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.

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.

Diapause is a fundamental component of the life cycle in the majority of insects living in environments characterized by strong seasonality. The present study addresses poorly understood associations and trade-offs between endogenous diapause duration, thermal sensitivity of development, energetic cost of development and cold tolerance. Diapause intensity, metabolic rate trajectories and lipid profiles of directly developing and diapausing animals were studied using pupae and adults of Pieris napi butterflies from a population in which endogenous diapause has been well studied. Endogenous diapause was terminated after 3 months and termination required chilling. Metabolic and post-diapause development rates increased with diapause duration, while the metabolic cost of post-diapause development decreased, indicating that once diapause is terminated, development proceeds at a low rate even at low temperature. Diapausing pupae had larger lipid stores than the directly developing pupae, and lipids constituted the primary energy source during diapause. However, during diapause, lipid stores did not decrease. Thus, despite lipid catabolism meeting the low energy costs of the diapausing pupae, primary lipid store utilization did not occur until the onset of growth and metamorphosis in spring. In line with this finding, diapausing pupae contained low amounts of mitochondria-derived cardiolipins, which suggests a low capacity for fatty acid β-oxidation. While ontogenic development had a large effect on lipid and fatty acid profiles, only small changes in these were seen during diapause. The data therefore indicate that the diapause lipidomic phenotype is developed early, when pupae are still at high temperature, and retained until post-diapause development.

The effect of spring temperature on spring phenology is well understood in a wide range of taxa. However, studies on how winter conditions may affect spring phenology are underrepresented. Previous work on Anthocharis cardamines (orange tip butterfly) has shown population‐specific reaction norms of spring development in relation to spring temperature and a speeding up of post‐winter development with longer winter durations. In this experiment, we examined the effects of a greater and ecologically relevant range of winter durations on post‐winter pupal development of A. cardamines of two populations from the United Kingdom and two from Sweden. By analyzing pupal weight loss and metabolic rate, we were able to separate the overall post‐winter pupal development into diapause duration and post‐diapause development. We found differences in the duration of cold needed to break diapause among populations, with the southern UK population requiring a shorter duration than the other populations. We also found that the overall post‐winter pupal development time, following removal from winter cold, was negatively related to cold duration, through a combined effect of cold duration on diapause duration and on post‐diapause development time. Longer cold durations also lead to higher population synchrony in hatching. For current winter durations in the field, the A. cardamines population of southern UK could have a reduced development rate and lower synchrony in emergence because of short winters. With future climate change, this might become an issue also for other populations. Differences in winter conditions in the field among these four populations are large enough to have driven local adaptation of characteristics controlling spring phenology in response to winter duration. The observed phenology of these populations depends on a combination of winter and spring temperatures; thus, both must be taken into account for accurate predictions of phenology.

Diapause is a deep resting stage facilitating temporal avoidance of unfavourable environmental conditions, and is used by many insects to adapt their life cycle to seasonal variation. Although considerable work has been invested in trying to understand each of the major diapause stages (induction, maintenance and termination), we know very little about the transitions between stages, especially diapause termination. Understanding diapause termination is crucial for modelling and predicting spring emergence and winter physiology of insects, including many pest insects. In order to gain these insights, we investigated metabolome dynamics across diapause development in pupae of the butterfly , which exhibits adaptive latitudinal variation in the length of endogenous diapause that is uniquely well characterized. By employing a time-series experiment, we show that the whole-body metabolome is highly dynamic throughout diapause and differs between pupae kept at a diapause-terminating (low) temperature and those kept at a diapause-maintaining (high) temperature. We show major physiological transitions through diapause, separate temperature-dependent from temperature-independent processes and identify significant patterns of metabolite accumulation and degradation. Together, the data show that although the general diapause phenotype (suppressed metabolism, increased cold tolerance) is established in a temperature-independent fashion, diapause termination is temperature dependent and requires a cold signal. This revealed several metabolites that are only accumulated under diapause-terminating conditions and degraded in a temperature-unrelated fashion during diapause termination. In conclusion, our findings indicate that some metabolites, in addition to functioning as cryoprotectants, for example, are candidates for having regulatory roles as metabolic clocks or time-keepers during diapause.

Isolated islands and their often unique biota continue to play key roles for understanding the importance of drift, genetic variation and adaptation in the process of population differentiation and speciation. One island system that has inspired and intrigued evolutionary biologists is the blue tit complex (Cyanistes spp.) in Europe and Africa, in particular the complex evolutionary history of the multiple genetically distinct taxa of the Canary Islands. Understanding Afrocanarian colonization events is of particular importance because of recent unconventional suggestions that these island populations acted as source of the widespread population in mainland Africa. We investigated the relationship between mainland and island blue tits using a combination of Sanger sequencing at a population level (20 loci; 12 500 nucleotides) and next-generation sequencing of single population representatives (>3 200 000 nucleotides), analysed in coalescence and phylogenetic frameworks. We found (i) that Afrocanarian blue tits are monophyletic and represent four major clades, (ii) that the blue tit complex has a continental origin and that the Canary Islands were colonized three times, (iii) that all island populations have low genetic variation, indicating low long-term effective population sizes and (iv) that populations on La Palma and in Libya represent relicts of an ancestral North African population. Further, demographic reconstructions revealed (v) that the Canary Islands, conforming to traditional views, hold sink populations, which have not served as source for back colonization of the African mainland. Our study demonstrates the importance of complete taxon sampling and an extensive multimarker study design to obtain robust phylogeographical inferences.

Many temperate insects survive harsh environmental conditions, such as winter, by entering a state of developmental arrest. This diapause state is predominantly induced by photoperiod. The photoperiod varies with latitude and has led to local adaptation in the photoperiodic induction of diapause in many insects. To understand the rapid evolution of the photoperiodic threshold, it is important to investigate and understand the underlying genetic mechanisms. In the present study, the genetic basis of photoperiodic diapause induction is investigated in the green-veined white butterfly Pieris napi (Lepidoptera, Pieridae) by assaying diapause induction in a range of conditions for a Swedish and Spanish population. Furthermore, the inheritance of diapause induction is assessed in reciprocal F1 hybrids and backcrosses between the two populations. The southern population shows a clear photoperiodic threshold determining diapause or direct development, whereas the northern populations show a high incidence of diapause, regardless of photoperiod. The hybrid crosses reveal that the inheritance of diapause induction is strongly sex-linked, and that diapause incidence in the genetic crosses is highly dependent on photoperiod. This emphasizes the importance of assaying a range of conditions in diapause inheritance studies. The results indicate a strongly heritable diapause induction with a major component on the Z-chromosome, as well as a minor effect of the autosomal background.

Recombination is responsible for breaking up haplotypes, influencing genetic variability, and the efficacy of selection. Bird genomes lack the protein PRDM9, a key determinant of recombination dynamics in most metazoans. The historical recombination maps in birds show an apparent stasis in the positioning of recombination events. This highly conserved recombination pattern over long timescales should constrain the evolution of recombination in birds, but extensive variation in recombination rate across the genome and between different species has been reported. Here, we characterise a fine-scale historical recombination map of an iconic migratory songbird, the European blackcap (Sylvia atricapilla) using a LD-based approach which accounts for population demography. We found variable recombination rates among and within chromosomes, which associate positively with nucleotide diversity and GC content, and a negatively with chromosome size. The recombination rates increased significantly at regulatory regions and not necessarily at high-dense gene regions. CpG islands associated strongly with recombination rates; however, their specific position and local DNA methylation patterns likely influenced this relationship. The association with retrotransposons varied according to specific family and location. Our results also provide evidence of a heterogeneous conservation of recombination maps between the blackcap and its closest sister taxon, the garden warbler at the intra-chromosomal level. These findings highlight the considerable variability of recombination rates at different scales and the role of specific genomic features at shaping this variation. This study opens the possibility of further investigating the impact of recombination in specific population-genomic features.