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.