Seasonal Polyphenism Research Articles

Larval growth rate is not a major determinant of adult wing shape and eyespot size in the seasonally polyphenic butterfly Melanitis leda.

Insects often show adaptive phenotypic plasticity where environmental cues during early stages are used to produce a phenotype that matches the environment experienced by adults. Many tropical satyrine butterflies (Nymphalidae: Satyrinae) are seasonally polyphenic and produce distinct wet- and dry-season form adults, providing tight environment-phenotype matching in seasonal environments. In studied Mycalesina butterflies, dry-season forms can be induced in the laboratory by growing larvae at low temperatures or on poor food quality. Since both these factors also tend to reduce larval growth rate, larval growth rate may be an internal cue that translates the environmental cues into the expression of phenotypes. If this is the case, we predict that slower-growing larvae would be more likely to develop a dry-season phenotype. We performed the first experimental study on seasonal polyphenism of a butterfly in the tribe Melanitini. We measured both larval growth rate and adult phenotype (eyespot size and wing shape) of common evening brown butterflies (Melanitis leda), reared at various temperatures and on various host-plant species. We constructed provisional reaction norms, and tested the hypothesis that growth rate mediates between external cues and adult phenotype. Reaction norms were similar to those found in Mycalesina butterflies. We found that both among and within treatments, larvae with lower growth rates (low temperature, particular host plants) were more likely to develop dry-season phenotypes (small eyespots, falcate wing tips). However, among temperature treatments, similar growth rates could lead to very different wing phenotypes, and within treatments the relationships were weak. Moreover, males and females responded differently, and eyespot size and wing shape were not strongly correlated with each other. Overall, larval growth rate seems to be weakly related to eyespot size and wing shape, indicating that seasonal plasticity in M. leda is primarily mediated by other mechanisms.

Transcription Factor CcFoxO Mediated the Transition from Summer Form to Winter Form in Cacopsylla chinensis.

Amid global climate change featuring erratic temperature fluctuations, insects adapt via seasonal polyphenism, essential for population sustainability and reproductive success. Cacopsylla chinensis, influenced by environment variations, displays a distinct summer form and winter form distinguished by significant morphological variations. Previous studies have highlighted the role of temperature receptor CcTPRM in orchestrating the transition in response to 10 °C temperature. Nevertheless, the contribution of the transcription factor FoxO in this process has remained ambiguous. Here, we aimed to explore the correlation between C. chinensis FoxO (CcFoxO) and cold stress responses, while identifying potential energetic substances for monitoring physiological shifts during this transition from summer to winter form under cold stress by using RNAi. Initially, CcFoxO emerges as responsive to low temperatures (10 °C) and is regulated by CcTRPM. Subsequent investigations reveal that CcFoxO facilitates the accumulation of triglycerides and glycogen, thereby influencing the transition from summer form to winter form by affecting cuticle pigment content, cuticle chitin levels, and cuticle thickness. Thus, the knockdown of CcFoxO led to high mortality and failed transition. Overall, our findings demonstrate that CcFoxO governs seasonal polyphenism by regulating energy storage. These insights not only enhance our comprehension of FoxO functionality but also offer avenues for environmentally friendly management strategies for C. chinensis.

Thermal plasticity in protective wing pigmentation is modulated by genotype and food availability in an insect model of seasonal polyphenism

Abstract Phenotypic variation in natural populations results from complex interactions between organisms and their changing environments. The environment shapes both phenotypic frequencies (during adaptation) and organismal phenotypes (through phenotypic plasticity). Developmental plasticity, in particular, refers to the phenomenon whereby an organism's phenotype depends on the environmental conditions during development. It can match phenotype to ecological conditions and help organisms to cope with environmental heterogeneity, including differences between alternating seasons. Experimental studies of developmental plasticity often focus on the impact of individual environmental cues and do not take explicit account of genetic variation. In contrast, natural environments are complex, comprising multiple variables with combined effects that are poorly understood and may vary among genotypes. We investigated the effects of multifactorial environments on the development of the seasonally plastic eyespots of Bicyclus anynana butterflies. Eyespot size depends on developmental temperature and is involved in alternative seasonal strategies for predator avoidance. In nature, both temperature and food availability undergo seasonal fluctuations. However, our understanding of how thermal plasticity in eyespot size varies in response to food availability and across genotypes remains limited. To address this, we investigated the combined effects of temperature (T; two levels: 20°C and 27°C) and food availability (N; two levels: control and limited) during development. We examined their impact on wing and eyespot size in adult males and females from multiple genotypes (G; 28 families). We found evidence of thermal and nutritional plasticity and temperature‐by‐nutrition interactions (significant T × N) on the size of eyespots in both sexes. Food limitation resulted in relatively smaller eyespots and tempered the effects of temperature. Additionally, we found differences among families for thermal plasticity (significant G × T effects), but not for nutritional plasticity (non‐significant G × N effects) nor for the combined effects of temperature and food limitation (non‐significant G × T × N effects). Our results reveal the context dependence of thermal plasticity, with the slope of thermal reaction norms varying across genotypes and across nutritional environments. We discuss these results in the light of the ecological significance of pigmentation and the value of considering thermal plasticity in studies of the biological impact of climate change. Read the free Plain Language Summary for this article on the Journal blog.

Phenotypic plasticity in tropical butterflies is linked to climatic seasonality on a macroevolutionary scale.

Phenotypic plasticity can be adaptive in fluctuating environments by providing rapid environment-phenotype matching and this applies particularly in seasonal environments. African Bicyclus butterflies have repeatedly colonized seasonal savannahs from ancestral forests around the late Miocene, and many species now exhibit seasonal polyphenism. On a macroevolutionary scale, it can be expected that savannah species will exhibit higher plasticity because of experiencing stronger environmental seasonality than forest species. We quantified seasonality using environmental niche modeling and surveyed the degree of plasticity in a key wing pattern element (eyespot size) using museum specimens. We showed that species occurring in highly seasonal environments display strong plasticity, while species in less seasonal or aseasonal environments exhibit surprisingly variable degrees of plasticity, including strong to no plasticity. Furthermore, eyespot size plasticity has a moderate phylogenetic signal and the ancestral Bicyclus likely exhibited some degree of plasticity. We propose hypotheses to explain the range of plasticity patterns seen in less seasonal environments and generate testable predictions for the evolution of plasticity in Bicyclus. Our study provides one of the most compelling cases showing links between seasonality and phenotypic plasticity on a macroevolutionary scale and the potential role of plasticity in facilitating the colonization of novel environments.

MiR-2765 Modulates the Seasonal Polyphenism in Cacopsylla chinensis by Targeting a Novel Cold Rreceptor CcTRPC3.

Polyphenism is a beneficial way in organisms to better cope with changing circumstances and is a hot topic in entomology, evolutionary biology, and ecology. Until now, this phenomenon has been proven to be season-, density-, and diet-dependent; however, there are very few reports on temperature regulation. Cacopsylla chinensis showed seasonal polyphenism, namely as summer- and winter-form, with obvious diversity in phenotypic characteristics in response to seasonal variation. Previous studies have found that low temperature in autumn is an extremely important element in inducing summer-form change to winter-form, but the underlying regulatory mechanism is still a mystery. Herein, we provided the initial evidence that the third instar of the summer-form is the critical period for developing to the winter-form, and 10 °C induces this transition by affecting the total pigment, chitin level, and thickness of the cuticle. Second, CcTPRC3 was proven to function as a novel cold receptor to control this seasonal polyphenism. Moreover, miR-2765 was found to mediate seasonal polyphenism by inhibiting CcTRPC3 expression. Last, we found that cuticle binding proteins CcCPR4 and CcCPR9 function as the downstream signals of CcTRPC3 to regulate the seasonal polyphenism in C. chinensis. In conclusion, our results displayed a novel signal pathway of miR-2765 and CcTRPC3 for the regulation of seasonal polyphenism in C. chinensis. These findings provide insights into the comprehensive analysis of insect polyphenism and are useful in developing potential strategies to block the phase transition for the pest control of C. chinensis.

High promiscuity among females of the invasive pest species Drosophila suzukii.

Drosophila suzukii (Matsumura, 1931), the spotted-wing drosophila, is a highly invasive fruit fly that spread from Southern Asia across most regions of Asia and, in the last 15 years, has invaded Europe and the Americas. It is an economically important pest of small fruits such as berries and stone fruits. Drosophila suzukii speciated by adapting to cooler, mountainous, and forest environments. In temperate regions, it evolved seasonal polyphenism traits which enhanced its survival during stressful winter population bottlenecks. Consequently, in these temperate regions, the populations undergo seasonal reproductive dynamics. Despite its economic importance, no data are available on the behavioural reproductive strategies of this fly. The presence of polyandry, for example, has not been determined despite the important role it might play in the reproductive dynamics of populations. We explored the presence of polyandry in an established population in Trentino, a region in northern Italy. In this area, D. suzukii overcomes the winter bottleneck and undergoes a seasonal reproductive fluctuation. We observed a high remating frequency in females during the late spring demographic explosion that led to the abundant summer population. The presence of a high degree of polyandry and shared paternity associated with the post-winter population increase raises the question of the possible evolutionary adaptive role of this reproductive behaviour in D. suzukii.

Life history traits of cycad blue butterfly, Luthrodes pandava Horsfield (Lepidoptera: Lycaenidae) on cycad hosts in an Island ecosystem

AbstractThe life history traits (egg, larva, and pupa stage) of the Lycaenidae butterfly, Luthrodes pandava Horsfield (Lepidoptera: Lycaenidae) observed and trapped on cycad plants are described from the Andaman and Nicobar Islands (ANI), India. The diversity of cycad host plants offered as larval food influences fecundity and conservation status. Zamia furfuracea is recorded for the first time as a cycad host plant and seasonal polyphenism in the ANI, India.

Winter Is (Not) Coming: Is Climate Change Helping Drosophila suzukii Overwintering?

Anthropogenic challenges, particularly climate change-associated factors, are strongly impacting the behavior, distribution, and survival of insects. Yet how these changes affect pests such as Drosophila suzukii, a cosmopolitan pest of soft-skinned small fruits, remains poorly understood. This polyphagous pest is chill-susceptible, with cold temperatures causing multiple stresses, including desiccation and starvation, also challenging the immune system. Since the invasion of Europe and the United States of America in 2009, it has been rapidly spreading to several European and American countries (both North and South American) and North African and Asian countries. However, globalization and global warming are allowing an altitudinal and latitudinal expansion of the species, and thus the colonization of colder regions. This review explores how D. suzukii adapts to survive during cold seasons. We focus on overwintering strategies of behavioral adaptations such as migration or sheltering, seasonal polyphenism, reproductive adaptations, as well as metabolic and transcriptomic changes in response to cold. Finally, we discuss how the continuation of climate change may promote the ability of this species to survive and spread, and what mitigation measures could be employed to overcome cold-adapted D. suzukii.

Comparative Transcriptome Analysis Reveals bmo-miR-6497-3p Regulate Circadian Clock Genes during the Embryonic Diapause Induction Process in Bivoltine Silkworm.

Simple SummaryDiapause is a type of seasonal polyphenism and is induced by multiple cues, such as light and temperature. However, the molecular mechanisms of how the environment induces diapause remains unclear. We are interested in the epigenetic regulation, especially the non-coding RNA, involved in environmentally induced diapause. The progeny eggs of bivoltine silkworm strains undergoing diapause or not depends on the incubation temperature during the maternal embryonic stage. In this research, we compared the differentially expressed microRNAs in embryos incubated under diapause-inducing temperature (25 °C) and non-diapause-inducing temperature (15 °C) in silkworm. Our results indicate that miRNAs respond to diapause-inducing temperature and regulate the expression of circadian clock genes. Our research not only provides reference for the study of the diapause induction process, but also gives insights into the role of epigenetic modification in diapause.Diapause is one of the survival strategies of insects for confronting adverse environmental conditions. Bombyx mori displays typical embryonic diapause, and offspring diapause depends on the incubation environment of the maternal embryo in the bivoltine strains of the silkworm. However, the molecular mechanisms of the diapause induction process are still poorly understood. In this study, we compared the differentially expressed miRNAs (DEmiRs) in bivoltine silkworm embryos incubated at diapause- (25 °C) and non-diapause (15 °C)-inducing temperatures during the blastokinesis (BK) and head pigmentation (HP) phases using transcriptome sequencing. There were 411 known miRNAs and 71 novel miRNAs identified during the two phases. Among those miRNAs, there were 108 and 74 DEmiRs in the BK and HP groups, respectively. By the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the predicted target genes of the DEmiRs, we found that aside from metabolism, the targets were also enriched in phototransduction-fly and insect hormone biosynthesis in the BK group and the HP group, respectively. Dual luciferase reporter assay illustrated that bmo-miR-6497-3p directly regulated Bmcycle and subsequently regulated the expression of circadian genes. These results imply that microRNAs, as vitally important regulators, respond to different temperatures and participate in the diapause induction process across species.

Predictability of temporal variation in climate and the evolution of seasonal polyphenism in tropical butterfly communities

Phenotypic plasticity in heterogeneous environments can provide tight environment-phenotype matching. However, the prerequisite is a reliable environmental cue(s) that enables organisms to use current environmental information to induce the development of a phenotype with high fitness in a forthcoming environment. Here, we quantify predictability in the timing of precipitation and temperature change to examine how this is associated with seasonal polyphenism in tropical Mycalesina butterflies. Seasonal precipitation in the tropics typically results in distinct selective environments, the wet and dry seasons, and changes in temperature can be a major environmental cue. We sampled communities of Mycalesina butterflies from two seasonal locations and one aseasonal location. Quantifying environmental predictability using wavelet analysis and Colwell's indices confirmed a strong periodicity of precipitation over a 12-month period at both seasonal locations compared to the aseasonal one. However, temperature seasonality and periodicity differed between the two seasonal locations. We further show that: (a) most females from both seasonal locations synchronize their reproduction with the seasons by breeding in the wet season but arresting reproduction in the dry season. In contrast, all species breed throughout the year in the aseasonal location and (b) species from the seasonal locations, but not those from the aseasonal location, exhibited polyphenism in wing pattern traits (eyespot size). We conclude that seasonal precipitation and its predictability are primary factors shaping the evolution of polyphenism in Mycalesina butterflies, and populations or species secondarily evolve local adaptations for cue use that depend on the local variation in the environment.

The European Map Butterfly Araschnia levana as a Model to Study the Molecular Basis and Evolutionary Ecology of Seasonal Polyphenism.

Simple SummaryThe European map butterfly looks different in spring and summer due to day length and temperature. If the butterfly’s caterpillars receive 16 h of light per day, the resulting butterfly hatches a few weeks later with blackish wings. However, if caterpillars receive less than 15.5 h of daylight, many overwinter as pupae. In the following spring, butterflies have orange wings. Overwintering and wing color are decided by hormones. If a certain hormone is released in the first days after the caterpillar has become a pupa, no overwintering takes place, and the wings are black. If this hormone is released later, overwintering occurs, and the wings are orange. Different genes are activated to make either of those two options happen. They guide what happens during overwintering and how long it lasts but also how the butterfly looks once it hatches. We do not yet fully understand how the caterpillars count the amount of light they receive and how this information leads to the differences described above. In addition, the butterfly’s whole body and its immune system are different in the two color types. Here we discuss how the butterfly probably makes these changes happen and which role the environment plays.The European map butterfly Araschnia levana is a well-known example of seasonal polyphenism. Spring and summer imagoes exhibit distinct morphological phenotypes. Key environmental factors responsible for the expression of different morphs are day length and temperature. Larval exposure to light for more than 16 h per day entails direct development and results in the adult f. prorsa summer phenotype. Less than 15.5 h per day increasingly promotes diapause and the adult f. levana spring phenotype. The phenotype depends on the timing of the release of 20-hydroxyecdysone in pupae. Release within the first days after pupation potentially inhibits the default “levana-gene-expression-profile” because pre-pupae destined for diapause or subitaneous development have unique transcriptomic programs. Moreover, multiple microRNAs and their targets are differentially regulated during the larval and pupal stages, and candidates for diapause maintenance, duration, and phenotype determination have been identified. However, the complete pathway from photoreception to timekeeping and diapause or subitaneous development remains unclear. Beside the wing polyphenism, the hormonal and epigenetic modifications of the two phenotypes also include differences in biomechanical design and immunocompetence. Here, we discuss research on the physiological and molecular basis of polyphenism in A. levana, including hormonal control, epigenetic regulation, and the effect of ecological parameters on developmental fate.

Ontogenetic Basis of Among-Generation Differences in Size-Related Traits in a Polyphenic Butterfly

Seasonal polyphenisms are cases in which individuals representing generations occurring in different times of the year systematically differ in their morphological, physiological, and/or behavioral traits. Such differences are often assumed to constitute adaptive responses to seasonally varying environments, but the evidence for this is still scarce. The adaptive character of the response would be corroborated by the pattern in which the decision about choosing a particular seasonal phenotype is made before the onset of respective environmental conditions (anticipatory plasticity). Alternatively, the between-generation differences can be caused by immediate effects of seasonally varying environments (responsive plasticity). Here we reared the larvae of the seasonally polymorphic map butterfly Araschnia levana under two different photoperiodic regimes, which provided different seasonal cues. These two treatments induced direct development and diapause pathways, respectively. Replicating the experiment at different temperatures and levels of host plant quality allowed us to evaluate both the anticipatory and the responsive components of the associated plastic changes in life-history traits. Larvae representing the direct development pathway invariably had higher growth rates and shorter development periods, although the difference between the developmental pathways was smaller at inferior host quality. Body size differences between the developmental pathways turned out to be less consistent, as the natural pattern of higher pupal mass of the directly developing individuals could only be reproduced at lower rearing temperature. Though being considerably modified by immediate environmental effects, the between-generation differences in size, growth rates, and larval are largely based on anticipatory plasticity (= responses to photoperiodic cues) and should be treated as seasonal adaptations in A. levana. In a more general context, we show how investigating the proximate basis of size differences can serve the purpose of identifying the limits of phenotypic plasticity in juvenile growth schedules.

Transcriptomic changes in the pea aphid, Acyrthosiphon pisum: Effects of the seasonal timer and photoperiod

Many insect species use photoperiod as a cue for induction of seasonal responses, including seasonal polyphenism. Although most aphid species viviparously produce parthenogenetic and sexual morphs under long and short days, respectively, a seasonal timer suppresses the sexual morph production over several successive generations during a few months following hatching of a sexually produced diapause egg. To reveal the relative influences of photoperiod and the seasonal timer on the reproductive polyphenism at the gene expression level, we performed RNA sequencing-based transcriptome analyses in the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae). Under short days, aphids with an expired seasonal timer showed a higher expression level in hundreds of genes than those with an operative seasonal timer. In contrast, aphids with an operative seasonal timer did not show upregulation in most of these genes. Functional annotations based on gene ontology showed that histone modifications and small non-coding RNA pathways were enriched in aphids with an expired seasonal timer under short-day conditions, suggesting that these epigenetic regulations on gene expression might be involved in a mechanism of maternal switching from the parthenogenetic to sexual morph production.

Seasonal morphotypes of Drosophila suzukii differ in key life-history traits during and after a prolonged period of cold exposure.

Seasonal polyphenism in Drosophila suzukii manifests itself in two discrete adult morphotypes, the “winter morph” (WM) and the “summer morph” (SM). These morphotypes are known to differ in thermal stress tolerance, and they co‐occur during parts of the year. In this study, we aimed to estimate morph‐specific survival and fecundity in laboratory settings simulating field conditions. We specifically analyzed how WM and SM D. suzukii differed in mortality and reproduction during and after a period of cold exposure resembling winter and spring conditions in temperate climates. The median lifespan of D. suzukii varied around 5 months for the WM flies and around 7 months for the SM flies. WM flies showed higher survival during the cold‐exposure period compared with SM flies, and especially SM males suffered high mortality under these conditions. In contrast, SM flies had lower mortality rates than WM flies under spring‐like conditions. Intriguingly, reproductive status (virgin or mated) did not impact the fly survival, either during the cold exposure or during spring‐like conditions. Even though the reproductive potential of WM flies was greatly reduced compared with SM flies, both WM and SM females that had mated before the cold exposure were able to continuously produce viable offspring for 5 months under spring‐like conditions. Finally, the fertility of the overwintered WM males was almost zero, while the surviving SM males did not suffer reduced fertility. Combined with other studies on D. suzukii monitoring and overwintering behavior, these results suggest that overwintered flies of both morphotypes could live long enough to infest the first commercial crops of the season. The high mortality of SM males and the low fertility of WM males after prolonged cold exposure also highlight the necessity for females to store sperm over winter to be able to start reproducing early in the following spring.

The complete mitochondrial genome and phylogenetic analysis of the European map butterfly Araschnia levana (Insecta: Lepidoptera: Nymphalidae)

The European map butterfly Araschnia levana (Linnaeus, 1758) is a species showing extreme seasonal polyphenism. The complete 15,207 bp circular A. levana mitogenome consisting of 81.6% AT nucleotides, was assembled by Illumina genome skimming. It includes 22 tRNAs, 13 protein-coding genes, 2 rRNAs, and a control region in the typical butterfly gene order. Araschnia levana COX1 features an atypical CGA start codon and ATP6, COX1, COX2, ND1, ND3, and ND4 have incomplete stop codons completed by 3′A residues added to the mRNA. Phylogenetic reconstruction places A. levana as a basal lineage within tribe Nymphalini, consistent with previous phylogenetic hypotheses.

Seasonal polyphenism of spotted-wing Drosophila is affected by variation in local abiotic conditions within its invaded range, likely influencing survival and regional population dynamics.

Overwintering Drosophila often display adaptive phenotypic differences beneficial for survival at low temperatures. However, it is unclear which morphological traits are the best estimators of abiotic conditions, how those traits are correlated with functional outcomes in cold tolerance, and whether there are regional differences in trait expression.We used a combination of controlled laboratory assays, and collaborative field collections of invasive Drosophila suzukii in different areas of the United States, to study the factors affecting phenotype variability of this temperate fruit pest now found globally.Laboratory studies demonstrated that winter morph (WM) trait expression is continuous within the developmental temperature niche of this species (10–25°C) and that wing length and abdominal melanization are the best predictors of the larval abiotic environment.However, the duration and timing of cold exposure also produced significant variation in development time, morphology, and survival at cold temperatures. During a stress test assay conducted at −5°C, although cold tolerance was greater among WM flies, long‐term exposure to cold temperatures as adults significantly improved summer morph (SM) survival, indicating that these traits are not controlled by a single mechanism.Among wild D. suzukii populations, we found that regional variation in abiotic conditions differentially affects the expression of morphological traits, although further research is needed to determine whether these differences are genetic or environmental in origin and whether thermal susceptibility thresholds differ among populations within its invaded range.

Effects of photoperiod and aging on the adult spermatogenesis of Polygonia c-aureum (Lepidoptera: Nymphalidae), in relation to adult diapause.

Adult spermatogenesis of Polygonia c-aureum was compared between non-diapausing and diapausing butterflies before overwintering. This butterfly has seasonal polyphenism, i.e., summer and autumnal forms. Summer form butterflies that emerged in summer reproduce shortly after emergence, while autumnal forms that emerged in autumn mate in spring. Immatures were reared under either a long photoperiod, which produced the summer form without diapause or under a short photoperiod, which produced the autumnal form with diapause. We found almost no differences in adult spermatogenesis between the two seasonal forms, indicating that adult spermatogenesis is not related to adult diapause. Although adult diapause in the autumnal form is maintained under short photoperiods and terminated under long photoperiods, such a photoperiod did not affect the spermatogenesis of the autumnal form. Our earlier studies indicate that relatively few eupyrene and apyrene sperm are produced after overwintering. Although apyrene spermatogenesis occurred in young adults, eupyrene spermatogenesis did in a small scale before overwintering. These results suggest strongly that male autumnal form butterflies prepare the sperm until overwintering, which had been formed during the larval, pupal and young adult stages.

To mate, or not to mate: The evolution of reproductive diapause facilitates insect radiation into African savannahs in the Late Miocene.

Many tropical environments experience cyclical seasonal changes, frequently with pronounced wet and dry seasons, leading to a highly uneven temporal distribution of resources. Short-lived animals inhabiting such environments often show season-specific adaptations to cope with alternating selection pressures. African Bicyclus butterflies show strong seasonal polyphenism in a suite of phenotypic and life-history traits, and their adults are thought to undergo reproductive diapause associated with the lack of available larval host plants during the dry season. Using 3years of longitudinal field data for three species in Malawi, dissections demonstrated that one forest species reproduces continuously, whereas two savannah species undergo reproductive diapause in the dry season, either with or without pre-diapause mating. Using additional data from field-collected and museum samples, we then documented the same three mating strategies for a further 37 species. Phylogenetic analyses indicated that the ancestral state was a non-diapausing forest species, and that habitat preference and mating strategy evolved in a correlated fashion. Bicyclus butterflies underwent rapid diversification during the Late Miocene, coinciding with expansions into more open savannah habitat. We conclude that the ability to undergo reproductive diapause was a key trait that facilitated colonization and eventual radiation into savannahs in the Late Miocene.

Seasonal phenotype-specific expression of microRNAs during metamorphosis in the European map butterfly Araschnia levana.

The European map butterfly (Araschnia levana) is a well-known example of seasonal polyphenism because the spring and summer imagoes exhibit distinct morphological phenotypes. The day length and temperature during larval and prepupal development determine whether spring or summer imagoes emerge after metamorphosis. Inspired by the fundamentally different transcriptomic profiles in prepupae developing from larvae exposed to long days or short days, we postulate that posttranscriptional epigenetic regulators such as microRNAs (miRNAs) may contribute to the epigenetic control of seasonal polyphenism in A. levana. To test this hypothesis, we used microarrays containing over 2,000 insect miRNAs to identify candidate regulators that are differentially expressed in last-instar larvae or pupae developing under long-day or short-day conditions. We used our transcriptomic database to identify potential 3'-untranslated regions of messenger RNAs to predict miRNA targets by considering both base pair complementarity and minimum free energy hybridization. This approach resulted in the identification of multiple targets of miRNAs that were differentially regulated in polyphenic morphs of A. levana including a candidate (miR-2856-3p) regulating the previously identified diapause bioclock protein gene. In conclusion, the expression profiling of miRNAs provided insights into their possible involvement in seasonal polyphenism of A. levana and offer an important resource for further studies.

Continent‐scale phenotype mapping using citizen scientists’ photographs

Field investigations of phenotypic variation in free‐living organisms are often limited in scope owing to time and funding constraints. By collaborating with online communities of amateur naturalists, investigators can greatly increase the amount and diversity of phenotypic data in their analyses while simultaneously engaging with a public audience. Here, we present a method for quantifying phenotypes of individual organisms in citizen scientists’ photographs. We then show that our protocol for measuring wing phenotypes from photographs yields accurate measurements in two species of Calopterygid damselflies. Next, we show that, while most observations of our target species were made by members of the large and established community of amateur naturalists at iNaturalist.org, our efforts to increase recruitment through various outreach initiatives were successful. Finally, we present results from two case studies: 1) an analysis of wing pigmentation in male smoky rubyspots Hetaerina titia showing previously undocumented geographical variation in a seasonal polyphenism, and 2) an analysis of variation in the relative size of the wing spots of male banded demoiselles Calopteryx splendens in Great Britain questioning previously documented evidence for character displacement. Our results demonstrate that our protocol can be used to create high quality phenotypic datasets using citizen scientists’ photographs, and, when combined with metadata (e.g. date and location), can greatly broaden the scope of studies of geographical and temporal variation in phenotypes. Our analyses of the recruitment and engagement process also demonstrate that collaborating with an online community of amateur naturalists can be a powerful way to conduct hypothesis‐driven research aiming to elucidate the processes that impact trait evolution at landscape scales.