Dominance mechanisms in supergene alleles controlling butterfly wing pattern variation: insights from gene expression in Heliconius numata.

Loci under balancing selection, where multiple alleles are maintained, offer a relevant opportunity to investigate the role of natural selection in shaping genetic dominance: the high frequency of heterozygotes at these loci has been shown to enable the evolution of dominance among alleles. In the butterfly Heliconius numata, mimetic wing color variations are controlled by an inversion polymorphism of a circa 2 Mb genomic region (supergene P), with strong dominance between sympatric alleles. To test how differences in dominance observed on wing patterns correlate with variations in expression levels throughout the supergene region, we sequenced the complete transcriptome of heterozygotes at the prepupal stage and compared it to corresponding homozygotes. By defining dominance based on non-overlapping ranges of transcript expression between genotypes, we found contrasting patterns of dominance between the supergene and the rest of the genome; the patterns of transcript expression in the heterozygotes were more similar to the expression observed in the dominant homozygotes in the supergene region. Dominance also differed among the three subinversions of the supergene, suggesting possible epistatic interactions among their gene contents underlying dominance evolution. We found the expression pattern of the melanization gene cortex located in the P-region to predict wing pattern phenotype in the heterozygote. We also identify new candidate genes that are potentially involved in mimetic color pattern variations highlighting the relevance of transcriptomic analyses in heterozygotes to pinpoint candidate genes in non-recombining regions.

Deep structure, long-distance migration and admixture in the colour polymorphic land snail Cepaea nemoralis.

Although snails of the genus Cepaea have historically been important in studying colour polymorphism, an ongoing issue is that there is a lack of knowledge of the underlying genetics of the polymorphism, as well as an absence of genomic data to put findings in context. We, therefore, used phylogenomic methods to begin to investigate the post-glacial history of Cepaea nemoralis, with a long-term aim to understand the roles that selection and drift have in determining both European-wide and local patterns of colour polymorphism. By combining prior and new mitochondrial DNA data from over 1500 individuals with ddRAD genomic data from representative individuals across Europe, we show that patterns of differentiation are primarily due to multiple deeply diverged populations of snails. Minimally, there is a widespread Central European population and additional diverged groups in Northern Spain, the Pyrenees, as well as likely Italy and South Eastern Europe. The genomic analysis showed that the present-day snails in Ireland and possibly some other locations are likely descendants of admixture between snails from the Pyrenees and the Central European group, an observation that is consistent with prior inferences from mitochondrial DNA alone. The interpretation is that C. nemoralis may have arrived in Ireland via long-distance migration from the Pyrenean region, subsequently admixing with arrivals from elsewhere. This work, therefore, provides a baseline expectation for future studies on the genetics of the colour polymorphism, as well as providing a comparator for similar species.

The draft genome sequence of the grove snail Cepaea nemoralis.

Studies on the shell color and banding polymorphism of the grove snail Cepaea nemoralis and the sister taxon Cepaea hortensis have provided compelling evidence for the fundamental role of natural selection in promoting and maintaining intraspecific variation. More recently, Cepaea has been the focus of citizen science projects on shell color evolution in relation to climate change and urbanization. C. nemoralis is particularly useful for studies on the genetics of shell polymorphism and the evolution of "supergenes," as well as evo-devo studies of shell biomineralization, because it is relatively easily maintained in captivity. However, an absence of genomic resources for C. nemoralis has generally hindered detailed genetic and molecular investigations. We therefore generated ∼23× coverage long-read data for the ∼3.5 Gb genome, and produced a draft assembly composed of 28,537 contigs with the N50 length of 333 kb. Genome completeness, estimated by BUSCO using the metazoa dataset, was 91%. Repetitive regions cover over 77% of the genome. A total of 43,519 protein-coding genes were predicted in the assembled genome, and 97.3% of these were functionally annotated from either sequence homology or protein signature searches. This first assembled and annotated genome sequence for a helicoid snail, a large group that includes edible species, agricultural pests, and parasite hosts, will be a core resource for identifying the loci that determine the shell polymorphism, as well as in a wide range of analyses in evolutionary and developmental biology, and snail biology in general.

Evo-devo of shell colour in gastropods and bivalves.

Recent technical innovations are revealing surprising patterns in mollusc shell pigmentation, such as an unexpectedly modest role for melanins and rapid divergences in the mix of pigments used to achieve similar colour patterns. The elucidation of the molecular genetic basis of shell pigmentation has been slow, probably because of the high genome complexity of gastropods and bivalves. Recent work within the old field of evolutionary ecology of shell pigmentation allows a greater role for the analysis of large-geographic-scale patterns (sometimes employing citizen-science data), as well as experimental field studies. However, the field remains dominated by land snails as model organisms, while colour pattern evolution in marine gastropods and bivalves, particularly those not exposed to visual predators, remains mysterious.

Unravelling the genes forming the wing pattern supergene in the polymorphic butterfly Heliconius numata.

BACKGROUND: Unravelling the genetic basis of polymorphic characters is central to our understanding of the origins and diversification of living organisms. Recently, supergenes have been implicated in a wide range of complex polymorphisms, from adaptive colouration in butterflies and fish to reproductive strategies in birds and plants. The concept of a supergene is now a hot topic in biology, and identification of its functional elements is needed to shed light on the evolution of highly divergent adaptive traits. Here, we apply different gene expression analyses to study the supergene P that controls polymorphism of mimetic wing colour patterns in the neotropical butterfly Heliconius numata. RESULTS: We performed de novo transcriptome assembly and differential expression analyses using high-throughput Illumina RNA sequencing on developing wing discs of different H. numata morphs. Within the P interval, 30 and 17 of the 191 transcripts were expressed differentially in prepupae and day-1 pupae, respectively. Among these is the gene cortex, known to play a role in wing pattern formation in Heliconius and other Lepidoptera. Our in situ hybridization experiments confirmed the relationship between cortex expression and adult wing patterns. CONCLUSIONS: This study found the majority of genes in the P interval to be expressed in the developing wing discs during the critical stages of colour pattern formation, and detect drastic changes in expression patterns in multiple genes associated with structural variants. The patterns of expression of cortex only partially recapitulate the variation in adult phenotype, suggesting that the remaining phenotypic variation could be controlled by other genes within the P interval. Although functional studies on cortex are now needed to determine its exact developmental role, our results are in accordance with the classical supergene hypothesis, whereby several genes inherited together due to tight linkage control a major developmental switch.

The gene cortex controls mimicry and crypsis in butterflies and moths.

The wing patterns of butterflies and moths (Lepidoptera) are diverse and striking examples of evolutionary diversification by natural selection. Lepidopteran wing colour patterns are a key innovation, consisting of arrays of coloured scales. We still lack a general understanding of how these patterns are controlled and whether this control shows any commonality across the 160,000 moth and 17,000 butterfly species. Here, we use fine-scale mapping with population genomics and gene expression analyses to identify a gene, cortex, that regulates pattern switches in multiple species across the mimetic radiation in Heliconius butterflies. cortex belongs to a fast-evolving subfamily of the otherwise highly conserved fizzy family of cell-cycle regulators, suggesting that it probably regulates pigmentation patterning by regulating scale cell development. In parallel with findings in the peppered moth (Biston betularia), our results suggest that this mechanism is common within Lepidoptera and that cortex has become a major target for natural selection acting on colour and pattern variation in this group of insects.

Amelanism in the corn snake is associated with the insertion of an LTR-retrotransposon in the OCA2 gene.

The corn snake (Pantherophis guttatus) is a new model species particularly appropriate for investigating the processes generating colours in reptiles because numerous colour and pattern mutants have been isolated in the last five decades. Using our captive-bred colony of corn snakes, transcriptomic and genomic next-generation sequencing, exome assembly, and genotyping of SNPs in multiple families, we delimit the genomic interval bearing the causal mutation of amelanism, the oldest colour variant observed in that species. Proceeding with sequencing the candidate gene OCA2 in the uncovered genomic interval, we identify that the insertion of an LTR-retrotransposon in its 11(th) intron results in a considerable truncation of the p protein and likely constitutes the causal mutation of amelanism in corn snakes. As amelanistic snakes exhibit white, instead of black, borders around an otherwise normal pattern of dorsal orange saddles and lateral blotches, our results indicate that melanocytes lacking melanin are able to participate to the normal patterning of other colours in the skin. In combination with research in the zebrafish, this work opens the perspective of using corn snake colour and pattern variants to investigate the generative processes of skin colour patterning shared among major vertebrate lineages.

Phylogeography and support vector machine classification of colour variation in panther chameleons.

Lizards and snakes exhibit colour variation of adaptive value for thermoregulation, camouflage, predator avoidance, sexual selection and speciation. Furcifer pardalis, the panther chameleon, is one of the most spectacular reptilian endemic species in Madagascar, with pronounced sexual dimorphism and exceptionally large intraspecific variation in male coloration. We perform here an integrative analysis of molecular phylogeography and colour variation after collecting high-resolution colour photographs and blood samples from 324 F. pardalis individuals in locations spanning the whole species distribution. First, mitochondrial and nuclear DNA sequence analyses uncover strong genetic structure among geographically restricted haplogroups, revealing limited gene flow among populations. Bayesian coalescent modelling suggests that most of the mitochondrial haplogroups could be considered as separate species. Second, using a supervised multiclass support vector machine approach on five anatomical components, we identify patterns in 3D colour space that efficiently predict assignment of male individuals to mitochondrial haplogroups. We converted the results of this analysis into a simple visual classification key that can assist trade managers to avoid local population overharvesting.

Photonic crystals cause active colour change in chameleons.

Many chameleons, and panther chameleons in particular, have the remarkable ability to exhibit complex and rapid colour changes during social interactions such as male contests or courtship. It is generally interpreted that these changes are due to dispersion/aggregation of pigment-containing organelles within dermal chromatophores. Here, combining microscopy, photometric videography and photonic band-gap modelling, we show that chameleons shift colour through active tuning of a lattice of guanine nanocrystals within a superficial thick layer of dermal iridophores. In addition, we show that a deeper population of iridophores with larger crystals reflects a substantial proportion of sunlight especially in the near-infrared range. The organization of iridophores into two superposed layers constitutes an evolutionary novelty for chameleons, which allows some species to combine efficient camouflage with spectacular display, while potentially providing passive thermal protection.

Precise colocalization of interacting structural and pigmentary elements generates extensive color pattern variation in Phelsuma lizards.

BACKGROUND: Color traits in animals play crucial roles in thermoregulation, photoprotection, camouflage, and visual communication, and are amenable to objective quantification and modeling. However, the extensive variation in non-melanic pigments and structural colors in squamate reptiles has been largely disregarded. Here, we used an integrated approach to investigate the morphological basis and physical mechanisms generating variation in color traits in tropical day geckos of the genus Phelsuma. RESULTS: Combining histology, optics, mass spectrometry, and UV and Raman spectroscopy, we found that the extensive variation in color patterns within and among Phelsuma species is generated by complex interactions between, on the one hand, chromatophores containing yellow/red pteridine pigments and, on the other hand, iridophores producing structural color by constructive interference of light with guanine nanocrystals. More specifically, we show that 1) the hue of the vivid dorsolateral skin is modulated both by variation in geometry of structural, highly ordered narrowband reflectors, and by the presence of yellow pigments, and 2) that the reflectivity of the white belly and of dorsolateral pigmentary red marks, is increased by underlying structural disorganized broadband reflectors. Most importantly, these interactions require precise colocalization of yellow and red chromatophores with different types of iridophores, characterized by ordered and disordered nanocrystals, respectively. We validated these results through numerical simulations combining pigmentary components with a multilayer interferential optical model. Finally, we show that melanophores form dark lateral patterns but do not significantly contribute to variation in blue/green or red coloration, and that changes in the pH or redox state of pigments provide yet another source of color variation in squamates. CONCLUSIONS: Precisely colocalized interacting pigmentary and structural elements generate extensive variation in lizard color patterns. Our results indicate the need to identify the developmental mechanisms responsible for the control of the size, shape, and orientation of nanocrystals, and the superposition of specific chromatophore types. This study opens up new perspectives on Phelsuma lizards as models in evolutionary developmental biology.

Evolutionary history of the recruitment of conserved developmental genes in association to the formation and diversification of a novel trait.

BACKGROUND: The origin and modification of novel traits are important aspects of biological diversification. Studies combining concepts and approaches of developmental genetics and evolutionary biology have uncovered many examples of the recruitment, or co-option, of genes conserved across lineages for the formation of novel, lineage-restricted traits. However, little is known about the evolutionary history of the recruitment of those genes, and of the relationship between them -for example, whether the co-option involves whole or parts of existing networks, or whether it occurs by redeployment of individual genes with de novo rewiring. We use a model novel trait, color pattern elements on butterfly wings called eyespots, to explore these questions. Eyespots have greatly diversified under natural and sexual selection, and their formation involves genetic circuitries shared across insects. RESULTS: We investigated the evolutionary history of the recruitment and co-recruitment of four conserved transcription regulators to the larval wing disc region where circular pattern elements develop. The co-localization of Antennapedia, Notch, Distal-less, and Spalt with presumptive (eye)spot organizers was examined in 13 butterfly species, providing the largest comparative dataset available for the system. We found variation between families, between subfamilies, and between tribes. Phylogenetic reconstructions by parsimony and maximum likelihood methods revealed an unambiguous evolutionary history only for Antennapedia, with a resolved single origin of eyespot-associated expression, and many homoplastic events for Notch, Distal-less, and Spalt. The flexibility in the (co-)recruitment of the targeted genes includes cases where different gene combinations are associated with morphologically similar eyespots, as well as cases where identical protein combinations are associated with very different phenotypes. CONCLUSIONS: The evolutionary history of gene (co-)recruitment is consistent with both divergence from a recruited putative ancestral network, and with independent co-option of individual genes. The diversity in the combinations of genes expressed in association with eyespot formation does not parallel diversity in characteristics of the adult phenotype. We discuss these results in the context of inferring homology. Our study underscores the importance of widening the representation of phylogenetic, morphological, and genetic diversity in order to establish general principles about the mechanisms behind the evolution of novel traits.

Involvement of the conserved Hox gene Antennapedia in the development and evolution of a novel trait.

BACKGROUND: Hox proteins specify segment identity during embryogenesis and have typical associated expression patterns. Changes in embryonic expression and activity of Hox genes were crucial in the evolution of animal body plans, but their role in the post-embryonic development of lineage-specific traits remains largely unexplored. Here, we focus on the insect Hox genes Ultrabithorax (Ubx) and Antennapedia (Antp), and implicate the latter in the formation and diversification of novel, butterfly-specific wing patterns. RESULTS: First, we describe a conserved pattern of Ubx expression and a novel pattern of Antp expression in wing discs of Bicyclus anynana butterflies. The discrete, reiterated domains of Antp contrast with the typical expression of Hox genes in single continuous regions in arthropod embryos. Second, we show that this pattern is associated with the establishment of the organizing centres of eyespots. Antp upregulation is the earliest event in organizer development described to date, and in contrast to all genes implicated in eyespot formation, is exclusive to those centres. Third, our comparative analysis of gene expression across nymphalids reveals unexpected differences in organizer determination. CONCLUSIONS: We show that the Antp's recruitment for the formation of novel traits in butterfly wing discs involved the evolution of new expression domains, and is restricted to a particular lineage. This study contributes novel insights into the evolution of Antp expression, as well as into the genetic mechanisms underlying morphological diversification. Our results also underscore how a wider representation of morphological and phylogenetic diversity is essential in evolutionary developmental biology.

Single locus affects embryonic segment polarity and multiple aspects of an adult evolutionary novelty.

BACKGROUND: The characterization of the molecular changes that underlie the origin and diversification of morphological novelties is a key challenge in evolutionary developmental biology. The evolution of such traits is thought to rely largely on co-option of a toolkit of conserved developmental genes that typically perform multiple functions. Mutations that affect both a universal developmental process and the formation of a novelty might shed light onto the genetics of traits not represented in model systems. Here we describe three pleiotropic mutations with large effects on a novel trait, butterfly eyespots, and on a conserved stage of embryogenesis, segment polarity. RESULTS: We show that three mutations affecting eyespot size and/or colour composition in Bicyclus anynana butterflies occurred in the same locus, and that two of them are embryonic recessive lethal. Using surgical manipulations and analysis of gene expression patterns in developing wings, we demonstrate that the effects on eyespot morphology are due to changes in the epidermal response component of eyespot induction. Our analysis of morphology and of gene expression in mutant embryos shows that they have a typical segment polarity phenotype, consistent with the mutant locus encoding a negative regulator of Wingless signalling. CONCLUSIONS: This study characterizes the segregation and developmental effects of alleles at a single locus that controls the morphology of a lineage-specific trait (butterfly eyespots) and a conserved process (embryonic segment polarity and, specifically, the regulation of Wingless signalling). Because no gene with such function was found in the orthologous, highly syntenic genomic regions of two other lepidopterans, we hypothesize that our locus is a yet undescribed, possibly lineage-specific, negative regulator of the conserved Wnt/Wg pathway. Moreover, the fact that this locus interferes with multiple aspects of eyespot morphology and maps to a genomic region containing key wing pattern loci in different other butterfly species suggests it might correspond to a 'hotspot' locus in the diversification of this novel trait.

Development of a wingless morph in the ladybird beetle, Adalia bipunctata.

Many taxa of winged insects have independently lost the ability to fly and often possess reduced wings. Species exhibiting natural variation in wing morphology provide opportunities to investigate the genetics and developmental processes underlying the evolution of alternative wing morphs. Although many wing dimorphic species of beetles are known, the underlying mechanisms of variation are not well understood in this insect order. Here, we examine wing development of wild type and natural wingless morphs of the two-spot ladybird beetle, Adalia bipunctata. We show that both pairs of wings are distally truncated in the wingless adults. A laboratory population of the wingless morph displays heritable variation in the degree of wing truncation, reflecting reduced growth of the larval wing discs. The coexistence of variable wingless morphs supports the idea that typical monomorphic wingless insects may be the result of a gradual evolution of wing loss. Gene expression patterns in wing discs suggest that the conserved gene network controlling wing development in wild-type Adalia is disrupted in the dorsoventral patterning pathway in the wingless morphs. Previous research on several species of ant has revealed that the anteroposterior wing patterning pathway is disrupted in wingless workers. Future investigations should confirm whether interruptions in both taxa are limited to the patterning pathways found thus far, or whether there are also shared interruption points. Nevertheless, our results highlight that diverse mechanisms of development are likely to underlie the evolution of wingless insects.

A gene-based linkage map for Bicyclus anynana butterflies allows for a comprehensive analysis of synteny with the lepidopteran reference genome.

Lepidopterans (butterflies and moths) are a rich and diverse order of insects, which, despite their economic impact and unusual biological properties, are relatively underrepresented in terms of genomic resources. The genome of the silkworm Bombyx mori has been fully sequenced, but comparative lepidopteran genomics has been hampered by the scarcity of information for other species. This is especially striking for butterflies, even though they have diverse and derived phenotypes (such as color vision and wing color patterns) and are considered prime models for the evolutionary and developmental analysis of ecologically relevant, complex traits. We focus on Bicyclus anynana butterflies, a laboratory system for studying the diversification of novelties and serially repeated traits. With a panel of 12 small families and a biphasic mapping approach, we first assigned 508 expressed genes to segregation groups and then ordered 297 of them within individual linkage groups. We also coarsely mapped seven color pattern loci. This is the richest gene-based map available for any butterfly species and allowed for a broad-coverage analysis of synteny with the lepidopteran reference genome. Based on 462 pairs of mapped orthologous markers in Bi. anynana and Bo. mori, we observed strong conservation of gene assignment to chromosomes, but also evidence for numerous large- and small-scale chromosomal rearrangements. With gene collections growing for a variety of target organisms, the ability to place those genes in their proper genomic context is paramount. Methods to map expressed genes and to compare maps with relevant model systems are crucial to extend genomic-level analysis outside classical model species. Maps with gene-based markers are useful for comparative genomics and to resolve mapped genomic regions to a tractable number of candidate genes, especially if there is synteny with related model species. This is discussed in relation to the identification of the loci contributing to color pattern evolution in butterflies.

Conserved developmental processes and the formation of evolutionary novelties: examples from butterfly wings.

The origin and diversification of evolutionary novelties-lineage-specific traits of new adaptive value-is one of the key issues in evolutionary developmental biology. However, comparative analysis of the genetic and developmental bases of such traits can be difficult when they have no obvious homologue in model organisms. The finding that the evolution of morphological novelties often involves the recruitment of pre-existing genes and/or gene networks offers the potential to overcome this challenge. Knowledge about shared developmental processes obtained from extensive studies in model organisms can then be used to understand the origin and diversification of lineage-specific structures. Here, we illustrate this approach in relation to eyespots on the wings of Bicyclus anynana butterflies. A number of spontaneous mutations isolated in the laboratory affect eyespots, lepidopteran-specific features, and also processes that are shared by most insects. We discuss how eyespot mutants with disturbed embryonic development may help elucidate the genetic pathways involved in eyespot formation, and how venation mutants with altered eyespot patterns might shed light on mechanisms of eyespot development.