Genetic constraints in genes exhibiting splicing plasticity in facultative diapause.

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.

Fine-Scale Map Reveals Highly Variable Recombination Rates Associated with Genomic Features in the Eurasian Blackcap.

Recombination is responsible for breaking up haplotypes, influencing genetic variability, and the efficacy of selection. Bird genomes lack the protein PR domain-containing protein 9, a key determinant of recombination dynamics in most metazoans. Historical recombination maps in birds show an apparent stasis in positioning recombination events. This highly conserved recombination pattern over long timescales may constrain the evolution of recombination in birds. At the same time, extensive variation in recombination rate is observed across the genome and between different species of birds. Here, we characterize the fine-scale historical recombination map of an iconic migratory songbird, the Eurasian blackcap (Sylvia atricapilla), using a linkage disequilibrium-based approach that accounts for population demography. Our results reveal variable recombination rates among and within chromosomes, which associate positively with nucleotide diversity and GC content and negatively with chromosome size. Recombination rates increased significantly at regulatory regions but not necessarily at gene bodies. CpG islands are associated strongly with recombination rates, though their specific position and local DNA methylation patterns likely influence this relationship. The association with retrotransposons varied according to specific family and location. Our results also provide evidence of heterogeneous intrachromosomal conservation of recombination maps between the blackcap and its closest sister taxon, the garden warbler. These findings highlight the considerable variability of recombination rates at different scales and the role of specific genomic features in shaping this variation. This study opens the possibility of further investigating the impact of recombination on specific population-genomic features.

The blackcap (Sylvia atricapilla) genome reveals a recent accumulation of LTR retrotransposons.

Transposable elements (TEs) are mobile genetic elements that can move around the genome, and as such are a source of genomic variability. Based on their characteristics we can annotate TEs within the host genome and classify them into specific TE types and families. The increasing number of available high-quality genome references in recent years provides an excellent resource that will enhance the understanding of the role of recently active TEs on genetic variation and phenotypic evolution. Here we showcase the use of a high-quality TE annotation to understand the distinct effect of recent and ancient TE insertions on the evolution of genomic variation, within our study species the Eurasian blackcap (Sylvia atricapilla). We investigate how these distinct TE categories are distributed along the genome and evaluate how their coverage across the genome is correlated with four genomic features: recombination rate, gene coverage, CpG island coverage and GC content. We found within the recent TE insertions an accumulation of LTRs previously not seen in birds. While the coverage of recent TE insertions was negatively correlated with both GC content and recombination rate, the correlation with recombination rate disappeared and turned positive for GC content when considering ancient TE insertions.

The fine-scale recombination rate variation and associations with genomic features in a butterfly.

Recombination is a key molecular mechanism that has profound implications on both micro- and macroevolutionary processes. However, the determinants of recombination rate variation in holocentric organisms are poorly understood, in particular in Lepidoptera (moths and butterflies). The wood white butterfly (Leptidea sinapis) shows considerable intraspecific variation in chromosome numbers and is a suitable system for studying regional recombination rate variation and its potential molecular underpinnings. Here, we developed a large whole-genome resequencing data set from a population of wood whites to obtain high-resolution recombination maps using linkage disequilibrium information. The analyses revealed that larger chromosomes had a bimodal recombination landscape, potentially caused by interference between simultaneous chiasmata. The recombination rate was significantly lower in subtelomeric regions, with exceptions associated with segregating chromosome rearrangements, showing that fissions and fusions can have considerable effects on the recombination landscape. There was no association between the inferred recombination rate and base composition, supporting a limited influence of GC-biased gene conversion in butterflies. We found significant but variable associations between the recombination rate and the density of different classes of transposable elements, most notably a significant enrichment of short interspersed nucleotide elements in genomic regions with higher recombination rate. Finally, the analyses unveiled significant enrichment of genes involved in farnesyltranstransferase activity in recombination coldspots, potentially indicating that expression of transferases can inhibit formation of chiasmata during meiotic division. Our results provide novel information about recombination rate variation in holocentric organisms and have particular implications for forthcoming research in population genetics, molecular/genome evolution, and speciation.

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.

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.

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.

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.

Metabolome dynamics of diapause in the butterfly Pieris napi: distinguishing maintenance, termination and post-diapause phases.

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 Pieris napi, 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.

Energy and lipid metabolism during direct and diapause development in a pierid butterfly.

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 ß-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.

Disentangling the complex evolutionary history of the Western Palearctic blue tits (Cyanistes spp.) - phylogenomic analyses suggest radiation by multiple colonization events and subsequent isolation.

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.

Effect of winter cold duration on spring phenology of the orange tip butterfly, Anthocharis cardamines.

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.