Project Psyche is a trans-national initiative to generate and study chromosome-level reference genomes of all ca. 11,000 described species of Lepidoptera (butterflies and moths) found in Europe. The Project Psyche community encompasses diverse researchers, amateur lepidopterists, practitioners, and industry experts united by a common vision of the importance of genomics for Lepidoptera. Lepidoptera are at the forefront of biodiversity genomics with genomes for over 1,000 species already generated - the highest number across all eukaryotic orders. Here, we outline how Project Psyche is generating and harnessing these genomes. This pan-European catalogue of openly accessible lepidopteran genomes will transform our understanding of evolution and ecology, inform conservation, and foster advances in pest management.

Parasitoid wasps are major causes of mortality of many species, making host immune defences a common target of adaptive evolution, though such targets outside model species are poorly understood. In this study, we used two tests of positive selection to compare across three closely related Galerucella leaf beetles that show substantial differences in their phenotypic response to the shared parasitoid wasp Asecodes parviclava, their main natural enemy. Using a codon-based test, which detects excess amino acid fixations per locus along each species’ lineage, we found more evidence of positive selection on parasitoid-relevant immune genes in the species with the strongest immunocompetence (G. pusilla) compared with the species having weaker immunocompetence (G. tenella and G. calmariensis). Moreover, genes coding for the early phases in the immune response cascade were predominantly among the positively selected immune genes, providing targets for future functional genomic study to pin-point connections between genotypic and phenotypic differences in defences towards a parasitoid wasp. In contrast, genome-wide analyses of the haplotype frequency spectrum, which quantify selection over recent evolutionary time scales, revealed similar signatures of positive selection on immune genes across species. These results advance the field of host-parasitoid dynamics by providing novel insights into the tempo and mode of insect host evolutionary dynamics, and offering a framework for making genotype to phenotype connections for immunocompetence phenotypes.

Summary:The protein-coding regions of eukaryotic genes are fragmented into exons that, like the genes within which they are situated, can be duplicated, deleted, or reorganized. Cataloging and organizing within-gene exon similarities is necessary for a systematic study of exon evolution and its consequences. To facilitate the study of exon duplications, we present Exonize, a computational tool that identifies and classifies coding exon duplications in annotated genomes. Exonize implements a graph-based framework to handle clusters of related exons resulting from repeated rounds of exon duplication. The interdependence between duplicated exons or groups of exons across transcripts is classified. By identifying duplication events between exonic and intronic regions, Exonize can detect unannotated or degenerate exons. To aid in data parsing and downstream analysis, the Python module exonize_analysis is provided. The application of Exonize to 20 eukaryote genomes identifies full-exon duplications in at least 4% of vertebrate genes, with more than 900 human genes having a full-exon duplication event. Availability and implementation:Exonize is available at https://github.com/msarrias/exonize.

In cases of recurrent colonizations of similar habitats from the same base population, it is commonly expected that repeated phenotypic adaptation is caused by parallel changes in genetic variation. However, it is becoming increasingly clear that similar phenotypic variation may also evolve by alternative genetic pathways. Here, we explore the repeated evolution of photoperiodic plasticity for diapause induction across Swedish populations of the speckled wood butterfly, Pararge aegeria. This species has colonized Scandinavia at least twice, and population genomic results show that one of the candidate regions associated with spatial variation in photoperiodism is situated on the Z-chromosome. Here, we assay hybrid crosses between several populations that differ in photoperiodic plasticity for sex-linked inheritance of the photoperiodic reaction norm. We find that while a cross between more distantly related populations from the two different colonization events shows strong sex-dependent inheritance of photoperiodic plasticity, a cross between two more closely related populations within the oldest colonization range shows no such effect. We conclude that the genotype-phenotype map for photoperiodic plasticity varies across these populations and that similar local phenotypic adaptation has evolved during recurrent colonization events by partly non-parallel genetic changes.

To date, the use of CRISPR/Cas9 technology in ecological-model species for validating genotype to phenotype connections has focused primarily on visual phenotypes using G0 mutations, coupled with analyses of resulting mosaic phenotypes. However, studies of physiological phenotypes necessitate germline mutations in order to assess non-visible phenotypic effects, and thus, dedicated efforts to develop efficient germline mutations in ecological model species are needed. Here, we applied the CRISPR/Cas9 technology to an ecological model species, the speckled wood butterfly (Pararge aegeria). We targeted yellow-y, which is required for the production of black melanin, as yellow-y loss of function (LOF) mutations are not lethal and easy to phenotype, affording efficient assessment of G0 and germline mutations. To explore what factors may affect the efficiency of transformation, we employed four alternative treatments, including variation in sgRNAs and their concentrations. Color changes in the head capsule of first larval instars, as well as adult wing color, were used as indicators of successful knockouts. Individuals with wings that were at least 50% transformed were mated, with their G1 offspring assessed for the presence of germline mutations. Our CRISPR/Cas9 technique was highly efficient at generating LOF mutations in yellow-y. Across all treatments, nearly 80% of adults exhibited mosaic LOF phenotypes, of which nearly 30% appeared to have 100% LOF phenotypes. Crosses between adults exhibiting at least 50% LOF phenotypes resulted in fully transformed offspring, revealing a high incidence of germline LOF mutations in yellow-y. We provide a detailed protocol on how to obtain high germline LOF mutation efficiency in order to advance the study of genotype-phenotype connections for non-visible physiological traits across natural populations of this and other model ecological species.

1. Plants and herbivorous insects are engaged in long-term coevolutionary arms races, where gains in novel plant defences and corresponding adaptations in herbivores may drive evolutionary change. One such recent innovation is the gain of cardenolide toxins in the Brassicaceae genus Erysimum , which resulted in the effective deterrence of most herbivores by these plants. Nonetheless, some herbivores continue to attack Erysimum , likely by tolerating cardenolide defences through general detoxification mechanisms that may serve as evolutionary stepping-stones for more specialized resistance. 2. We investigated the interaction between diamondback moth (DBM, Plutella xylostella ) and its occasional host plant E. cheiranthoides , by first screening the standing variation in DBM performance, and second by experimentally evolving DBM populations for increased performance on Erysimum . Despite considerable variation among wild DBM populations, larvae consistently avoided Erysimum leaves for feeding, and when constrained to Erysimum , they exhibited reduced growth rate, survival, and lower adult size compared to individuals feeding on control broccoli plants. 3. Surprisingly, cardenolides could only partly explain the reduced performance of DBM on Erysimum , and experimental evolution failed to improve overall performance. Instead, phenotypes of evolved lines converged on what appears to be a pre-existing, highly plastic phenotype found among wild DBM ancestors and which is characterized by rapid development and high weight gain on control plants, but slow development and low weight gain on Erysimum . 4. Although DBM failed to evolve improved performance on Erysimum , our results demonstrate that its existing genetic variation and phenotypic plasticity are evidently sufficient to support long-term development on Erysimum , thereby fulfilling a key condition for the future evolution of specialized adaptations. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, 950319 Swiss National Science Foundation, PCEFP3_194590

The neritid snail Theodoxus fluviatilis is found across habitats differing in salinity, from shallow waters along the coast of the Baltic Sea to lakes throughout Europe. Living close to the water surface makes this species vulnerable to changes in salinity in their natural habitat, and the lack of a free-swimming larval stage limits this species’ dispersal. Together, these factors have resulted in a patchy distribution of quite isolated populations differing in their salinity tolerances. In preparation for investigating the mechanisms underlying the physiological differences in osmoregulation between populations that cannot be explained solely by phenotypic plasticity, we present here an annotated draft genome assembly for T. fluviatilis, generated using PacBio long reads, Illumina short reads, and transcriptomic data. While the total assembly size (1045 kb) is similar to those of related species, it remains highly fragmented (N scaffolds = 35,695; N50 = 74 kb) though moderately high in complete gene content (BUSCO single copy complete: 74.3%, duplicate: 2.6%, fragmented: 10.6%, missing: 12.5% using metazoa n = 954). Nevertheless, we were able to generate gene annotations of 21,220 protein-coding genes (BUSCO single copy complete: 65.1%, duplicate: 16.7%, fragmented: 9.1%, missing: 9.1% using metazoa n = 954). Not only will this genome facilitate comparative evolutionary studies across Gastropoda, as this is the first genome assembly for the basal snail family Neritidae, it will also greatly facilitate the study of salinity tolerance in this species. Additionally, we discuss the challenges of working with a species where high molecular weight DNA isolation is very difficult.

Generalist plant-feeding insects are characterised by a broad host repertoire that can comprise several families or even different orders of plants. The genetic and physiological mechanisms underlying the use of such a wide host range are still not fully understood. Earlier studies indicate that the consumption of different host plants is associated with host-specific gene expression profiles. It remained, however, unclear if and how larvae can alter these profiles in the case of a changing host environment. Using the polyphagous comma butterfly (Polygonia c-album) we show that larvae can adjust their transcriptional profiles in response to a new host plant. The switch to some of the host plants, however, resulted in a larger transcriptional response and, thus, seems to be more challenging. At a physiological level, no correspondence for these patterns could be found in larval performance. This suggests that a high transcriptional but also phenotypic flexibility are essential for the use of a broad and diverse host range. We furthermore propose that host switch tests in the laboratory followed by transcriptomic investigations can be a valuable tool to examine not only plasticity in host use but also subtle and/or transient trade-offs in the evolution of host plant repertoires.

Sexual dimorphisms represent a source of phenotypic variation and result from differences in how natural and sexual selection act on males and females within a species. Identifying the genetic basis of dimorphism can be challenging, especially once it is fixed within a species. However, studying polymorphisms, even when fixed within a population, can provide insights into the genetic basis of sexual dimorphisms. In this study, we investigate the genetic basis of a regionally isolated sexual dimorphism in the wings of \emph{Pieris napi adalvinda}, a subspecies of \emph{P. napi} found in northernmost Scandinavia, where females exhibit heavily melanized wings. By using a combination of male and female informative crosses, genomic sequencing of melanic outliers, and a population genomic analysis with a new reference genome for the melanic morph, we demonstrate that the female-limited morph adalvinda is caused by a single dominant allele at an autosomal locus upstream of the gene \emph{cortex}. This novel finding adds to the growing body of literature that connects repeated mutations in and near the cortex gene to the regulation of butterfly wing melanization, providing insights into the evolution of sexual dimorphisms and the recruitment of genes into monomorphic or sex-limited forms. This study thus highlights the significance of cortex as a basis for a female-limited trait and lays the foundation for future comparative analyses of dimorphism genetic underpinnings.

Evolutionary changes in geographic distribution and larval host plants may promote the rapid diversification of montane insects, but this scenario has been rarely investigated. We studied the rapid radiation of the butterfly genus Colias, which has diversified in mountain ecosystems in Eurasia, Africa, and the Americas. Based on a data set of 150 nuclear protein-coding genetic loci and mitochondrial genomes, we constructed a time-calibrated phylogenetic tree of Colias species with broad taxon sampling. We then inferred their ancestral geographic ranges, historical diversification rates, and the evolution of host use. We found that the most recent common ancestor of Colias was likely geographically widespread and originated similar to 3.5 Ma. The group subsequently diversified in different regions across the world, often in tandem with geographic expansion events. No aspect of elevation was found to have a direct effect on diversification. The genus underwent a burst of diversification soon after the divergence of the Neotropical lineage, followed by an exponential decline in diversification rate toward the present. The ancestral host repertoire included the legume genera Astragalus and Trifolium but later expanded to include a wide range of Fabaceae genera and plants in more distantly related families, punctuated with periods of host range expansion and contraction. We suggest that the widespread distribution of the ancestor of all extant Colias lineages set the stage for diversification by isolation of populations that locally adapted to the various different environments they encountered, including different host plants. In this scenario, elevation is not the main driver but might have accelerated diversification by isolating populations.

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.

Quantifying the tempo and mode via modern phylogenetic comparative methods can provide crucial insights into the role of selection and constraints in trait evolution. Here we elucidate the evolution of diapause, a complex and defining life-history trait that allows temporal escape from unfavorable conditions in many insects, including our model system, butterflies. Using a thorough literature survey, we first scored the developmental stage of diapause (egg, larva, pupa, adult) vs. absence of diapause. We find that larval diapause is most common in temperate lineages while pupal, egg, and adult diapause are relatively rare. Next, we determined that the loss of diapause occurred at a much higher rate than the gain, and its gain primarily occurred from the non-diapause state. While ancestral state estimation at deeper nodes remained uncertain, we found consistent patterns for some families and strong evidence for the convergent evolution of diapause across butterflies. We found no support for the hypothesis that the rate of the gain of diapause should be higher during the Eocene-Oligocene glacial maximum event (~35 MYA). Overall, the evolution of diapause in butterflies has a complex history, has evolved convergently, and has likely evolved much earlier than the Eocene-Oligocene glaciation event consistent with the deep history of diapause evolution in insects. These findings fill a deep gap in much-needed studies for future comparative research.

Organisms inhabiting highly seasonal environments must cope with a wide range of environmentally induced challenges. Many seasonal challenges require extensive physiological modification to survive. In winter, to survive extreme cold and limited resources, insects commonly enter diapause, which is an endogenously derived dormant state associated with minimized cellular processes and low energetic expenditure. Due to the high degree of complexity involved in diapause, substantial cellular regulation is required, of which our understanding primarily derives from the transcriptome via messenger RNA expression dynamics. Here we aim to advance our understanding of diapause by investigating microRNA (miRNA) expression in diapausing and direct developing pupae of the butterfly Pieris napi. We identified coordinated patterns of miRNA expression throughout diapause in both head and abdomen tissues of pupae, and via miRNA target identification, found several expression patterns to be enriched for relevant diapause-related physiological processes. We also identified two candidate miRNAs, miR-14-5p and miR-2a-3p, that are likely involved in diapause progression through their activity in the ecdysone pathway, a critical regulator of diapause termination. miR-14-5p targets phantom, a gene in the ecdysone synthesis pathway, and is upregulated early in diapause. miR-2a-3p has been found to be expressed in response to ecdysone, and is upregulated during diapause termination. Together, the expression patterns of these two miRNAs match our current understanding of the timing of hormonal regulation of diapause in P. napi and provide interesting candidates to further explore the mechanistic role of microRNAs in diapause regulation.

Phenotypic plasticity is produced and maintained by processes regulating the transcriptome. While differential gene expression is among the most important of these processes, relatively little is known about other sources of transcriptional variation. Previous work suggests that alternative splicing plays an extensive and functionally unique role in transcriptional plasticity, though plastically spliced genes may be more constrained than the remainder of expressed genes. In this study, we explore the relationship between expression and splicing plasticity, along with the genetic diversity in those genes, in an ecologically consequential polyphenism: facultative diapause. Using 96 samples spread over two tissues and 10 timepoints, we compare the extent of differential splicing and expression between diapausing and direct developing pupae of the butterfly Pieris napi . Splicing differs strongly between diapausing and direct developing trajectories but alters a smaller and functionally unique set of genes compared to differential expression. We further test the hypothesis that among these expressed loci, plastically spliced genes are likely to experience the strongest purifying selection to maintain seasonally plastic phenotypes. Genes with unique transcriptional changes through diapause consistently had the lowest nucleotide diversity, and this effect was consistently stronger among genes that were differentially spliced compared to those with just differential expression through diapause. Further, the strength of negative selection was higher in the population expressing diapause every generation. Our results suggest that maintenance of the molecular mechanisms involved in diapause progression, including post-transcriptional modifications, are highly conserved and likely to experience genetic constraints, especially in northern populations of P. napi .

Abstract 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.

The ithomiine butterflies (Nymphalidae: Danainae) represent the largest known radiation of Mullerian mimetic butterflies. They dominate by number the mimetic butterfly communities, which include species such as the iconic neotropical Heliconius genus. Despite recent studies carried out on ithomiine ecology and genetic structure, no reference genome was available for the tribe. Here, we generated high-quality, chromosome-scale genome assemblies of two Melinaea species, Melinaea marsaeus and Melinaea menophilus , and a draft genome of Ithomia salapia . We obtained genomes with a size ranging from 396 Mb to 503 Mb across the three species and scaffold N50 of 40.5 Mb and 23.2 Mb for the two chromosome-scale assemblies. Using collinearity analyses we identified massive rearrangements between the two closely related Melinaea species. A detailed annotation of transposable elements and genes was performed, resulting in the identification of 24,341, 31,081 and 31,976 genes in I. salapia , M. marsaeus and M. menophilus , respectively. We used a specialist annotation to target chemosensory genes, which is crucial for host plant detection and mate recognition in mimetic species. A comparative genomic approach revealed independent gene expansions in ithomiines and particularly in gustatory receptor genes. These first three genomes of ithomiine mimetic butterflies constitute a valuable addition and a welcome comparison to existing biological models of mimicry, such as Heliconius , and will enable further understanding of the mechanisms of adaptation and the genetic bases underpinning mimicry.

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.

Understanding the evolutionary origins and factors maintaining alternative life history strategies (ALHS) within species is a major goal of evolutionary research. While alternative alleles causing discrete ALHS are expected to purge or fix over time, one-third of the ~90 species of Colias butterflies are polymorphic for a female-limited ALHS called Alba. Whether Alba arose once, evolved in parallel, or has been exchanged among taxa is currently unknown. Using comparative genome-wide association study (GWAS) and population genomic analyses, we placed the genetic basis of Alba in time-calibrated phylogenomic framework, revealing that Alba evolved once near the base of the genus and has been subsequently maintained via introgression and balancing selection. CRISPR-Cas9 mutagenesis was then used to verify a putative cis-regulatory region of Alba, which we identified using phylogenetic foot printing. We hypothesize that this cis-regulatory region acts as a modular enhancer for the induction of the Alba ALHS, which has likely facilitated its long evolutionary persistence.

Although miRNA regulation of protein production is a likely target of adaptive evolution, high false-positive rates in the identification of mRNAs targeted by miRNAs in non-model species’ complicates interpretation of recent advances. Here we document the challenges and then outline steps for the community to address these challenges.

Parasitoid wasps are major causes of mortality of many species, resulting in host immune defences commonly being the target of adaptive evolution, though such targets outside model species are poorly understood. Here we compare the power of different molecular tests of selection to provide such insights in novel species. We combined our understanding of variation in immune defence capacity among three closely related Galerucella leaf beetles with a shared parasitoid wasp, with information on genomic targets of parasitoid attacks from exemplar insect species. Based on this, we predicted that these genomic targets would vary in their evolutionary history across three closely related leaf beetle species, such that genomic targets would experience stronger positive selection in the species with strongest immune response to attack. Codon based tests revealed variation among species in positive selection genome wide, and showed that parasitoid-relevant immune genes experienced more positive selection in the species with the greatest immunocompetence (G. pusilla), while almost no immune genes were under positive selection in the species with the least immunocompetence (G. calmariensis). Genome wide analyses of the haplotype frequency spectrum also identified genes experiencing positive selection across the species, though few were parasitoid-relevant immune genes and no species was particularly enriched for them. Thus, our codon based test, which summarizes all sweep events since the last common ancestor, found results consistent with our a priori hypothesis, providing a series of targets for future functional genomic study.