DNA barcodes from over-a-century-old type specimens shed light on the taxonomy of a group of rare butterflies (Lepidoptera: Nymphalidae: Calinaginae).

We analyzed COI barcode sequences from 138 over-a-century old specimens of Calinaga including 36 name-bearing type specimens stored at the Natural History Museum London. These new data, combined with previously available RPS5 sequences, divide the Calinaga samples into four well-supported mitochondrial lineages that together with a novel wing-pattern analysis, support the recognition of six species (lhatso, buddha, brahma, aborica, formosana and davidis), with all other names subsumed either as subspecies or synonyms. One new taxon is described, Calinaga aborica naima Vane-Wright, ssp. n.

Male and female contributions to diversity among birdwing butterfly images.

Machine learning (ML) newly enables tests for higher inter-species diversity in visible phenotype (disparity) among males versus females, predictions made from Darwinian sexual selection versus Wallacean natural selection, respectively. Here, we use ML to quantify variation across a sample of > 16,000 dorsal and ventral photographs of the sexually dimorphic birdwing butterflies (Lepidoptera: Papilionidae). Validation of image embedding distances, learnt by a triplet-trained, deep convolutional neural network, shows ML can be used for automated reconstruction of phenotypic evolution achieving measures of phylogenetic congruence to genetic species trees within a range sampled among genetic trees themselves. Quantification of sexual disparity difference (male versus female embedding distance), shows sexually and phylogenetically variable inter-species disparity. Ornithoptera exemplify high embedded male image disparity, diversification of selective optima in fitted multi-peak OU models and accelerated divergence, with cases of extreme divergence in allopatry and sympatry. However, genus Troides shows inverted patterns, including comparatively static male embedded phenotype, and higher female than male disparity - though within an inferred selective regime common to these females. Birdwing shapes and colour patterns that are most phenotypically distinctive in ML similarity are generally those of males. However, either sex can contribute majoritively to observed phenotypic diversity among species.

Bottom-up butterfly model with thorax-pitch control and wing-pitch flexibility.

The diversity in butterfly morphology has attracted many people around the world since ancient times. Despite morphological diversity, the wing and body kinematics of butterflies have several common features. In the present study, we constructed a bottom-up butterfly model, whose morphology and kinematics are simplified while preserving the important features of butterflies. The present bottom-up butterfly model is composed of two trapezoidal wings and a rod-shaped body with a thorax and abdomen. Its wings are flapped downward in the downstroke and backward in the upstroke by changing the geometric angle of attack (AOA). The geometric AOA is determined by the thorax-pitch and wing-pitch angles. The thorax-pitch angle is actively controlled by abdominal undulation, and the wing-pitch angle is passively determined because of a rotary spring representing the basalar and subalar muscles connecting the wings and thorax. We investigated the effectiveness of abdominal undulation for thorax-pitch control and how wing-pitch flexibility affects aerodynamic-force generation and thorax-pitch control, through numerical simulations using the immersed boundary-lattice Boltzmann method. As a result, the thorax-pitch angle perfectly follows the desired angle through abdominal undulation. In addition, there is an optimal wing-pitch flexibility that maximizes the flying speed in both the forward and upward directions, but the effect of wing-pitch flexibility on thorax-pitch control is not significant. Finally, we compared the flight behavior of the present bottom-up butterfly model with that of an actual butterfly. It was found that the present model does not reproduce reasonable body kinematics but can provide reasonable aerodynamics in butterfly flights.

Selection drives divergence of eye morphology in sympatric Heliconius butterflies.

When populations experience different sensory conditions, natural selection may favor sensory system divergence, affecting peripheral structures and/or downstream neural pathways. We characterized the outer eye morphology of sympatric Heliconius butterflies from different forest types and their first-generation reciprocal hybrids to test for adaptive visual system divergence and hybrid disruption. In Panama, Heliconius cydno occurs in closed forests, whereas Heliconius melpomene resides at the forest edge. Among wild individuals, H. cydno has larger eyes than H. melpomene, and there are heritable, habitat-associated differences in the visual brain structures that exceed neutral divergence expectations. Notably, hybrids have intermediate neural phenotypes, suggesting disruption. To test for similar effects in the visual periphery, we reared both species and their hybrids in common garden conditions. We confirm that H. cydno has larger eyes and provide new evidence that this is driven by selection. Hybrid eye morphology is more H. melpomene-like despite body size being intermediate, contrasting with neural trait intermediacy. Overall, our results suggest that eye morphology differences between H. cydno and H. melpomene are adaptive and that hybrids may suffer fitness costs due to a mismatch between the peripheral visual structures and previously described neural traits that could affect visual performance.

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.

Not all apparently gynandromorphic butterflies are gynandrous: The case of Polyommatus icarus and its relatives (Lepidoptera: Lycaenidae).

Beside the more than two thousand normal specimens of Polyommatus icarus (Rottemburg, 1775) yielded by rearing experiments, there was one perfectly bilateral dichromatic individual first considered to be gynandrous. On the basis of analysing genitalia traits, wing surface covering scale micromorphology, and the spectral characteristics of the blue colour generated by the cover scales, the gender of the specimen has been identified as female. This exemplar was investigated in comparison with gynandrous specimens from the collections of the Hungarian Natural History Museum exhibiting various degrees of intermixing of blue and brown coloration. Focus stacking microscopy for detailed scale morphology and UV-visible reflectance spectroscopy was used for the characterization of the optical properties. Inspecting literature references and the Lycaenidae collection of the museum, further examples have been found for female bilateral dichromatism in the closely related polyommatine lycaenid species Lysandra bellargus (Rottemburg, 1775) and Lysandra coridon (Poda, 1761) what suggests that polyommatine female dichromaticity may be displayed by the manner of bilaterality and mosaicism, phenomena hitherto solely connected to gynandromorphy.

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.

Surprise RNA paints colorful patterns on butterfly wings.

Understudied means of regulating genes is likely widespread in butterflies-and perhaps other animals.

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.

Immature Stages, Natural History and Population Biology of Opoptera syme (Hübner, 1821), (Lepidoptera: Nymphalidae: Satyrinae) from Southeastern Brazil.

The present study describes the immature stages of Opoptera syme (Hübner, [1821]) using SEM and Micro-CT to generate 2D and 3D models to study the morphology and chaetotaxy, and present information on the natural history, behavior, and population biology. In laboratory, eggs were laid singly, and the isolated larvae passed through six to seven instars. Host plant in the study site is unknown, but larvae are known to feed on bamboos; in laboratory, larvae accepted the ornamental bamboo Bambusa textilis McClure, 1940. Adults are diurnal and univoltine, flying from December to April (the austral summer). The present study adds information to the biology and natural history of Brassolini, an iconic and still poorly known tribe of Neotropical butterflies.

Ultramorphology of the proboscis and sensilla of the Asian corn borer Ostrinia furnacalis (Lepidoptera: Crambidae).

The Asian corn borer, Ostrinia furnacalis (Guenée), is one of the most destructive lepidopteran pests affecting maize in Asia. Previous research on the feeding behavior of O. furnacalis has mostly focused on larvae, but the adult feeding preference has been unclear hitherto. In this study, we investigated the ultramorphology of the proboscis and sensilla of O. furnacalis using scanning electron microscopy and made morphological comparisons between the sexes, attempting to make predictions on the food preference and feeding behavior of the adults. The proboscis of O. furnacalis is divided into zones 1 and 2 with three types of sensilla, namely chaetica, basiconica, and styloconica. The entire proboscis and zone 2 of the females are significantly longer than those of the males. The main sexual difference in the sensilla is shown in the number of sensilla styloconica, which are eight in females but nine in males. The feeding preference of adult O. furnacalis is discussed based on the morphology of the proboscis and sensilla.

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.

Early origin and diverse phenotypic implementation of iridescent UV patterns for sexual signaling in pierid butterflies.

Iridescent ultraviolet (IUV) patterns on pierid butterfly wings are phenotypic adaptations commonly used as sexual signals, generated by scales with ultrastructural modifications. Pierid IUV patterns are sexually dichromatic, with reduced size in females, where conspicuous sexual signaling balances courtship against ecological predation. There have been no phylogenetic reconstructions of IUV within Pieridae and little morphological characterization of phenotypic diversity. Our genus-wide characterization of IUV revealed the uniform similarity of stacked lamellar ridges on the dorsal surface of cover scales. We tested a hypothesis of single versus multiple origins by reconstructing a phylogeny of 534 species (~43.2% described species), with all genera represented, and a trait matrix of 734 species (~59.4%) screened for IUV. A single, early dimorphic origin of IUV followed by several losses and gains received strong support, concluding that IUV patterns and structural coloration are old traits. Collectively, these results support the homology of IUV scales and patterns that diversified within several lineages, suggesting an interplay between female-mediated sexual selection and ecological predatory selection.

A review of the South American metalmark genus Alesa Doubleday, 1847 (Lepidoptera: Riodinidae: Eurybiini) with the description of two new species.

The taxonomy of the South American metalmark butterflies of the genus Alesa Doubleday, 1847 is reviewed. A new taxonomic arrangement for Alesa is supported after the analysis of about 600 specimens, including the recognition of two new species, described herein, A. juliae sp. nov. and A. mariae sp. nov.. Alesa negra Röber, 1931, so far recognized as a junior subjective synonym of A. telephae (Boisduval, 1836), is here recognized as a valid species (stat. rev.) and A. amethystina Gallard & Fernandez, 2015 as a junior subjective synonym (syn. nov.) of A. negra, and A. fournierae Lathy, 1958 is recognized as a junior subjective synonym (syn. nov.) of A. amesis (Cramer, 1777). Neotypes are designated for Papilio amesis Cramer, 1777, Erycina prema Godart, [1824] and Mimocastinia egeria Biedermann, 1936. Lectotypes are designated for Erycina priolas Godart, [1824], Eurybia telephae Boisduval, 1836, A. smaragdifera Westwood, 1851, A. lipara Bates, 1868, A. hemiurga Bates, 1868, A. thelydrias Bates, 1868, and Mimocastnia rothschildi Seitz, 1913. Three previously established species groups are here supported by morphological characters including the following species, "prema group": A. prema, A. esmeralda Salazar & Constantino, 2007, A. beneluzi Gallard & Fernandez, 2015, A. rothschildi and A. juliae sp. nov.; "telephae group": A. telephae, A. humilis Brévignon, 2015, A. negra stat. rev. and A. suzana Ahrenholz & Hall, 2010; and "amesis group": A. amesis, A. mariae sp. nov., A. lipara Bates, 1867, A. hemiurga Bates, 1867 and A. thelydrias Bates, 1867. Adults and their genitalia are illustrated for all species available to examination. The main diagnostic characters used to distinguish the species belonging to the "amesis group" are illustrated, and pictures of adults images in situ and distribution maps are provided. Additionally, all species are diagnosed and discussed based on their morphology.

High level of novelty under the hood of convergent evolution.

Little is known about the extent to which species use homologous regulatory architectures to achieve phenotypic convergence. By characterizing chromatin accessibility and gene expression in developing wing tissues, we compared the regulatory architecture of convergence between a pair of mimetic butterfly species. Although a handful of color pattern genes are known to be involved in their convergence, our data suggest that different mutational paths underlie the integration of these genes into wing pattern development. This is supported by a large fraction of accessible chromatin being exclusive to each species, including the de novo lineage-specific evolution of a modular optix enhancer. These findings may be explained by a high level of developmental drift and evolutionary contingency that occurs during the independent evolution of mimicry.

Early Stages and Notes on the Biology of Melitaea gina Higgins, 1941 (Lepidoptera: Nymphalidae) from Northern Zagros Mts (Iran) with the Special Remarks on Taxonomy.

This article presents characteristics of the habitats of Melitaea gina Higgins, 1941 and describes the behavior of adults under natural conditions, their host plant, egg chorion, caterpillars, and pupa morphology. The morphology of the early stages and the taxonomy of M. gina are discussed.

Evolutionary functional morphology of the proboscis and feeding apparatus of hawk moths (Sphingidae: Lepidoptera).

The morphology of the proboscis and associated feeding organs was studied in several nectar-feeding hawk moths, as well as a specialized honey-feeder and two supposedly nonfeeding species. The proboscis lengths ranged from a few millimeters to more than 200 mm. Despite the variation in proboscis length and feeding strategy, the principle external and internal composition of the galeae, the stipes pump, and the suction pump were similar across all species. The morphology of the smooth and slender proboscis is highly conserved among all lineages of nectar-feeding Sphingidae. Remarkably, they share a typical arrangement of the sensilla at the tip. The number and length of sensilla styloconica are independent from proboscis length. A unique proboscis morphology was found in the honey-feeding species Acherontia atropos. Here, the distinctly pointed apex displays a large subterminal opening of the food canal, and thus characterizes a novel type of piercing proboscis in Lepidoptera. In the probably nonfeeding species, the rudimentary galeae are not interlocked and the apex lacks sensilla styloconica; galeal muscles, however, are present. All studied species demonstrate an identical anatomy of the stipes, and suction pump, regardless of proboscis length and diet. Even supposedly nonfeeding Sphingidae possess all organs of the feeding apparatus, suggesting that their proboscis rudiments might still be functional. The morphometric analyses indicate significant positive correlations between galea lumen volume and stipes muscle volume as well as the volume of the food canal and the muscular volume of the suction pump. Size correlations of these functionally connected organs reflect morphological fine-tuning in the evolution of proboscis length and function.

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.

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.

Immature stages and new host plant records for four satyrine species feeding on herbaceous bamboos in southeastern Peru (Lepidoptera: Nymphalidae: Satyrinae: Satyrini).

We here document the immature stages of three euptychiine butterflies, Nhambikuara mima (Butler, 1867), Splendeuptychia furina (Hewitson, 1862), and Paryphthimoides brixius (Godart, [1824]), all found feeding on a species of herbaceous bamboo, Taquara micrantha (Kunth) I.L.C.Oliveira R.P.Oliveira (Poaceae: Bambusoideae: Olyreae) in Madre de Dios, Peru. This study is the first to report the life history of these three taxa with their natural host plant. We provide illustrations of immatures, head capsules, and the host plant for each of these three species. The immature morphology of these taxa supports recent generic arrangements of these three species in comparison with their close relatives, namely Splendeuptychia furina to Nhambikuara mima and Paryphthimoides brixius to Paryphthimoides terrestris (Butler, 1867), a species documented in our successive study. Thus, the present study includes taxonomic implications based on immature stages by discovering putative synapomorphic characters of larvae and pupae. These pairs of closely related species occur in micro-sympatry at the study site in southeastern Peru, and our observations possibly suggest niche partitioning between sibling species. Additionally, we report two herbaceous bamboo species, Olyra latifolia L. and Taquara micrantha (Kunth) I.L.C.Oliveira R.P.Oliveira as the first known natural host plants for Magneuptychia harpyia (C. Felder R. Felder, 1867).