Hybrid speciation driven by multilocus introgression of ecological traits.

Hybridization allows adaptations to be shared among lineages and may trigger the evolution of new species1,2. However, convincing examples of homoploid hybrid speciation remain rare because it is challenging to demonstrate that hybridization was crucial in generating reproductive isolation3. Here we combine population genomic analysis with quantitative trait locus mapping of species-specific traits to examine a case of hybrid speciation in Heliconius butterflies. We show that Heliconius elevatus is a hybrid species that is sympatric with both parents and has persisted as an independently evolving lineage for at least 180,000 years. This is despite pervasive and ongoing gene flow with one parent, Heliconius pardalinus, which homogenizes 99% of their genomes. The remaining 1% introgressed from the other parent, Heliconius melpomene, and is scattered widely across the H. elevatus genome in islands of divergence from H. pardalinus. These islands contain multiple traits that are under disruptive selection, including colour pattern, wing shape, host plant preference, sex pheromones and mate choice. Collectively, these traits place H. elevatus on its own adaptive peak and permit coexistence with both parents. Our results show that speciation was driven by introgression of ecological traits, and that speciation with gene flow is possible with a multilocus genetic architecture.

Wing pattern diversity in Eunica butterflies (Nymphalidae: Biblidinae): phylogenetic analysis implies decoupled adaptive trends in dorsal sexual dimorphism and ventral eyespot evolution.

Butterfly eyespots are wing patterns reminiscent of vertebrate eyes, formed by concentric rings of contrastingly coloured scales. Eyespots are usually located close to the wing margin and often regarded as the single most conspicuous pattern element of butterfly wing colour displays. Recent efforts to understand the processes involved in the formation of eyespots have been driven mainly by evo-devo approaches focused on model species. However, patterns of change implied by phylogenetic relationships can also inform hypotheses about the underlying developmental mechanisms associated with the formation or disappearance of eyespots, and the limits of phenotypic diversity occurring in nature. Here we present a combined evidence phylogenetic hypothesis for the genus Eunica, a prominent member of diverse Neotropical butterfly communities, that features notable variation among species in eyespot patterns on the ventral hind wing surface. The data matrix consists of one mitochondrial gene region (COI), four nuclear gene regions (GAPDH, RPS5, EF1a and Wingless) and 68 morphological characters. A combined cladistic analysis with all the characters concatenated produced a single most parsimonious tree that, although fully resolved, includes many nodes with modest branch support. The phylogenetic hypothesis presented corroborates a previously proposed morphological trend leading to the loss of eyespots, together with an increase in the size of the conserved eyespots, relative to outgroup taxa. Furthermore, wing colour pattern dimorphism and the presence of androconia suggest that the most remarkable instances of sexual dimorphism are present in the species of Eunica with the most derived eyespot patterns, and are in most cases accompanied by autapomorphic combinations of scent scales and "hair pencils". We discuss natural and sexual selection as potential adaptive explanations for dorsal and ventral wing patterns.

Blind measurements did not confirm effects of forest fragmentation on fluctuating asymmetry of a tropical butterfly Morpho helenor.

Re-evaluation of photographs of the tropical butterfly Morpho helenor from a previous study (Pignataro et al. 2023) revealed that its conclusion regarding increased wing fluctuating asymmetry in forest edge habitats compared to forest interior habitats could not be replicated. This discrepancy likely arises from (i) original measurements not being conducted blindly, (ii) insufficient photograph quality hindering accurate landmark selection, and (iii) a lack of detailed description of the measurement protocol. The likelihood of false positive discoveries within the published data concerning the impacts of environmental stress on the fluctuating asymmetry of plants and animals is probably higher than previously assumed.

Neural divergence and hybrid disruption between ecologically isolated Heliconius butterflies.

The importance of behavioral evolution during speciation is well established, but we know little about how this is manifest in sensory and neural systems. A handful of studies have linked specific neural changes to divergence in host or mate preferences associated with speciation. However, the degree to which brains are adapted to local environmental conditions, and whether this contributes to reproductive isolation between close relatives that have diverged in ecology, remains unknown. Here, we examine divergence in brain morphology and neural gene expression between closely related, but ecologically distinct, Heliconius butterflies. Despite ongoing gene flow, sympatric species pairs within the melpomene-cydno complex are consistently separated across a gradient of open to closed forest and decreasing light intensity. By generating quantitative neuroanatomical data for 107 butterflies, we show that Heliconius melpomene and Heliconius cydno clades have substantial shifts in brain morphology across their geographic range, with divergent structures clustered in the visual system. These neuroanatomical differences are mirrored by extensive divergence in neural gene expression. Differences in both neural morphology and gene expression are heritable, exceed expected rates of neutral divergence, and result in intermediate traits in first-generation hybrid offspring. Strong evidence of divergent selection implies local adaptation to distinct selective optima in each parental microhabitat, suggesting the intermediate traits of hybrids are poorly matched to either condition. Neural traits may therefore contribute to coincident barriers to gene flow, thereby helping to facilitate speciation.

In vivo visualization of butterfly scale cell morphogenesis in Vanessa cardui.

During metamorphosis, the wings of a butterfly sprout hundreds of thousands of scales with intricate microstructures and nano-structures that determine the wings' optical appearance, wetting characteristics, thermodynamic properties, and aerodynamic behavior. Although the functional characteristics of scales are well known and prove desirable in various applications, the dynamic processes and temporal coordination required to sculpt the scales' many structural features remain poorly understood. Current knowledge of scale growth is primarily gained from ex vivo studies of fixed scale cells at discrete time points; to fully understand scale formation, it is critical to characterize the time-dependent morphological changes throughout their development. Here, we report the continuous, in vivo, label-free imaging of growing scale cells of Vanessa cardui using speckle-correlation reflection phase microscopy. By capturing time-resolved volumetric tissue data together with nanoscale surface height information, we establish a morphological timeline of wing scale formation and gain quantitative insights into the underlying processes involved in scale cell patterning and growth. We identify early differences in the patterning of cover and ground scales on the young wing and quantify geometrical parameters of growing scale features, which suggest that surface growth is critical to structure formation. Our quantitative, time-resolved in vivo imaging of butterfly scale development provides the foundation for decoding the processes and biomechanical principles involved in the formation of functional structures in biological materials.

Two centuries of monarch butterfly collections reveal contrasting effects of range expansion and migration loss on wing traits.

Migratory animals exhibit traits that allow them to exploit seasonally variable habitats. In environments where migration is no longer beneficial, such as oceanic islands, migration-association traits may be selected against or be under relaxed selection. Monarch butterflies are best known for their continent-scale migration in North America but have repeatedly become established as nonmigrants in the tropical Americas and on Atlantic and Pacific Islands. These replicated nonmigratory populations provide natural laboratories for understanding the rate of evolution of migration-associated traits. We measured >6,000 museum specimens of monarch butterflies collected from 1856 to the present as well as contemporary wild-caught monarchs from around the world. We determined 1) how wing morphology varies across the monarch's global range, 2) whether initial long-distance founders were particularly suited for migration, and 3) whether recently established nonmigrants show evidence for contemporary phenotypic evolution. We further reared >1,000 monarchs from six populations around the world under controlled conditions and measured migration-associated traits. Historical specimens show that 1) initial founders are well suited for long-distance movement and 2) loss of seasonal migration is associated with reductions in forewing size and elongation. Monarch butterflies raised in a common garden from four derived nonmigratory populations exhibit genetically based reductions in forewing size, consistent with a previous study. Our findings provide a compelling example of how migration-associated traits may be favored during the early stages of range expansion, and also the rate of reductions in those same traits upon loss of migration.

A release from developmental bias accelerates morphological diversification in butterfly eyespots.

Development can bias the independent evolution of traits sharing ontogenetic pathways, making certain evolutionary changes less likely. The eyespots commonly found on butterfly wings each have concentric rings of differing colors, and these serially repeated pattern elements have been a focus for evo-devo research. In the butterfly family Nymphalidae, eyespots have been shown to function in startling or deflecting predators and to be involved in sexual selection. Previous work on a model species of Mycalesina butterfly, Bicyclus anynana, has provided insights into the developmental control of the size and color composition of individual eyespots. Experimental evolution has also shown that the relative size of a pair of eyespots on the same wing surface is highly flexible, whereas they are resistant to diverging in color composition, presumably due to the underlying shared developmental process. This fixed color composition has been considered as a prime example of developmental bias with significant consequences for wing pattern evolution. Here, we test this proposal by surveying eyespots across the whole subtribe of Mycalesina butterflies and demonstrate that developmental bias shapes evolutionary diversification except in the genus Heteropsis which has gained independent control of eyespot color composition. Experimental manipulations of pupal wings reveal that the bias has been released through a novel regional response of the wing tissue to a conserved patterning signal. Our study demonstrates that development can bias the evolutionary independence of traits, but it also shows how bias can be released through developmental innovations, thus, allowing rapid morphological change, facilitating evolutionary diversification.

Exposure to elevated temperature during development affects eclosion and morphology in the temperate Pieris napi butterfly (Lepidoptera: Pieridae).

Global warming has been identified as one of the main drivers of population decline in insect pollinators. One aspect of the insect life cycle that would be particularly sensitive to elevated temperatures is the developmental transition from larva to adult. Temperature-induced modifications to the development of body parts and sensory organs likely have functional consequences for adult behaviour. To date, we have little knowledge about the effect of sub-optimal temperature on the development and functional morphology of different body parts, particularly sensory organs, in ectothermic solitary pollinators such as butterflies. To address this knowledge gap, we exposed the pupae of the butterfly Pieris napi to either 23 °C or 32 °C and measured the subsequent effects on eclosion, body size and the development of the wings, proboscis, eyes and antennae. In comparison to individuals that developed at 23 °C, we found that exposure to 32 °C during the pupal stage increased mortality and decreased time to eclose. Furthermore, both female and male butterflies that developed at 32 °C were smaller and had shorter proboscides, while males had shorter antennae. In contrast, we found no significant effect of rearing temperature on wing and eye size or wing deformity. Our findings suggest that increasing global temperatures and its corresponding co-stressors, such as humidity, will impact the survival of butterflies by impairing eclosion and the proper development of body and sensory organs.

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.

Cretaceous lophocoronids with short proboscis and retractable female genitalia provide the earliest evidence for their feeding and oviposition habits.

We describe two new species of Lophocoronidae: Acanthocorona hedida Zhang, Shih and Engel sp. n. and Acanthocorona venulosa Zhang, Shih and Engel sp. n., and an undetermined specimen from mid-Cretaceous Kachin amber. Phylogenetic analysis of basal lepidopteran lineages, including three extinct families, was undertaken. The analysis supported monophyly of Glossata although internal relationships remain controversial. Acanthocorona and Lophocorona form a monophyletic group. It is likely that short and simply structured proboscides of Acanthocorona were used to sip water droplets, pollination drops from gymnosperms, nectar from early flowers, or sap from injured leaves. Both retracted and extended ovipositors are preserved in the material reported here, revealing their morphology and indicating that these Cretaceous lophocoronids inserted eggs into the tissues of their host plants.

Molecular logic behind the three-way stochastic choices that expand butterfly colour vision.

Butterflies rely extensively on colour vision to adapt to the natural world. Most species express a broad range of colour-sensitive Rhodopsin proteins in three types of ommatidia (unit eyes), which are distributed stochastically across the retina. The retinas of Drosophila melanogaster use just two main types, in which fate is controlled by the binary stochastic decision to express the transcription factor Spineless in R7 photoreceptors. We investigated how butterflies instead generate three stochastically distributed ommatidial types, resulting in a more diverse retinal mosaic that provides the basis for additional colour comparisons and an expanded range of colour vision. We show that the Japanese yellow swallowtail (Papilio xuthus, Papilionidae) and the painted lady (Vanessa cardui, Nymphalidae) butterflies have a second R7-like photoreceptor in each ommatidium. Independent stochastic expression of Spineless in each R7-like cell results in expression of a blue-sensitive (Spineless(ON)) or an ultraviolet (UV)-sensitive (Spineless(OFF)) Rhodopsin. In P. xuthus these choices of blue/blue, blue/UV or UV/UV sensitivity in the two R7 cells are coordinated with expression of additional Rhodopsin proteins in the remaining photoreceptors, and together define the three types of ommatidia. Knocking out spineless using CRISPR/Cas9 (refs 5, 6) leads to the loss of the blue-sensitive fate in R7-like cells and transforms retinas into homogeneous fields of UV/UV-type ommatidia, with corresponding changes in other coordinated features of ommatidial type. Hence, the three possible outcomes of Spineless expression define the three ommatidial types in butterflies. This developmental strategy allowed the deployment of an additional red-sensitive Rhodopsin in P. xuthus, allowing for the evolution of expanded colour vision with a greater variety of receptors. This surprisingly simple mechanism that makes use of two binary stochastic decisions coupled with local coordination may prove to be a general means of generating an increased diversity of developmental outcomes.

Doublesex Mediates the Development of Sex-Specific Pheromone Organs in Bicyclus Butterflies via Multiple Mechanisms.

The Bicyclus lineage of satyrid butterflies exhibits male-specific traits, the scent organs, used for chemical communication during courtship. These organs consist of tightly packed brush-like scales (hair-pencils) that rub against scent patches to disperse pheromones, but the evolution and molecular basis of these organ's male-limited development remains unknown. Here, we examine the evolution of the number and location of the scent patches and hair-pencils within 53 species of Bicyclus butterflies, and the involvement of the sex determinant gene doublesex (dsx) in scent organ development in Bicyclus anynana using CRISPR/Cas9. We show that scent patches and hair-pencils arose via multiple, independent gains, in a correlated manner. Further, an initially nonsex-specific Dsx protein expression pattern in developing wing discs becomes male-specific and spatially refined to areas that develop the scent patches. Functional perturbations of dsx show that this gene activates patch development in males whereas hair-pencils develop in both sexes without Dsx input. Dsx in females is, instead, required to repress hair-pencils whereas Dsx in males regulates minor aspects of its development. These findings suggest that the patches and hair-pencils evolve as correlated composite organs presumably due to their functional integration. Divergence in the function of dsx isoforms occurred across the sexes, where the male isoform promotes patch development in males and the female isoform represses hair-pencil development in females, both leading to the development of male-limited traits. Furthermore, evolution in number of patches in males is due to the evolution of spatial regulation of dsx.

Genomics of altitude-associated wing shape in two tropical butterflies.

Understanding how organisms adapt to their local environment is central to evolution. With new whole-genome sequencing technologies and the explosion of data, deciphering the genomic basis of complex traits that are ecologically relevant is becoming increasingly feasible. Here, we studied the genomic basis of wing shape in two Neotropical butterflies that inhabit large geographical ranges. Heliconius butterflies at high elevations have been shown to generally have rounder wings than those in the lowlands. We reared over 1,100 butterflies from 71 broods of H. erato and H. melpomene in common-garden conditions and showed that wing aspect ratio, that is, elongatedness, is highly heritable in both species and that elevation-associated wing aspect ratio differences are maintained. Genome-wide associations with a published data set of 666 whole genomes from across a hybrid zone, uncovered a highly polygenic basis to wing aspect ratio variation in the wild. We identified several genes that have roles in wing morphogenesis or wing aspect ratio variation in Drosophila flies, making them promising candidates for future studies. There was little evidence for molecular parallelism in the two species, with only one shared candidate gene, nor for a role of the four known colour pattern loci, except for optix in H. erato. Thus, we present the first insights into the heritability and genomic basis of within-species wing aspect ratio in two Heliconius species, adding to a growing body of evidence that polygenic adaptation may underlie many ecologically relevant traits.

Random lasers from photonic crystal wings of butterfly and moth for speckle-free imaging.

Several biological membranes have been served as scattering materials of random lasers, but few of them include natural photonic crystals. Here, we propose and demonstrate a facile approach to fabricating high-performance biological photonic crystal random lasers, which is cost-effective and reproducible for mass production. As a benchmark, optical and lasing properties of dye-coated Lepidoptera wings, including Papilio ulysses butterfly and Chrysiridia rhipheus moth, are characterized and show a stable laser emission with a superior threshold of 0.016 mJ/cm2, as compared to previous studies. To deploy the proposed devices in practical implementation, we have applied the as-fabricated biological devices to bright speckle-free imaging applications, which is a more sustainable and more accessible imaging strategy.

Comparative Phylogenetics of Papilio Butterfly Wing Shape and Size Demonstrates Independent Hindwing and Forewing Evolution.

The complex forces that shape butterfly wings have long been a subject of experimental and comparative research. Butterflies use their wings for flight, camouflage, mate recognition, warning, and mimicry. However, general patterns and correlations among wing shape and size evolution are still poorly understood. We collected geometric morphometric measurements from over 1400 digitized museum specimens of Papilio swallowtails and combined them with phylogenetic data to test two hypotheses: 1) forewing shape and size evolve independently of hindwing shape and size and 2) wing size evolves more quickly than wing shape. We also determined the major axes of wing shape variation and discovered that most shape variability occurs in hindwing tails and adjacent areas. We conclude that forewing shape and size are functionally and biomechanically constrained, whereas hindwings are more labile, perhaps in response to disruptive selective pressure for Batesian mimicry or against predation. The development of a significant, re-usable, digitized data resource will enable further investigation on tradeoffs between flight performance and ecological selective pressures, along with the degree to which intraspecific, local-scale selection may explain macroevolutionary patterns. [Batesian mimicry; Lepidoptera; geometric morphometrics; museum specimens.].

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.

Secondary contact zones of closely-related Erebia butterflies overlap with narrow phenotypic and parasitic clines.

Zones of secondary contact between closely related taxa are a common legacy of the Quaternary ice ages. Despite their abundance, the factors that keep species apart and prevent hybridization are often unknown. Here, we study a very narrow contact zone between three closely related butterfly species of the Erebia tyndarus species complex. Using genomic data, we first determined whether gene flow occurs and then assessed whether it might be hampered by differences in chromosome number between some species. We found interspecific gene flow between sibling species that differ in karyotype by one chromosome. Conversely, only F1 hybrids occurred between two species that have the same karyotype, forming a steep genomic cline. In a second step, we fitted clines to phenotypic, ecological and parasitic data to identify the factors associated with the genetic cline. We found clines for phenotypic data and the prevalence of the endosymbiont parasite Wolbachia to overlap with the genetic cline, suggesting that they might be drivers for separating the two species. Overall, our results highlight that some gene flow is possible between closely related species despite different chromosome numbers, but that other barriers restrict such gene flow.

Molecular and morphometric divergence of four species of butterflies (Nymphalidae and Pieridae) from the Western Himalaya, India.

Morphometric and molecular divergence among four butterfly species of the families Nymphalidae and Pieridae from the western Himalaya region were investigated using molecular tools, traditional morphometric measures and a truss network system. The considered species were Danaus chrysippus, Vanessa cardui, Pieris brassicae and Pieris canidia. Traditional taxonomy is sometimes unable to discriminate cryptic species or species that have close morphological features. Although taxonomists carefully examine external body features to differentiate the species; however, there is a risk for misidentification during a visual assessment of cryptic species. Therefore, we aimed to use the truss network system of 14 morphological landmarks interconnected to yield 90 variables about molecular taxonomy. Principal component analysis (PCA), discriminant function analysis (DFA) and cluster analysis (CA) were employed to determine morphometric variations. In the traditional analysis, 79, 68, 16 and 5 characters out of 90 were found significant (p < 0.05) for D. chrysippus, V. cardui, P. brassicae and P. canidia, respectively. One to seven principal components were extracted through PCA; they explained 87.5-100% of the total variance in samples. Notably, DFA correctly classified 100% of the original grouped cases and 100% of the cross-validated grouped cases. However, the variations were not the same for the two different methods (truss and traditional) employed for the analysis. We correctly identified all the species; the interspecies sequence divergence was between 0.1034 and 0.1398, and the intra-species sequence divergence range was 0.0001 to 0.0128 using the Cytochrome c oxidase subunit-I (COI) gene. The present study provides useful information about the application and complementary role of traditional with truss morphometric analysis for the precise identification and classification of the selected species.

doublesex is a mimicry supergene.

One of the most striking examples of sexual dimorphism is sex-limited mimicry in butterflies, a phenomenon in which one sex--usually the female--mimics a toxic model species, whereas the other sex displays a different wing pattern. Sex-limited mimicry is phylogenetically widespread in the swallowtail butterfly genus Papilio, in which it is often associated with female mimetic polymorphism. In multiple polymorphic species, the entire wing pattern phenotype is controlled by a single Mendelian 'supergene'. Although theoretical work has explored the evolutionary dynamics of supergene mimicry, there are almost no empirical data that address the critical issue of what a mimicry supergene actually is at a functional level. Using an integrative approach combining genetic and association mapping, transcriptome and genome sequencing, and gene expression analyses, we show that a single gene, doublesex, controls supergene mimicry in Papilio polytes. This is in contrast to the long-held view that supergenes are likely to be controlled by a tightly linked cluster of loci. Analysis of gene expression and DNA sequence variation indicates that isoform expression differences contribute to the functional differences between dsx mimicry alleles, and protein sequence evolution may also have a role. Our results combine elements from different hypotheses for the identity of supergenes, showing that a single gene can switch the entire wing pattern among mimicry phenotypes but may require multiple, tightly linked mutations to do so.

Polymorphism at a mimicry supergene maintained by opposing frequency-dependent selection pressures.

Explaining the maintenance of adaptive diversity within populations is a long-standing goal in evolutionary biology, with important implications for conservation, medicine, and agriculture. Adaptation often leads to the fixation of beneficial alleles, and therefore it erodes local diversity so that understanding the coexistence of multiple adaptive phenotypes requires deciphering the ecological mechanisms that determine their respective benefits. Here, we show how antagonistic frequency-dependent selection (FDS), generated by natural and sexual selection acting on the same trait, maintains mimicry polymorphism in the toxic butterfly Heliconius numata Positive FDS imposed by predators on mimetic signals favors the fixation of the most abundant and best-protected wing-pattern morph, thereby limiting polymorphism. However, by using mate-choice experiments, we reveal disassortative mate preferences of the different wing-pattern morphs. The resulting negative FDS on wing-pattern alleles is consistent with the excess of heterozygote genotypes at the supergene locus controlling wing-pattern variation in natural populations of H. numata The combined effect of positive and negative FDS on visual signals is sufficient to maintain a diversity of morphs displaying accurate mimicry with other local prey, although some of the forms only provide moderate protection against predators. Our findings help understand how alternative adaptive phenotypes can be maintained within populations and emphasize the need to investigate interactions between selective pressures in other cases of puzzling adaptive polymorphism.