International policy recently adopted commitments to maintain genetic diversity in wild populations to secure their adaptive potential, including metrics to monitor temporal trends in genetic diversity – so-called indicators. A national programme for assessing trends in genetic diversity was recently initiated in Sweden. Relating to this effort, we systematically assess contemporary genome-wide temporal trends (40 years) in wild populations using the newly adopted indicators and whole genome sequencing (WGS). We use pooled and individual WGS data from brown trout (Salmo trutta) in eight alpine lakes in protected areas. Observed temporal trends in diversity metrics (nucleotide diversity, Watterson's ϴ and heterozygosity) lie within proposed acceptable threshold values for six of the lakes, but with consistently low values in lakes above the tree line and declines observed in these northern-most lakes. Local effective population size is low in all lakes, highlighting the importance of continued protection of interconnected systems to allow genetic connectivity for long-term viability of these populations. Inbreeding (F_ROH) spans 10%–30% and is mostly represented by ancient (<1 Mb) runs of homozygosity, with observations of little change in mutational load. We also investigate adaptive dynamics over evolutionarily short time frames (a few generations); identifying putative parallel selection across all lakes within a gene pertaining to skin pigmentation as well as candidates of selection unique to specific lakes and lake systems involved in reproduction and immunity. We demonstrate the utility of WGS for systematic monitoring of natural populations, a priority concern if genetic diversity is to be protected.

The sympatric existence of genetically distinguishable populations of the same species remains a puzzle in ecology. Coexisting salmonid fish populations are known from over 100 freshwater lakes. Most studies of sympatric populations have used limited numbers of genetic markers making it unclear if genetic divergence involves certain parts of the genome. We returned to the first reported case of salmonid sympatry, initially detected through contrasting homozygosity at a single allozyme locus (coding for lactate dehydrogenase A) in brown trout in the small Lakes Bunnersjöarna, Sweden. First, we verified the existence of the two coexisting demes using a 96-SNP fluidigm array. We then applied whole-genome resequencing of pooled DNA to explore genome-wide diversity within and between these demes; nucleotide diversity was higher in deme I than in deme II. Strong genetic divergence is observed with genome-wide F_ST ≈ 0.2. Compared with data from populations of similar small lakes, this divergence is of similar magnitude as that between reproductively isolated populations. Individual whole-genome resequencing of two individuals per deme suggests higher inbreeding in deme II versus deme I, indicating different degree of isolation. We located two gene-copies for LDH-A and found divergence between demes in a regulatory section of one of these genes. However, we did not find a perfect fit between the sequence data and previous allozyme results, and this will require further research. Our data demonstrates genome-wide divergence governed mostly by genetic drift but also by diversifying selection in coexisting populations. This type of hidden biodiversity needs consideration in conservation management.

Insects rely on their innate immune system to successfully mediate complex interactions with their internal microbiota, as well as the microbes present in the environment. Given the variation in microbes across habitats, the challenges to respond to them are likely to result in local adaptations in the immune system. Here we focus upon phagocytosis, a mechanism by which pathogens and foreign particles are engulfed in order to be contained, killed, and processed. We investigated the phenotypic and genetic variation related to phagocytosis in two allopatric populations of the butterfly Pieris napi. Populations were found to differ in their hemocyte composition and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type. Yet, genes annotated to phagocytosis showed no large genomic signal of divergence. However, a gene set enrichment analysis on significantly divergent genes identified loci involved in glutamine metabolism, which recently have been linked to immune cell differentiation in mammals. Together these results suggest that heritable variation in phagocytic capacity arises via a quantitative trait architecture with variation in genes affecting the activation and/or differentiation of phagocytic cells, suggesting them as potential candidate genes underlying these phenotypic differences.

Understanding the tradeoffs that result from successful infection responses is central to understanding how life histories evolve. Gaining such insights, however, can be challenging, as they may be pathogen specific and confounded with experimental design. Here, we investigated whether infection from gram positive or negative bacteria results in different physiological tradeoffs, and whether these infections impact life history later in life (post-diapause development), in the butterfly Pieris napi. During the first 24 h after infection (3, 6, 12, and 24 h), after removing effects due to injection, larvae infected with Micrococcus luteus showed a strong suppression of all non-immunity related processes while several types of immune responses were upregulated. In contrast, this tradeoff between homeostasis and immune response was much less pronounced in Escherichia coli infections. These differences were also visible long after infection, via weight loss and slower development, as well as an increased mortality at higher infection levels during later stages of development. Individuals infected with M. luteus, compared to E. coli, had a higher mortality rate, and a lower pupal weight, developmental rate and adult weight. Further, males exhibited a more negative impact of infection than females. Thus, immune responses come at a cost even when the initial infection has been overcome, and these costs are likely to affect later life history parameters with fitness consequences.

The biology of parasitoids in natural ecosystems remains very poorly studied, though they are key species for their functioning. Here we focused on Phobocampe confusa, a Nymphalini specialist, responsible for high mortality rates in charismatic butterfly species in Europe (genus Aglais). We studied its ecology and genetic structure in connection with those of its host butterflies in Sweden. To this aim, we gathered data from 428 P. confusa individuals reared from 6094 butterfly larvae (of A. urticae, A. io, and in two occasions of Araschnia levana) collected over two years (2017 and 2018) and across 19 sites distributed along a 500 km latitudinal gradient. We found that P. confusa is widely distributed along the latitudinal gradient. Its distribution seems constrained over time by the phenology of its hosts. The large variation in climatic conditions between sampling years explains the decrease in phenological overlap between P. confusa and its hosts in 2018 and the 33.5% decrease in the number of butterfly larvae infected. At least in this study, P. confusa seems to favour A. urticae as host. While it parasitized nests of A. urticae and A. io equally, the proportion of larvae parasitized is significantly higher for A. urticae. At the landscape scale, P. confusa is almost exclusively found in vegetated open land and near deciduous forests, whereas artificial habitats are negatively correlated with the likelihood of a nest to be parasitized. The genetic analyses on 89 adult P. confusa and 87 adult A. urticae using CO1 and AFLP markers reveal a low genetic diversity in P. confusa and a lack of genetic structure in both species, at the scale of our sampling. Further genetic studies using high-resolution genomics tools will be required to better understand the population genetic structure of P. confusa, its biotic interactions with its hosts, and ultimately the stability and the functioning of natural ecosystems.

Butterflies are ubiquitous and abundant, occurring in a wide variety of environments that contain diverse microbial communities with varied pathogenic pressures. These pathogens and parasites present a constant threat to organisms, and have led to the evolution of complex and intricate immune responses. Despite strong selection against immunological threats, organisms display great variation in their immune capabilities, both on the genetic and physiological level. Investigating this variation remains challenging, since differences in immune responses might arise from changes in the amount, size or performance of cells or organs. Disentangling these relative contributions is important, as the targets of selection are expected to differ, ranging from immune genes directly related to the phenotype to genes indirectly involved via cell proliferation. This thesis focuses on characterizing the immune system of the butterfly Pieris napi and investigating its remarkable variation across populations by using both phenotypic and genotypic measurements. By integrating RNA-seq with life history measurements, I found that the cost of infection and wounding in the final larval stage carries over the metamorphic boundary in P. napi (Paper II). Using population comparisons, I identified both the action and potential targets of natural selection in wild populations within their respective immune responses (Paper I, III & IV). The immune genes in P. napi show increased genetic variation compared to the rest of the genome, and microevolutionary selection dynamics act on these genes between and among populations (Paper I). I measured the cellular immune responses related to phagocytosis and melanization in common garden reared larvae originating from two allopatric populations (Spain, Sweden) (Paper III & IV). The two populations were found to differ in their blood cell composition, and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type (Paper III). However, genome wide analysis of divergence between these populations found no excess genetic differentiation in genes annotated to phagocytic capacity, suggesting that our observed population differences might arise from genes affecting the activation or transdifferentiation of cells, which currently lack functional annotation. Interestingly, genes involved in glutamine metabolism, which have been linked to immune cell differentiation in mammals, did show divergence between the populations. In addition, the populations also differed in prophenoloxidase activity, a common method for quantifying immune related melanization in insects, along with the abundance of the cell-type (oenocytoids) related to this important immune function (Paper IV). Integrative analysis using both transcriptomic and genomic data revealed that the genes involved in this phenotype showed no significant differentiation between the populations. However, a gene involved with proper trafficking of melanogenic enzymes in vertebrates was found to be highly expressed and highly diverged between the two populations, providing an interesting candidate for future studies. This thesis demonstrates the advantages of integrating several genomic tools with lab experiments to quantify natural variation in the immune system of butterflies.

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.

Insects rely on their innate immune system to successfully mediate complex interactions with their microbiota, as well as the microbes present in the environment. Previous work has shown that components of the canonical immune gene repertoire evolve rapidly and have evolutionary characteristics originating from interactions with fast-evolving microorganisms. Although these interactions are likely to vary among populations, there is a poor understanding of the microevolutionary dynamics of immune genes, especially in non-Dipteran insects. Here, we use the full set of canonical insect immune genes to investigate microevolutionary dynamics acting on these genes between and among populations by comparing three allopatric populations of the green-veined white butterfly, Pieris napi (Linne; Lepidoptera, Pieridae). Immune genes showed increased genetic diversity compared to genes from the rest of the genome and various functional categories exhibited different types of signatures of selection, at different evolutionary scales, presenting a complex pattern of selection dynamics. Signatures of balancing selection were identified in 10 genes, and 17 genes appear to be under positive selection. Genes involved with the cellular arm of the immune response as well as the Toll pathway appear to be enriched among our outlier loci, regardless of functional category. This suggests that the targets of selection might focus upon an entire pathway, rather than functional subsets across pathways. Our microevolutionary results are similar to previously observed macroevolutionary patterns from diverse taxa, suggesting that either the immune system is robust to dramatic differences in life history and microbial communities, or that diverse microbes exert similar selection pressures.

We propose that an evolutionary and phenotype-driven approach, harnessing current technological developments, has much to offer for our understanding of insect immunity. After briefly reviewing the history of the discovery of canonical immune system, the current understanding of its components is reviewed and then we argue that the current paradigm of research may be biassed due to (a) its limited taxonomic perspective, (b) the evolutionary time scale being studied, and (c) a focus primarily if not exclusively, upon the canonical, humoural gene set. For the rest of the review, we then discuss the importance of a phenotype down approach as an understudied perspective, exemplified by the need for understanding the basis of cellular responses and wounding as a source of selection on immunity in the wild. We propose that research on those topics almost certainly will provide new insights into the evolution of the insect immune system.