Means of ABC Random Forest.

Deciphering invasion routes from molecular data is crucial to understanding biological invasions, including identifying bottlenecks in population size and admixture among distinct populations. Here, we unravel the invasion routes of the invasive pest Drosophila suzukii using a multi-locus microsatellite dataset (25 loci on 23 worldwide sampling locations). To do this, we use approximate Bayesian computation (ABC), which has improved the reconstruction of invasion routes, but can be computationally expensive. We use our study to illustrate the use of a new, more efficient, ABC method, ABC random forest (ABC-RF) and compare it to a standard ABC method (ABC-LDA). We find that Japan emerges as the most probable source of the earliest recorded invasion into Hawaii. Southeast China and Hawaii together are the most probable sources of populations in western North America, which then in turn served as sources for those in eastern North America. European populations are genetically more homogeneous than North American populations, and their most probable source is northeast China, with evidence of limited gene flow from the eastern US as well. All introduced populations passed through bottlenecks, and analyses reveal five distinct admixture events. These findings can inform hypotheses concerning how this species evolved between different and independent source and invasive populations. Methodological comparisons indicate that ABC-RF and ABC-LDA show concordant results if ABC-LDA is based on a large number of simulated datasets but that ABC-RF out-performs ABC-LDA when using a comparable and more manageable number of simulated datasets, especially when analyzing complex introduction scenarios.

Phenotypic plasticity of Drosophila suzukii wing to developmental temperature: implications for flight.

Phenotypic plasticity has been proposed as a mechanism that facilitates the success of biological invasions. In order to test the hypothesis of an adaptive role for plasticity in invasions, particular attention should be paid to the relationship between the focal plastic trait, the environmental stimulus and the functional importance of the trait. The Drosophila wing is particularly amenable to experimental studies of phenotypic plasticity. Wing morphology is known for its plastic variation under different experimental temperatures, but this plasticity has rarely been investigated in a functional context of flight. Here, we investigate the effect of temperature on wing morphology and flight in the invasive pest species Drosophila suzukii Although the rapid invasion of both Europe and North America was most likely facilitated by human activities, D. suzukii is also expected to disperse actively. By quantifying wing morphology and individual flight trajectories of flies raised under different temperatures, we tested whether (1) invasive populations of D. suzukii show higher phenotypic plasticity than their native counterparts, and (2) wing plasticity affects flight parameters. Developmental temperature was found to affect both wing morphology and flight parameters (in particular speed and acceleration), leaving open the possibility of an adaptive value for wing plasticity. Our results show no difference in phenotypic plasticity between invasive and native populations, rejecting a role for wing plasticity in the invasion success.

Fluctuating asymmetry of meristic traits: an isofemale line analysis in an invasive drosophilid, Zaprionus indianus.

Metric (e.g., body size) and meristic (e.g., bristle number) traits are of general use in quantitative genetic studies, and the phenotypic variance is subdivided into a genetic and a non-genetic environmental component. The non-genetic variance may have two origins: a common garden effect between individuals and a developmental instability within the same individual. Developmental instability may be studied by considering the fluctuating asymmetry (FA) between the two sides of the body. The isofemale line technique is a convenient method for investigating the architecture of natural populations but has been rarely implemented for investigating FA. In this paper, we use this experimental design for analyzing four meristic traits in eight populations of the cosmopolitan Zaprionus indianus. A study of the correlation between left and right side of each line revealed that almost 90% of the variability was due to a developmental noise, while a much higher correlation among the means of the lines from the same population was observed. A slight trend toward a directional asymmetry was observed: more thoracic bristles on the left side. Four kinds of indices, scaled or non-scaled to the mean were used for comparing the different traits. Unscaled values (mean absolute values or standard deviation of each line) revealed a linear increase with the means. Interestingly the results of ovariole number were included in the same regression. With the scaled indices (mean absolute divided by each individual value or stadard deviation devided by the mean), the differences among traits were considerably decreased, but still remained significant. The mean FA of the various traits were not correlated, suggesting that each trait harbors its own developmental stability. The CVs of FA were high with a magnitude similar to those of the trait themselves, slightly less than 10%. Finally, even with the isofemale line design, which is a powerful means for unravelling slight genetic variations, we did not to find any clear indication of a genetic component of FA under the optimal environmental conditions used in this study.

Phenotypic defects in newborn Gammarus fossarum (Amphipoda) following embryonic exposure to fenoxycarb.

During morphogenesis numerous morphogenetic factors ensure the production of a target phenotype. By disrupting these processes, a toxic exposure during this period could cause an increase of phenotypic defects. In the present study, embryos of the freshwater amphipod Gammarus fossarum were exposed throughout the embryogenesis to increasing concentrations of fenoxycarb (0, 0.5µgL-1, 5µgL-1 and 50µgL-1), a growth regulator insecticide analog of the insect juvenile hormone. In addition, to identify morphogenesis' sensitive period, embryos were exposed during either early or late embryonic development to 5µgL-1 of fenoxycarb. In newborn individuals from exposed embryos, three phenotypes were investigated: i) eye pigmentation, ii) length of the antenna and gnathopod of both left and right sides and iii) midgut tissue state. Developmental homeostasis was assessed by measuring fluctuating asymmetry and inter-individual variance of both the antenna and gnathopod. Exposure to 5µgL-1 and 50µgL-1 fenoxycarb throughout the embryonic development induced a delayed hatching and altered appendages size. Moreover, exposure to 5µgL-1 throughout the embryogenesis and during the gastrulation phase impaired eye pigmentation, while exposure to 50µgL-1 resulted in increased tissue damages of the midgut. No significant increase of fluctuating asymmetry was observed in exposed individuals, neither for the antenna nor for the gnathopod. These results demonstrate that fenoxycarb can alter embryonic development of G. fossarum without disrupting developmental homeostasis.

Modularity and developmental stability in segmented animals: variation in translational asymmetry in geophilomorph centipedes.

Does a modular body organization present a challenge for developmental control? We investigate the idea of a possible developmental cost of modularity by examining the relationship between modularity and developmental stability in a multi-segmented arthropod taxon: the geophilomorph centipedes. In a sample of eight species, we tested the correlation between developmental stability, estimated from measures of translational fluctuating asymmetry, and the number of trunk segments and some other morphological traits, both at the species and individual levels. We found sizeable differences in size and shape patterns of variation at the level of species. However, we did not find any clear evidence of correlation between fluctuating asymmetry and the number of trunk segments or the other morphological traits considered. Thus, our results provide no support to the idea of a possible trade-off between the cardinality of a modular system and the level of developmental precision in the phenotypic expression of its modules. The results of this exploratory study invite further investigations of patterns of translational fluctuating asymmetry in segmented animals and other modular organisms, as these have the potential to reveal features of developmental stability that cannot be captured by the study of bilateral asymmetry alone.

Developmental stability: a major role for cyclin G in drosophila melanogaster.

Morphological consistency in metazoans is remarkable given the pervasive occurrence of genetic variation, environmental effects, and developmental noise. Developmental stability, the ability to reduce developmental noise, is a fundamental property of multicellular organisms, yet its genetic bases remains elusive. Imperfect bilateral symmetry, or fluctuating asymmetry, is commonly used to estimate developmental stability. We observed that Drosophila melanogaster overexpressing Cyclin G (CycG) exhibit wing asymmetry clearly detectable by sight. Quantification of wing size and shape using geometric morphometrics reveals that this asymmetry is a genuine-but extreme-fluctuating asymmetry. Overexpression of CycG indeed leads to a 40-fold increase of wing fluctuating asymmetry, which is an unprecedented effect, for any organ and in any animal model, either in wild populations or mutants. This asymmetry effect is not restricted to wings, since femur length is affected as well. Inactivating CycG by RNAi also induces fluctuating asymmetry but to a lesser extent. Investigating the cellular bases of the phenotypic effects of CycG deregulation, we found that misregulation of cell size is predominant in asymmetric flies. In particular, the tight negative correlation between cell size and cell number observed in wild-type flies is impaired when CycG is upregulated. Our results highlight the role of CycG in the control of developmental stability in D. melanogaster. Furthermore, they show that wing developmental stability is normally ensured via compensatory processes between cell growth and cell proliferation. We discuss the possible role of CycG as a hub in a genetic network that controls developmental stability.

Patterns of morphological variation highlight the effect of natural selection on eyespots modularity in the butterfly Morpho telemachus.

Morphological correlations can not only stem from developmental constraints but also from selective pressures. Butterfly eyespots are repeated wing color pattern elements, widespread across species. As developmental serial homologs, they are controlled by similar developmental pathways imposing correlations among eyespots: selection on a single eyespot may induce correlated responses in all eyespots. We study the variations in the ventral eyespots of Morpho telemachus, where two different selective regimes are likely to act: while most eyespots are always-visible, two eyespots are conditionally displayed: hidden at rest, they can be exposed when the butterflies are threatened, or during sexual interactions. We investigate how such contrasted selection across eyespots can alter the covariations imposed by their shared developmental origin. We quantified eyespots covariations within a large population of M. telemachus and compared the observed patterns to those found in M. helenor, where all eyespots are always-visible and thus probably affected by a similar selection regime. We found that M. telemachus conditionally displayed eyespots are less variable than always-visible eyespots and that these two eyespots form a separate variational module in this species, in contrast to M. helenor. Our results suggest that eyespots covariations were shaped by selection, highlighting how natural selection may promote the evolution of modularity.

Genome assembly of 3 Amazonian Morpho butterfly species reveals Z-chromosome rearrangements between closely related species living in sympatry.

The genomic processes enabling speciation and species coexistence in sympatry are still largely unknown. Here we describe the whole-genome sequencing and assembly of 3 closely related species from the butterfly genus Morpho: Morpho achilles (Linnaeus, 1758), Morpho helenor (Cramer, 1776), and Morpho deidamia (Höbner, 1819). These large blue butterflies are emblematic species of the Amazonian rainforest. They live in sympatry in a wide range of their geographical distribution and display parallel diversification of dorsal wing color pattern, suggesting local mimicry. By sequencing, assembling, and annotating their genomes, we aim at uncovering prezygotic barriers preventing gene flow between these sympatric species. We found a genome size of  480 Mb for the 3 species and a chromosomal number ranging from 2n = 54 for M. deidamia to 2n = 56 for M. achilles and M. helenor. We also detected inversions on the sex chromosome Z that were differentially fixed between species, suggesting that chromosomal rearrangements may contribute to their reproductive isolation. The annotation of their genomes allowed us to recover in each species at least 12,000 protein-coding genes and to discover duplications of genes potentially involved in prezygotic isolation like genes controlling color discrimination (L-opsin). Altogether, the assembly and the annotation of these 3 new reference genomes open new research avenues into the genomic architecture of speciation and reinforcement in sympatry, establishing Morpho butterflies as a new eco-evolutionary model.