The evolution and diversification of oakleaf butterflies.

Oakleaf butterflies in the genus Kallima have a polymorphic wing phenotype, enabling these insects to masquerade as dead leaves. This iconic example of protective resemblance provides an interesting evolutionary paradigm that can be employed to study biodiversity. We integrated multi-omic data analyses and functional validation to infer the evolutionary history of Kallima species and investigate the genetic basis of their variable leaf wing patterns. We find that Kallima butterflies diversified in the eastern Himalayas and dispersed to East and Southeast Asia. Moreover, we find that leaf wing polymorphism is controlled by the wing patterning gene cortex, which has been maintained in Kallima by long-term balancing selection. Our results provide macroevolutionary and microevolutionary insights into a model species originating from a mountain ecosystem.

A learning experience elicits sex-dependent neurogenomic responses in Bicyclus anynana butterflies.

Sexually dimorphic behaviour is pervasive across animals, with males and females exhibiting different mate selection, parental care, foraging, dispersal, and territorial strategies. However, the genetic underpinnings of sexually dimorphic behaviours are poorly understood. Here we investigate gene networks and expression patterns associated with sexually dimorphic imprinting-like learning in the butterfly Bicyclus anynana. In this species, both males and females learn visual preferences, but learn preferences for different traits and use different signals as salient, unconditioned cues. To identify genes and gene networks associated with this behaviour, we examined gene expression profiles of the brains and eyes of male and female butterflies immediately post training and compared them to the same tissues of naïve individuals. We found more differentially expressed genes and a greater number of associated gene networks in the eyes, indicating a role of the peripheral nervous system in visual imprinting-like learning. Females had higher chemoreceptor expression levels than males, supporting the hypothesized sexual dimorphic use of chemical cues during the learning process. In addition, genes that influence B. anynana wing patterns (sexual ornaments), such as invected, spalt, and apterous, were also differentially expressed in the brain and eye, suggesting that these genes may influence both sexual ornaments and the preferences for these ornaments. Our results indicate dynamic and sex-specific responses to social scenario in both the peripheral and central nervous systems and highlight the potential role of wing patterning genes in mate preference and learning across the Lepidoptera.

Evolutionary history limits species' ability to match colour sensitivity to available habitat light.

The spectrum of light that an animal sees-from ultraviolet to far red light-is governed by the number and wavelength sensitivity of a family of retinal proteins called opsins. It has been hypothesized that the spectrum of light available in an environment influences the range of colours that a species has evolved to see. However, invertebrates and vertebrates use phylogenetically distinct opsins in their retinae, and it remains unclear whether these distinct opsins influence what animals see, or how they adapt to their light environments. Systematically using published visual sensitivity data from across animal phyla, we found that terrestrial animals are more sensitive to shorter and longer wavelengths of light than aquatic animals and that invertebrates are more sensitive to shorter wavelengths of light than vertebrates. Using phylogenetically controlled analyses, we found that closed and open canopy habitat species have different spectral sensitivities when comparing across the Metazoa and excluding habitat generalists, while deepwater animals are no more sensitive to shorter wavelengths of light than shallow-water animals. Our results suggest that animals do adapt to their light environment; however, the invertebrate-vertebrate evolutionary divergence may limit the degree to which animals can perform visual tuning.

Searching for the genes driving assortative mating.

Animals display an astonishing array of diverse colors and patterns, and animals also exhibit preferences for these diverse, species-specific traits when choosing a mate (i.e., assortative mate preference). It is hypothesized that in order for both preference and trait to be species specific, alleles for a trait and the preference for that trait must be inherited together and hence maintained as linked loci. This linkage could be maintained by three different genetic architectures: (A) the genes responsible for a species-specific preferred trait also directly influence preference for that trait; (B) genes producing preference and the preferred trait are not identical but are instead in close physical proximity in the genome; and (C) genes for preference and the preferred trait are nonadjacent but are inherited together due to selection. Merrill and colleagues test these hypotheses by performing large-scale genetic mapping of mating behavior using hybrids of two sympatric species of Heliconius butterflies, Heliconius melpomene and H. cydno. They identified three small genomic regions highly associated with mate preference, one of which was adjacent to a gene for the preferred trait, and two of which were not. Their findings illustrate that mate preference may be influenced by a small number of genes, while providing support for multiple hypotheses for the genetic architecture of assortative mate preferences.

Stage- and sex-specific transcriptome analyses reveal distinctive sensory gene expression patterns in a butterfly.

BACKGROUND: Animal behavior is largely driven by the information that animals are able to extract and process from their environment. However, the function and organization of sensory systems often change throughout ontogeny, particularly in animals that undergo indirect development. As an initial step toward investigating these ontogenetic changes at the molecular level, we characterized the sensory gene repertoire and examined the expression profiles of genes linked to vision and chemosensation in two life stages of an insect that goes through metamorphosis, the butterfly Bicyclus anynana. RESULTS: Using RNA-seq, we compared gene expression in the heads of late fifth instar larvae and newly eclosed adults that were reared under identical conditions. Over 50 % of all expressed genes were differentially expressed between the two developmental stages, with 4,036 genes upregulated in larval heads and 4,348 genes upregulated in adult heads. In larvae, upregulated vision-related genes were biased toward those involved with eye development, while phototransduction genes dominated the vision genes that were upregulated in adults. Moreover, the majority of the chemosensory genes we identified in the B. anynana genome were differentially expressed between larvae and adults, several of which share homology with genes linked to pheromone detection, host plant recognition, and foraging in other species of Lepidoptera. CONCLUSIONS: These results revealed promising candidates for furthering our understanding of sensory processing and behavior in the disparate developmental stages of butterflies and other animals that undergo metamorphosis.

Biased learning affects mate choice in a butterfly.

Early acquisition of mate preferences or mate-preference learning is associated with signal diversity and speciation in a wide variety of animal species. However, the diversity of mechanisms of mate-preference learning across taxa remains poorly understood. Using the butterfly Bicyclus anynana we uncover a mechanism that can lead to directional sexual selection via mate-preference learning: a bias in learning enhanced ornamentation, which is independent of preexisting mating biases. Naïve females mated preferentially with wild-type males over males with enhanced wing ornamentation, but females briefly exposed to enhanced males mated significantly more often with enhanced males. In contrast, females exposed to males with reduced wing ornamentation did not learn to prefer drab males. Thus, we observe both a learned change of a preexisting mating bias, and a bias in ability to learn enhanced male ornaments over reduced ornaments. Our findings demonstrate that females are able to change their preferences in response to a single social event, and suggest a role for biased learning in the evolution of visual sexual ornamentation.

Behavioural changes in aposematic Heliconius melpomene butterflies in response to their predatory bird calls.

Prey-predator interactions have resulted in the evolution of many anti-predatory traits. One of them is the ability for prey to listen to predators and avoid them. Although prey anti-predatory behavioural responses to predator auditory cues are well described in a wide range of taxa, studies on whether butterflies change their behaviours in response to their predatory calls are lacking. Heliconius butterflies are unpalatable and form Müllerian mimicry rings as morphological defence strategies against their avian predators. Like many other butterflies in the Nymphalidae family, some Heliconius butterflies possess auditory organs, which are hypothesized to assist with predator detection. Here we test whether Heliconius melpomene change their behaviour in response to their predatory bird calls by observing the behaviour of male and female H. m. plessini exposed to calls of Heliconius avian predators: rufous-tailed jacamar, migratory Eastern kingbird, and resident tropical kingbird. We also exposed them to the calls of the toco toucan, a frugivorous bird as a control bird call, and an amplified greenhouse background noise as a noise control. We found that individuals changed their behaviour in response to jacamar calls only. Males increased their walking and fluttering behaviour, while females did not change their behaviour during the playback of the jacamar call. Intersexual behaviours like courtship, copulation, and abdomen lifting did not change in response to bird calls. Our findings suggest that despite having primary predatory defences like toxicity and being in a mimicry ring, H. m. plessini butterflies changed their behaviour in response to predator calls. Furthermore, this response was predator specific, as H. m. plesseni did not respond to either the Eastern kingbird or the tropical kingbird calls. This suggests that Heliconius butterflies may be able to differentiate predatory calls, and potentially the birds associated with those calls.

Digest: Recurrence of sexually dimorphic neurological structure in neotropical butterflies.

Although many traits can be gained or lost over evolutionary time, it has long been unclear whether complex sensory processing structures, such as brain neuropils, can be regained after having been lost. Morris et al. show that a part of the brain once lost in butterflies, a macro-glomeruli complex (MGC) in the antennal lobe, is prevalent and has diversified in the Ithomiini tribe. This structure is sexually dimorphic in some species. This re-emergence of a complex sensory processing structure is likely driven by ecological factors.

Engaging the Community in Pollinator Research: The Effect of Wing Pattern and Weather on Butterfly Behavior.

Community science, which engages students and the public in data collection and scientific inquiry, is often integrated into conservation and long-term monitoring efforts. However, it has the potential to also introduce the public to, and be useful for, sensory ecology and other fields of study. Here we describe a community science project that exposes participants to animal behavior and sensory ecology using the rich butterfly community of Northwest Arkansas, United States. Butterflies use visual signals to communicate and to attract mates. Brighter colors can produce stronger signals for mate attraction but can also unintentionally attract negative attention from predators. Environmental conditions such as weather can affect visual signaling as well, by influencing the wavelengths of light available and subsequent signal detection. However, we do not know whether the signals butterflies present correlate broadly with how they behave. In this study, we collaborated with hundreds of students and community members at the University of Arkansas (UARK) and the Botanical Gardens of the Ozarks (BGO) for over 3.5 years to examine relationships among wing pattern, weather, time of day, behavior, and flower choice. We found that both weather and wing color influenced general butterfly behavior. Butterflies were seen feeding more on cloudy days than on sunny or partly cloudy days. Brown butterflies fed or sat more often, while white butterflies flew more often relative to other butterfly colors. We also found that there was an interaction between the effects of weather and wing color on butterfly behavior. Furthermore, butterfly color predicted the choice of flower colors that butterflies visited, though this effect was influenced by the observer group (UARK student or BGO participant). These results suggest that flower choice may be associated with butterfly wing pattern, and that different environmental conditions may influence butterfly behavior in wing-pattern-specific ways. They also illustrate one way that public involvement in behavioral studies can facilitate the identification of coarse-scale, community-wide behavioral patterns, and lay the groundwork for future studies of sensory niches.

Nocturnal Pollinators Significantly Contribute to Apple Production.

Agricultural dependency on insect-mediated pollination is increasing at the same time that pollinator populations are experiencing declines in diversity and abundance. Current pollinator research in agriculture focuses largely on diurnal pollinators, yet the evidence for pollination by moths and other nocturnal pollinators is growing. Apples are one of the most valuable and important fruits produced globally, and apple production is dependent on insect-mediated cross-pollination to generate a profitable crop. We examined contributions to apple production provided by nocturnal insects via an exclusion experiment. We compared the relative contributions to apple production provided by nocturnal and diurnal pollinators using fruit set, the likelihood of cluster pollination, and seed set. We found nocturnal pollinators capable of facilitating the production of as many apples at similar levels of pollination as diurnal pollinators. We further found evidence that nocturnal and diurnal pollinators pollinate synergistically, with pollination contributions being additive in one year of our study. Our research identifies significant contributions to apple production provided by nocturnal pollinators, which may interact with diurnal pollinators in ways that are currently unrecognized. Expansions of this research into additional pollinator-dependent crops and focused investigations on specific nocturnal insects will provide more accurate assessments of nocturnal-pollinator roles in agriculture and improve our overall understanding of pollination in agriculture.

A widely diverged locus involved in locomotor adaptation in Heliconius butterflies.

Heliconius butterflies have undergone adaptive radiation and therefore serve as an excellent system for exploring the continuum of speciation and adaptive evolution. However, there is a long-lasting paradox between their convergent mimetic wing patterns and rapid divergence in speciation. Here, we characterize a locus that consistently displays high divergence among Heliconius butterflies and acts as an introgression hotspot. We further show that this locus contains multiple genes related to locomotion and conserved in Lepidoptera. In light of these findings, we consider that locomotion traits may be under selection, and if these are heritable traits that are selected for, then they might act as species barriers.

Lack of sibling avoidance during mate selection in the butterfly Bicyclus anynana.

Species susceptible to inbreeding depression are hypothesized to combat this problem through a number of different mechanisms, including kin recognition. For species with kin recognition, it is unknown if filial recognition is innate or due to prior juvenile experience with siblings. Here, we first test for the presence of kin recognition, and then test these two hypotheses for the development of filial recognition, in the butterfly Bicyclus anynana, a species that suffers from inbreeding depression when forcibly inbred but recovers within a few generations when allowed to breed freely. We evaluate whether the rapid recovery from inbreeding depression is associated with either innate or learned filial recognition. First, we determined whether females innately prefer unrelated males over sibling males using females reared in isolation and then given a choice between an unrelated and a sibling male. Then, we determined if females raised with siblings learned to detect and avoid mating with siblings as adults when provided a choice between an unrelated male and a sibling male. Finally, we determined if females raised with siblings could learn to detect and avoid mating with familiar siblings when given a choice between familiar and unfamiliar siblings. We found that females mated randomly in all three choice combinations. Observed male behavior also did not influence female mating outcome. Our results suggest that adult females do not innately avoid or learn to avoid siblings during mate selection, and that filial detection may not be as critical to reproductive fitness in B. anynana as previously thought.

Deploying Big Data to Crack the Genotype to Phenotype Code.

Mechanistically connecting genotypes to phenotypes is a longstanding and central mission of biology. Deciphering these connections will unite questions and datasets across all scales from molecules to ecosystems. Although high-throughput sequencing has provided a rich platform on which to launch this effort, tools for deciphering mechanisms further along the genome to phenome pipeline remain limited. Machine learning approaches and other emerging computational tools hold the promise of augmenting human efforts to overcome these obstacles. This vision paper is the result of a Reintegrating Biology Workshop, bringing together the perspectives of integrative and comparative biologists to survey challenges and opportunities in cracking the genotype to phenotype code and thereby generating predictive frameworks across biological scales. Key recommendations include promoting the development of minimum "best practices" for the experimental design and collection of data; fostering sustained and long-term data repositories; promoting programs that recruit, train, and retain a diversity of talent; and providing funding to effectively support these highly cross-disciplinary efforts. We follow this discussion by highlighting a few specific transformative research opportunities that will be advanced by these efforts.

Multiple Loci Control Eyespot Number Variation on the Hindwings of Bicyclus anynana Butterflies.

The underlying genetic changes that regulate the appearance and disappearance of repeated traits, or serial homologs, remain poorly understood. One hypothesis is that variation in genomic regions flanking master regulatory genes, also known as input-output genes, controls variation in trait number, making the locus of evolution almost predictable. Another hypothesis implicates genetic variation in up- or downstream loci of master control genes. Here, we use the butterfly Bicyclus anynana, a species that exhibits natural variation in eyespot number on the dorsal hindwing, to test these two hypotheses. We first estimated the heritability of dorsal hindwing eyespot number by breeding multiple butterfly families differing in eyespot number and regressing eyespot numbers of offspring on midparent values. We then estimated the number and identity of independent genetic loci contributing to eyespot number variation by performing a genome-wide association study with restriction site-associated DNA sequencing from multiple individuals varying in number of eyespots sampled across a freely breeding laboratory population. We found that dorsal hindwing eyespot number has a moderately high heritability of ∼0.50 and is characterized by a polygenic architecture. Previously identified genomic regions involved in eyespot development, and novel ones, display high association with dorsal hindwing eyespot number, suggesting that homolog number variation is likely determined by regulatory changes at multiple loci that build the trait, and not by variation at single master regulators or input-output genes.

Behaviour before beauty: signal weighting during mate selection in the butterfly Papilio polytes.

Mating displays often contain multiple signals. Different combinations of these signals may be equally successful at attracting a mate, as environment and signal combination may influence relative signal weighting by choosy individuals. This variation in signal weighting among choosy individuals may facilitate the maintenance of polymorphic displays and signalling behaviour. One group of animals known for their polymorphic patterning are Batesian mimetic butterflies, where the interaction of sexual selection and predation pressures are hypothesized to influence the maintenance of polymorphic wing patterning and behaviour. Males in the female-limited polymorphic Batesian mimetic butterfly Papilio polytes use female wing pattern and female activity levels when determining whom to court. They court stationary females with mimetic wing patterns more often than stationary females with non-mimetic, male-like wing patterns, and active females more often than inactive females. It is unclear whether females modify their behaviour to increase (or decrease) their likelihood of receiving male courtship, or whether non-mimetic females spend more time in cryptic environments than mimetic females, to compensate for their lack of mimicry-driven predation protection (at the cost of decreased visibility to males). In addition, relative signal weighting of female wing pattern and activity to male mate selection is unknown. To address these questions, we conducted a series of observational studies of a polymorphic P. polytes population in a large butterfly enclosure. We found that males exclusively courted active females, irrespective of female wing pattern. However, males did court active non-mimetic females significantly more often than expected given their relative abundance in the population. Females exhibited similar activity levels, and selected similar resting environments, irrespective of wing pattern. Our results suggest that male preference for non-mimetic females may play an active role in the maintenance of the non-mimetic female form in natural populations, where males are likely to be in the presence of active, as well as inactive, mimetic and non-mimetic females.

United States National Postdoc Survey results and the interaction of gender, career choice and mentor impact.

The postdoctoral community is an essential component of the academic and scientific workforce, but a lack of data about this community has made it difficult to develop policies to address concerns about salaries, working conditions, diversity and career development, and to evaluate the impact of existing policies. Here we present comprehensive survey results from 7,603 postdocs based at 351 US academic and non-academic (e.g. hospital, industry and government lab) institutions in 2016. In addition to demographic and salary information, we present multivariate analyses on factors influencing postdoc career plans and satisfaction with mentorship. We further analyze gender dynamics and expose wage disparities. Academic research positions remain the predominant career choice, although women and US citizens are less likely than their male and non-US citizen counterparts to choose academic research positions. Receiving mentorship training has a significant positive effect on postdoc satisfaction with mentorship. Quality of and satisfaction with postdoc mentorship also appear to heavily influence career choice.

Aristaless Controls Butterfly Wing Color Variation Used in Mimicry and Mate Choice.

Neotropical Heliconius butterflies display a diversity of warningly colored wing patterns, which serve roles in both Müllerian mimicry and mate choice behavior. Wing pattern diversity in Heliconius is controlled by a small number of unlinked, Mendelian "switch" loci [1]. One of these, termed the K locus, switches between yellow and white color patterns, important mimicry signals as well as mating cues [2-4]. Furthermore, mate preference behavior is tightly linked to this locus [4]. K controls the distribution of white versus yellow scales on the wing, with a dominant white allele and a recessive yellow allele. Here, we combine fine-scale genetic mapping, genome-wide association studies, gene expression analyses, population and comparative genomics, and genome editing with CRISPR/Cas9 to characterize the molecular basis of the K locus in Heliconius and to infer its evolutionary history. We show that white versus yellow color variation in Heliconius cydno is due to alternate haplotypes at a putative cis-regulatory element (CRE) downstream of a tandem duplication of the homeodomain transcription factor aristaless. Aristaless1 (al1) and aristaless2 (al2) are differentially regulated between white and yellow wings throughout development with elevated expression of al1 in developing white wings, suggesting a role in repressing pigmentation. Consistent with this, knockout of al1 causes white wings to become yellow. The evolution of wing color in this group has been marked by retention of the ancestral yellow color in many lineages, a single origin of white coloration in H. cydno, and subsequent introgression of white color from H. cydno into H. melpomene.

Tracing the origin and evolution of supergene mimicry in butterflies.

Supergene mimicry is a striking phenomenon but we know little about the evolution of this trait in any species. Here, by studying genomes of butterflies from a recent radiation in which supergene mimicry has been isolated to the gene doublesex, we show that sexually dimorphic mimicry and female-limited polymorphism are evolutionarily related as a result of ancient balancing selection combined with independent origins of similar morphs in different lineages and secondary loss of polymorphism in other lineages. Evolutionary loss of polymorphism appears to have resulted from an interaction between natural selection and genetic drift. Furthermore, molecular evolution of the supergene is dominated not by adaptive protein evolution or balancing selection, but by extensive hitchhiking of linked variants on the mimetic dsx haplotype that occurred at the origin of mimicry. Our results suggest that chance events have played important and possibly opposing roles throughout the history of this classic example of adaptation.

Rearing Temperature Influences Adult Response to Changes in Mating Status.

Rearing environment can have an impact on adult behavior, but it is less clear how rearing environment influences adult behavior plasticity. Here we explore the effect of rearing temperature on adult mating behavior plasticity in the butterfly Bicyclus anynana, a species that has evolved two seasonal forms in response to seasonal changes in temperature. These seasonal forms differ in both morphology and behavior. Females are the choosy sex in cohorts reared at warm temperatures (WS butterflies), and males are the choosy sex in cohorts reared at cooler temperatures (DS butterflies). Rearing temperature also influences mating benefits and costs. In DS butterflies, mated females live longer than virgin females, and mated males live shorter than virgin males. No such benefits or costs to mating are present in WS butterflies. Given that choosiness and mating costs are rearing temperature dependent in B. anynana, we hypothesized that temperature may also impact male and female incentives to remate in the event that benefits and costs of second matings are similar to those of first matings. We first examined whether lifespan was affected by number of matings. We found that two matings did not significantly increase lifespan for either WS or DS butterflies relative to single matings. However, both sexes of WS but not DS butterflies experienced decreased longevity when mated to a non-virgin relative to a virgin. We next observed pairs of WS and DS butterflies and documented changes in mating behavior in response to changes in the mating status of their partner. WS but not DS butterflies changed their mating behavior in response to the mating status of their partner. These results suggest that rearing temperature influences adult mating behavior plasticity in B. anynana. This developmentally controlled behavioral plasticity may be adaptive, as lifespan depends on the partner's mating status in one seasonal form, but not in the other.

Mate preference for a phenotypically plastic trait is learned, and may facilitate preference-phenotype matching.

Fixed, genetically determined, mate preferences for species whose adult phenotype varies with rearing environment may be maladaptive, as the phenotype that is most fit in the parental environment may be absent in the offspring environment. Mate preference in species with polyphenisms (environmentally dependent alternative phenotypes) should therefore either not focus on polyphenic traits, be polyphenic themselves, or learned each generation. Here, we test these alternative hypotheses by first describing a female-limited seasonal polyphenism in a sexually dimorphic trait in the butterfly Bicyclus anynana, dorsal hindwing spot number (DHSN), and then testing whether male and female mate preferences for this trait exist, and whether they are seasonally polyphenic, or learned. Neither naïve males nor naïve females in either seasonal form exhibited mating preferences for DHSN. However, males, but not females, noticed DHSN variation and learned mate preferences for DHSN. These results suggest that individuals may accommodate environmentally dependent variation in morphological traits via learned mate preferences in each generation, and that learned mate preference plasticity can be sexually dimorphic.