Testing for variation in photoperiodic plasticity in a butterfly: Inconsistent effects of circadian genes between geographic scales.

The genetic components of the circadian clock have been implicated as involved in photoperiodic regulation of winter diapause across various insect groups, thereby contributing to adaptation to adverse seasonal conditions. So far, the effects of within-population variation in these genes have not been well explored. Here, we present an experimental test of the effects of within-population variation at two circadian genes, timeless and period, on photoperiodic responses in the butterfly Pararge aegeria. While nonsynonymous candidate SNPs in both of these genes have previously shown to be associated with diapause induction on a between-population level, in the present experiment no such effect was found on a within-population level. In trying to reconcile these results, we examine sequence data, revealing considerable, previously unknown protein-level variation at both timeless and period across Scandinavian populations, including variants unique to the population studied here. Hence, we hypothesize that these variants may counteract the previously observed diapause-averting effect of the candidate SNPs, possibly explaining the difference in results between the experiments. Whatever the cause, these results highlight how the effects of candidate SNPs may sometimes vary across genetic backgrounds, which complicates evolutionary interpretations of geographic patterns of genetic variation.

Genome-Wide Gene Birth-Death Dynamics Are Associated with Diet Breadth Variation in Lepidoptera.

Comparative analyses of gene birth-death dynamics have the potential to reveal gene families that played an important role in the evolution of morphological, behavioral, or physiological variation. Here, we used whole genomes of 30 species of butterflies and moths to identify gene birth-death dynamics among the Lepidoptera that are associated with specialist or generalist feeding strategies. Our work advances this field using a uniform set of annotated proteins for all genomes, investigating associations while correcting for phylogeny, and assessing all gene families rather than a priori subsets. We discovered that the sizes of several important gene families (e.g. those associated with pesticide resistance, xenobiotic detoxification, and/or protein digestion) are significantly correlated with diet breadth. We also found 22 gene families showing significant shifts in gene birth-death dynamics at the butterfly (Papilionoidea) crown node, the most notable of which was a family of pheromone receptors that underwent a contraction potentially linked with a shift to visual-based mate recognition. Our findings highlight the importance of uniform annotations, phylogenetic corrections, and unbiased gene family analyses in generating a list of candidate genes that warrant further exploration.

Evolution of butterfly seasonal plasticity driven by climate change varies across life stages.

Photoperiod is a common cue for seasonal plasticity and phenology, but climate change can create cue-environment mismatches for organisms that rely on it. Evolution could potentially correct these mismatches, but phenology often depends on multiple plastic decisions made during different life stages and seasons that may evolve separately. For example, Pararge aegeria (Speckled wood butterfly) has photoperiod-cued seasonal life history plasticity in two different life stages: larval development time and pupal diapause. We tested for climate change-associated evolution of this plasticity by replicating common garden experiments conducted on two Swedish populations 30 years ago. We found evidence for evolutionary change in the contemporary larval reaction norm-although these changes differed between populations-but no evidence for evolution of the pupal reaction norm. This variation in evolution across life stages demonstrates the need to consider how climate change affects the whole life cycle to understand its impacts on phenology.

Larval transcriptomes reflect the evolutionary history of plant-insect associations.

In this study, we investigated whether patterns of gene expression in larvae feeding on different plants can explain important aspects of the evolution of insect-plant associations, such as phylogenetic conservatism of host use and re-colonization of ancestral hosts that have been lost from the host repertoire. To this end, we performed a phylogenetically informed study comparing the transcriptomes of 4 nymphalid butterfly species in Polygonia and the closely related genus Nymphalis. Larvae were reared on Urtica dioica, Salix spp., and Ribes spp. Plant-specific gene expression was found to be similar across butterfly species, even in the case of host plants that are no longer used by two of the butterfly species. These results suggest that plant-specific transcriptomes can be robust over evolutionary time. We propose that adaptations to particular larval food plants can profitably be understood as an evolved set of modules of co-expressed genes, promoting conservatism in host use and facilitating re-colonization. Moreover, we speculate that the degree of overlap between plant-specific transcriptomes may correlate with the strength of trade-offs between plants as resources and hence to the probability of colonizing hosts and complete host shifts.

Extensive transcriptomic profiling of pupal diapause in a butterfly reveals a dynamic phenotype.

Diapause is a common adaptation for overwintering in insects that is characterized by arrested development and increased tolerance to stress and cold. While the expression of specific candidate genes during diapause have been investigated, there is no general understanding of the dynamics of the transcriptional landscape as a whole during the extended diapause phenotype. Such a detailed temporal insight is important as diapause is a vital aspect of life cycle timing. Here, we performed a time-course experiment using RNA-Seq on the head and abdomen in the butterfly Pieris napi. In both body parts, comparing diapausing and nondiapausing siblings, differentially expressed genes are detected from the first day of pupal development and onwards, varying dramatically across these formative stages. During diapause there are strong gene expression dynamics present, revealing a preprogrammed transcriptional landscape that is active during the winter. Different biological processes appear to be active in the two body parts. Finally, adults emerging from either the direct or diapause pathways do not show large transcriptomic differences, suggesting the adult phenotype is strongly canalized.

Local adaptation of life cycles in a butterfly is associated with variation in several circadian clock genes.

Many insects exhibit geographical variation in voltinism, the number of generations produced per year. This includes high-latitude species in previously glaciated areas, meaning that divergent selection on life cycle traits has taken place during or shortly after recent colonization. Here, we use a population genomics approach to compare a set of nine Scandinavian populations of the butterfly Pararge aegeria that differ in life cycle traits (diapause thresholds and voltinism) along both north-south and east-west clines. Using a de novo-assembled genome, we reconstruct colonization histories and demographic relationships. Based on the inferred population structure, we then scan the genome for candidate loci showing signs of divergent selection potentially associated with population differences in life cycle traits. The identified candidate genes include a number of components of the insect circadian clock (timeless, timeless2, period, cryptochrome and clockwork orange). Most notably, the gene timeless, which has previously been experimentally linked to life cycle regulation in P. aegeria, is here found to contain a novel 97-amino acid deletion unique to, and fixed in, a single population. These results add to a growing body of research framing circadian gene variation as a potential mechanism for generating local adaptation of life cycles.

Conserved ancestral tropical niche but different continental histories explain the latitudinal diversity gradient in brush-footed butterflies.

The global increase in species richness toward the tropics across continents and taxonomic groups, referred to as the latitudinal diversity gradient, stimulated the formulation of many hypotheses to explain the underlying mechanisms of this pattern. We evaluate several of these hypotheses to explain spatial diversity patterns in a butterfly family, the Nymphalidae, by assessing the contributions of speciation, extinction, and dispersal, and also the extent to which these processes differ among regions at the same latitude. We generate a time-calibrated phylogeny containing 2,866 nymphalid species (~45% of extant diversity). Neither speciation nor extinction rate variations consistently explain the latitudinal diversity gradient among regions because temporal diversification dynamics differ greatly across longitude. The Neotropical diversity results from low extinction rates, not high speciation rates, and biotic interchanges with other regions are rare. Southeast Asia is also characterized by a low speciation rate but, unlike the Neotropics, is the main source of dispersal events through time. Our results suggest that global climate change throughout the Cenozoic, combined with tropical niche conservatism, played a major role in generating the modern latitudinal diversity gradient of nymphalid butterflies.

Phylogenetic reconstruction of ancestral ecological networks through time for pierid butterflies and their host plants.

The study of herbivorous insects underpins much of the theory that concerns the evolution of species interactions. In particular, Pieridae butterflies and their host plants have served as a model system for studying evolutionary arms races. To learn more about the coevolution of these two clades, we reconstructed ancestral ecological networks using stochastic mappings that were generated by a phylogenetic model of host-repertoire evolution. We then measured if, when, and how two ecologically important structural features of the ancestral networks (modularity and nestedness) evolved over time. Our study shows that as pierids gained new hosts and formed new modules, a subset of them retained or recolonised the ancestral host(s), preserving connectivity to the original modules. Together, host-range expansions and recolonisations promoted a phase transition in network structure. Our results demonstrate the power of combining network analysis with Bayesian inference of host-repertoire evolution to understand changes in complex species interactions over time.

Chromosome Level Assembly of the Comma Butterfly (Polygonia c-album).

The comma butterfly (Polygonia c-album, Nymphalidae, Lepidoptera) is a model insect species, most notably in the study of phenotypic plasticity and plant-insect coevolutionary interactions. In order to facilitate the integration of genomic tools with a diverse body of ecological and evolutionary research, we assembled the genome of a Swedish comma using 10X sequencing, scaffolding with matepair data, genome polishing, and assignment to linkage groups using a high-density linkage map. The resulting genome is 373 Mb in size, with a scaffold N50 of 11.7 Mb and contig N50 of 11,2Mb. The genome contained 90.1% of single-copy Lepidopteran orthologs in a BUSCO analysis of 5,286 genes. A total of 21,004 gene-models were annotated on the genome using RNA-Seq data from larval and adult tissue in combination with proteins from the Arthropoda database, resulting in a high-quality annotation for which functional annotations were generated. We further documented the quality of the chromosomal assembly via synteny assessment with Melitaea cinxia. The resulting annotated, chromosome-level genome will provide an important resource for investigating coevolutionary dynamics and comparative analyses in Lepidoptera.

A Population Genomic Investigation of Immune Cell Diversity and Phagocytic Capacity in a Butterfly.

Insects rely on their innate immune system to successfully mediate complex interactions with their internal microbiota, as well as the microbes present in the environment. Given the variation in microbes across habitats, the challenges to respond to them are likely to result in local adaptations in the immune system. Here we focus upon phagocytosis, a mechanism by which pathogens and foreign particles are engulfed in order to be contained, killed, and processed. We investigated the phenotypic and genetic variation related to phagocytosis in two allopatric populations of the butterfly Pieris napi. Populations were found to differ in their hemocyte composition and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type. Yet, genes annotated to phagocytosis showed no large genomic signal of divergence. However, a gene set enrichment analysis on significantly divergent genes identified loci involved in glutamine metabolism, which recently have been linked to immune cell differentiation in mammals. Together these results suggest that heritable variation in phagocytic capacity arises via a quantitative trait architecture with variation in genes affecting the activation and/or differentiation of phagocytic cells, suggesting them as potential candidate genes underlying these phenotypic differences.

A region of the sex chromosome associated with population differences in diapause induction contains highly divergent alleles at clock genes.

Developmental plasticity describes the capacity of individuals with the same genotype to induce permanent change in a phenotype depending on a specific external input. One well-studied example of adaptive developmental plasticity is the induction of facultative diapause in insects. Studies investigating the inheritance of diapause induction have suggested diverse genetic origins. However, only few studies have performed genome-wide scans to identify genes affecting the induction decision. Here we compare two populations of the butterfly Pieris napi that differ in the propensity to enter diapause, and despite showing a low genome-wide divergence, we identify a few genomic regions that show high divergence between populations. We then identified a single genomic region associated with diapause induction by genotyping diapausing and directly developing siblings from backcrosses of these populations. This region is located on the Z chromosome and contained three circadian clock genes, cycle, clock, and period. Additionally, period harbored the largest number of SNPs showing complete fixation between populations. We conclude that the heritable basis of between-population variation in the plasticity that determines diapause induction resides on the Z chromosome, with the period gene being the prime candidate for the genetic basis of adaptive plasticity.

Bayesian Inference of Ancestral Host-Parasite Interactions under a Phylogenetic Model of Host Repertoire Evolution.

Intimate ecological interactions, such as those between parasites and their hosts, may persist over long time spans, coupling the evolutionary histories of the lineages involved. Most methods that reconstruct the coevolutionary history of such interactions make the simplifying assumption that parasites have a single host. Many methods also focus on congruence between host and parasite phylogenies, using cospeciation as the null model. However, there is an increasing body of evidence suggesting that the host ranges of parasites are more complex: that host ranges often include more than one host and evolve via gains and losses of hosts rather than through cospeciation alone. Here, we develop a Bayesian approach for inferring coevolutionary history based on a model accommodating these complexities. Specifically, a parasite is assumed to have a host repertoire, which includes both potential hosts and one or more actual hosts. Over time, potential hosts can be added or lost, and potential hosts can develop into actual hosts or vice versa. Thus, host colonization is modeled as a two-step process that may potentially be influenced by host relatedness. We first explore the statistical behavior of our model by simulating evolution of host-parasite interactions under a range of parameter values. We then use our approach, implemented in the program RevBayes, to infer the coevolutionary history between 34 Nymphalini butterfly species and 25 angiosperm families. Our analysis suggests that host relatedness among angiosperm families influences how easily Nymphalini lineages gain new hosts. [Ancestral hosts; coevolution; herbivorous insects; probabilistic modeling.].

Physiological Tradeoffs of Immune Response Differs by Infection Type in Pieris napi.

Understanding the tradeoffs that result from successful infection responses is central to understanding how life histories evolve. Gaining such insights, however, can be challenging, as they may be pathogen specific and confounded with experimental design. Here, we investigated whether infection from gram positive or negative bacteria results in different physiological tradeoffs, and whether these infections impact life history later in life (post-diapause development), in the butterfly Pieris napi. During the first 24 h after infection (3, 6, 12, and 24 h), after removing effects due to injection, larvae infected with Micrococcus luteus showed a strong suppression of all non-immunity related processes while several types of immune responses were upregulated. In contrast, this tradeoff between homeostasis and immune response was much less pronounced in Escherichia coli infections. These differences were also visible long after infection, via weight loss and slower development, as well as an increased mortality at higher infection levels during later stages of development. Individuals infected with M. luteus, compared to E. coli, had a higher mortality rate, and a lower pupal weight, developmental rate and adult weight. Further, males exhibited a more negative impact of infection than females. Thus, immune responses come at a cost even when the initial infection has been overcome, and these costs are likely to affect later life history parameters with fitness consequences.

Structural plasticity of olfactory neuropils in relation to insect diapause.

Many insects that live in temperate zones spend the cold season in a state of dormancy, referred to as diapause. As the insect must rely on resources that were gathered before entering diapause, keeping a low metabolic rate is of utmost importance. Organs that are metabolically expensive to maintain, such as the brain, can therefore become a liability to survival if they are too large.Insects that go through diapause as adults generally do so before entering the season of reproduction. This order of events introduces a conflict between maintaining low metabolism during dormancy and emerging afterward with highly developed sensory systems that improve fitness during the mating season.We investigated the timing of when investments into the olfactory system are made by measuring the volumes of primary and secondary olfactory neuropils in the brain as they fluctuate in size throughout the extended diapause life-period of adult Polygonia c-album butterflies.Relative volumes of both olfactory neuropils increase significantly during early adult development, indicating the importance of olfaction to this species, but still remain considerably smaller than those of nondiapausing conspecifics. However, despite butterflies being kept under the same conditions as before the dormancy, their olfactory neuropil volumes decreased significantly during the postdormancy period.The opposing directions of change in relative neuropil volumes before and after diapause dormancy indicate that the investment strategies governing structural plasticity during the two life stages could be functionally distinct. As butterflies were kept in stimulus-poor conditions, we find it likely that investments into these brain regions rely on experience-expectant processes before diapause and experience-dependent processes after diapause conditions are broken.As the shift in investment strategies coincides with a hard shift from premating season to mating season, we argue that these developmental characteristics could be adaptations that mitigate the trade-off between dormancy survival and reproductive fitness.

Climate-induced phenology shifts linked to range expansions in species with multiple reproductive cycles per year.

Advances in phenology (the annual timing of species' life-cycles) in response to climate change are generally viewed as bioindicators of climate change, but have not been considered as predictors of range expansions. Here, we show that phenology advances combine with the number of reproductive cycles per year (voltinism) to shape abundance and distribution trends in 130 species of British Lepidoptera, in response to ~0.5 °C spring-temperature warming between 1995 and 2014. Early adult emergence in warm years resulted in increased within- and between-year population growth for species with multiple reproductive cycles per year (n = 39 multivoltine species). By contrast, early emergence had neutral or negative consequences for species with a single annual reproductive cycle (n = 91 univoltine species), depending on habitat specialisation. We conclude that phenology advances facilitate polewards range expansions in species exhibiting plasticity for both phenology and voltinism, but may inhibit expansion by less flexible species.

Insect brain plasticity: effects of olfactory input on neuropil size.

Insect brains are known to express a high degree of experience-dependent structural plasticity. One brain structure in particular, the mushroom body (MB), has been attended to in numerous studies as it is implicated in complex cognitive processes such as olfactory learning and memory. It is, however, poorly understood to what extent sensory input per se affects the plasticity of the mushroom bodies. By performing unilateral blocking of olfactory input on immobilized butterflies, we were able to measure the effect of passive sensory input on the volumes of antennal lobes (ALs) and MB calyces. We showed that the primary and secondary olfactory neuropils respond in different ways to olfactory input. ALs show absolute experience-dependency and increase in volume only if receiving direct olfactory input from ipsilateral antennae, while MB calyx volumes were unaffected by the treatment and instead show absolute age-dependency in this regard. We therefore propose that cognitive processes related to behavioural expressions are needed in order for the calyx to show experience-dependent volumetric expansions. Our results indicate that such experience-dependent volumetric expansions of calyces observed in other studies may have been caused by cognitive processes rather than by sensory input, bringing some causative clarity to a complex neural phenomenon.

Unprecedented reorganization of holocentric chromosomes provides insights into the enigma of lepidopteran chromosome evolution.

Chromosome evolution presents an enigma in the mega-diverse Lepidoptera. Most species exhibit constrained chromosome evolution with nearly identical haploid chromosome counts and chromosome-level gene collinearity among species more than 140 million years divergent. However, a few species possess radically inflated chromosomal counts due to extensive fission and fusion events. To address this enigma of constraint in the face of an exceptional ability to change, we investigated an unprecedented reorganization of the standard lepidopteran chromosome structure in the green-veined white butterfly (Pieris napi). We find that gene content in P. napi has been extensively rearranged in large collinear blocks, which until now have been masked by a haploid chromosome number close to the lepidopteran average. We observe that ancient chromosome ends have been maintained and collinear blocks are enriched for functionally related genes suggesting both a mechanism and a possible role for selection in determining the boundaries of these genome-wide rearrangements.

Local adaptation of photoperiodic plasticity maintains life cycle variation within latitudes in a butterfly.

The seasonal cycle varies geographically and organisms are under selection to express life cycles that optimally exploit their spatiotemporal habitats. In insects, this often means producing an annual number of generations (voltinism) appropriate to the local season length. Variation in voltinism may arise from variation in environmental factors (e.g., temperature or photoperiod) acting on a single reaction norm shared across populations, but it may also result from local adaptation of reaction norms. However, such local adaptation is poorly explored at short geographic distances, especially within latitudes. Using a combination of common-garden rearing and life cycle modeling, we have investigated the causal factors behind voltinism variation in Swedish populations of the butterfly Pararge aegeria, focusing on a set of populations that lie within a single degree of latitude but nonetheless differ in season length and voltinism. Despite considerable differences in ambient temperature between populations, modeling suggested that the key determinant of local voltinism was in fact interpopulation differences in photoperiodic response. These include differences in the induction thresholds for winter diapause, as well as differences in photoperiodic regulation of larval development, a widespread but poorly studied phenomenon. Our results demonstrate previously neglected ways that photoperiodism may mediate insect phenological responses to temperature, and emphasize the importance of local adaptation in shaping phenological patterns in general, as well as for predicting the responses of populations to changes in climate.

Unifying host-associated diversification processes using butterfly-plant networks.

Explaining the exceptional diversity of herbivorous insects is an old problem in evolutionary ecology. Here we focus on the two prominent hypothesised drivers of their diversification, radiations after major host switch or variability in host use due to continuous probing of new hosts. Unfortunately, current methods cannot distinguish between these hypotheses, causing controversy in the literature. Here we present an approach combining network and phylogenetic analyses, which directly quantifies support for these opposing hypotheses. After demonstrating that each hypothesis produces divergent network structures, we then investigate the contribution of each to diversification in two butterfly families: Pieridae and Nymphalidae. Overall, we find that variability in host use is essential for butterfly diversification, while radiations following colonisation of a new host are rare but can produce high diversity. Beyond providing an important reconciliation of alternative hypotheses for butterfly diversification, our approach has potential to test many other hypotheses in evolutionary biology.

Genetic variation underlying local adaptation of diapause induction along a cline in a butterfly.

Diapause is a life history strategy allowing individuals to arrest development until favourable conditions return, and it is commonly induced by shortened day length that is latitude specific for local populations. Although understanding the evolutionary dynamics of a threshold trait like diapause induction provides insights into the adaptive process and adaptive potential of populations, the genetic mechanism of variation in photoperiodic induction of diapause is not well understood. Here, we investigate genetic variation underlying latitudinal variation in diapause induction and the selection dynamics acting upon it. Using a genomewide scan for divergent regions between two populations of the butterfly Pararge aegeria that differ strongly in their induction thresholds, we identified and investigated the patterns of variation in those regions. We then tested the association of these regions with diapause induction using between-population crosses, finding significant SNP associations in four genes present in two chromosomal regions, one with the gene period, and the other with the genes kinesin, carnitine O-acetyltransferase and timeless. Patterns of allele frequencies in these two regions in population samples along a latitudinal cline suggest strong selection against heterozygotes at two genes within these loci (period, timeless). Evidence for additional loci modifying the diapause decision was found in patterns of allelic change in relation to induction thresholds over the cline, as well as in backcross analyses. Taken together, population-specific adaptations of diapause induction appear to be due to a combination of alleles of larger and smaller effect size, consistent with an exponential distribution of effect sizes involved in local adaption.