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.

Local adaptation enables species to expand their ranges and enhance survival in their core distributions. To uncover the genetic basis of such adaptation, population genomic association studies have identified candidate genes. However, natural variation in these genes is rarely functionally validated, especially for polygenic traits. Here, we functionally validate a genomic region containing the core circadian clock gene timeless, previously associated with clinal variation in diapause timing in Pararge aegeria in Sweden. One isolated island population carries a large deletion in timeless that spans   ̴1.5 exons and is fixed in the population. By back crossing and selective breeding, we introgressed this deletion into the genetic background of an alternative mainland population. Using this introgressed population, we compared the diapause response of two genotypes: one homozygous for the island deletion and one homozygous for the alternative mainland allele. Despite sharing most of their genome, the genotype homozygous for the deletion largely recovered the seasonal timing phenotype of its island source population. While it remains unclear whether this effect stems from timeless alone or linked loci, our results demonstrate that this genomic region contributes substantially to variation in seasonal timing between these populations. This finding is significant, as the photoperiodic reaction norm of a population strongly influences both its voltinism and overall life history. 

Pleiotropic effects of naturally occurring polymorphisms can constrain adaptive evolution by generating correlated phenotypic responses, making them key to understanding the genetic basis of local adaptation and predicting evolutionary responses to climate change. However, few studies have functionally tested the causal roles of candidate loci and their potential pleiotropic effects. Diapause, a type of hibernation in insects, is a crucial adaptation for surviving unfavorable seasonal conditions and requires precise timing of both induction and termination. Genetic correlations between these traits may strongly influence fitness under climate change. In P. aegeria, the circadian clock gene timeless appears to be associated with variation in diapause induction timing across many Swedish populations, and in one particular population, this association has been shown to result from a single structural mutation. That population is fixed for a large deletion in timeless and differs from other Swedish populations in both induction and termination timing. In a previous study, we confirmed that this deletion causally influences induction timing between this and a closely related population. Here, we used homozygous genotypes from an introgressed line to test if the same deletion also affects termination timing. We found no significant difference between the genotypes, suggesting that variation in termination timing is not driven by pleiotropic effects of timeless but may instead reflect local adaptation at other loci. The apparent absence of pleiotropy may allow induction and termination timing to evolve independently in response to climate change. Given the independent evolution of diapause across insects, further functional studies of naturally occurring polymorphisms in circadian clock genes are needed to assess their general pleiotropic role in diapause and related life-history traits.       

In their simulation study, Garcia-Costoya et al. (2023) conclude that evolutionary constraints might aid populations facing climate change. However, we are concerned that this conclusion is largely a consequence of the simulated temperature variation being too small, and, most importantly, that uneven limitations to standing variation disadvantage unconstrained populations. In their simulation study, Garcia-Costoya et al. (2023) conclude that evolutionary constraints might aid populations facing climate change. However, we are concerned that this conclusion is largely a consequence of the simulated temperature variation being too small, and, most importantly, that uneven limitations to standing variation disadvantage unconstrained populations.image