Chromosome-scale Genome Assembly of the Rough Periwinkle Littorina saxatilis.

The intertidal gastropod Littorina saxatilis is a model system to study speciation and local adaptation. The repeated occurrence of distinct ecotypes showing different levels of genetic divergence makes L. saxatilis particularly suited to study different stages of the speciation continuum in the same lineage. A major finding is the presence of several large chromosomal inversions associated with the divergence of ecotypes and, specifically, the species offers a system to study the role of inversions in this divergence. The genome of L. saxatilis is 1.35 Gb and composed of 17 chromosomes. The first reference genome of the species was assembled using Illumina data, was highly fragmented (N50 of 44 kb), and was quite incomplete, with a BUSCO completeness of 80.1% on the Metazoan dataset. A linkage map of one full-sibling family enabled the placement of 587 Mbp of the genome into 17 linkage groups corresponding to the haploid number of chromosomes, but the fragmented nature of this reference genome limited the understanding of the interplay between divergent selection and gene flow during ecotype formation. Here, we present a newly generated reference genome that is highly contiguous, with a N50 of 67 Mb and 90.4% of the total assembly length placed in 17 super-scaffolds. It is also highly complete with a BUSCO completeness of 94.1% of the Metazoa dataset. This new reference will allow for investigations into the genomic regions implicated in ecotype formation as well as better characterization of the inversions and their role in speciation.

The genetic basis of a recent transition to live-bearing in marine snails.

Key innovations are fundamental to biological diversification, but their genetic basis is poorly understood. A recent transition from egg-laying to live-bearing in marine snails (Littorina spp.) provides the opportunity to study the genetic architecture of an innovation that has evolved repeatedly across animals. Individuals do not cluster by reproductive mode in a genome-wide phylogeny, but local genealogical analysis revealed numerous small genomic regions where all live-bearers carry the same core haplotype. Candidate regions show evidence for live-bearer-specific positive selection and are enriched for genes that are differentially expressed between egg-laying and live-bearing reproductive systems. Ages of selective sweeps suggest that live-bearer-specific alleles accumulated over more than 200,000 generations. Our results suggest that new functions evolve through the recruitment of many alleles rather than in a single evolutionary step.

Impact of Z chromosome inversions on gene expression in testis and liver tissues in the zebra finch.

Chromosomal inversions have been identified in many natural populations and can be responsible for novel traits and rapid adaptation. In zebra finch, a large region on the Z chromosome has been subject to multiple inversions, which have pleiotropic effects on multiple traits but especially on sperm phenotypes, such as midpiece and flagellum length. To understand the effect, the Z inversion has on these traits, we examined testis and liver transcriptomes of young males at different maturation times. We compared gene expression differences among three inversion karyotypes: AA, B*B* and AB*, where B* denotes the inverted regions on Z with respect to A. In testis, 794 differentially expressed genes were found and most of them were located on chromosome Z. They were functionally enriched for sperm-related traits. We also identified clusters of co-expressed genes that matched with the inversion-related sperm phenotypes. In liver, there were some enriched functions and some overrepresentation on chromosome Z with similar location as in testis. In both tissues, the overrepresented genes were located near the distal end of Z but also in the middle of the chromosome. For the heterokaryotype, we observed several genes with one allele being dominantly expressed, similar to expression patterns in one or the other homokaryotype. This was confirmed with SNPs for three genes, and interestingly one gene, DMGDH, had allele-specific expression originating mainly from one inversion haplotype in the testis, yet both inversion haplotypes were expressed equally in the liver. This karyotype-specific difference in tissue-specific expression suggests a pleiotropic effect of the inversion and thus suggests a mechanism for divergent phenotypic effects resulting from an inversion.

Rapid sperm length divergence in a polygynandrous passerine: a mechanism of cryptic speciation?

When populations become geographically isolated, they begin to diverge in various traits and at variable rates. The dynamics of such trait divergences are relevant for understanding evolutionary processes such as local adaptation and speciation. Here we examine divergences in sperm and body structures in a polygynandrous songbird, the alpine accentor (Prunella collaris) between two allopatric high-altitude populations, in Morocco and Spain. The populations diverged around 82,000 years ago, as estimated with a coalescence-based phylogenetic analysis of genome-wide single-nucleotide polymorphisms. We found that birds in the two areas had nonoverlapping sperm lengths, which suggests adaptation to divergent female reproductive tract environments. Sperm length also showed an exceptionally low coefficient of among-male variation, a signal of strong stabilizing selection imposed by sperm competition. The evolutionary rate of sperm length was almost twice the rates for the most divergent morphological traits and more than three times higher than expected from literature data over a similar generational timescale. This rapid evolution of a key reproductive trait has implications for reproductive isolation and ultimately for speciation. Strong selection for different sperm length optima in allopatry predicts conspecific sperm precedence and disruptive selection in sympatry, hence a possible postcopulatory prezygotic barrier to gene flow.

Responsiveness to long days for flowering is reduced in Arabidopsis by yearly variation in growing season temperatures.

Conservative flowering behaviours, such as flowering during long days in summer or late flowering at a high leaf number, are often proposed to protect against variable winter and spring temperatures which lead to frost damage if premature flowering occurs. Yet, due the many factors in natural environments relative to the number of individuals compared, assessing which climate characteristics drive these flowering traits has been difficult. We applied a multidisciplinary approach to 10 winter-annual Arabidopsis thaliana populations from a wide climactic gradient in Norway. We used a variable reduction strategy to assess which of 100 climate descriptors from their home sites correlated most to their flowering behaviours when tested for responsiveness to photoperiod after saturation of vernalization; then, assessed sequence variation of 19 known environmental-response flowering genes. Photoperiod responsiveness inversely correlated with interannual variation in timing of growing season onset. Time to flowering appeared driven by growing season length, curtailed by cold fall temperatures. The distribution of FLM, TFL2 and HOS1 haplotypes, genes involved in ambient temperature response, correlated with growing-season climate. We show that long-day responsiveness and late flowering may be driven not by risk of spring frosts, but by growing season temperature and length, perhaps to opportunistically maximize growth.

Ten years of marine evolutionary biology-Challenges and achievements of a multidisciplinary research initiative.

The Centre for Marine Evolutionary Biology (CeMEB) at the University of Gothenburg, Sweden, was established in 2008 through a 10-year research grant of 8.7 m€ to a team of senior researchers. Today, CeMEB members have contributed >500 scientific publications, 30 PhD theses and have organised 75 meetings and courses, including 18 three-day meetings and four conferences. What are the footprints of CeMEB, and how will the centre continue to play a national and international role as an important node of marine evolutionary research? In this perspective article, we first look back over the 10 years of CeMEB activities and briefly survey some of the many achievements of CeMEB. We furthermore compare the initial goals, as formulated in the grant application, with what has been achieved, and discuss challenges and milestones along the way. Finally, we bring forward some general lessons that can be learnt from a research funding of this type, and we also look ahead, discussing how CeMEB's achievements and lessons can be used as a springboard to the future of marine evolutionary biology.

Molecular, behavioural and morphological comparisons of sperm adaptations in a fish with alternative reproductive tactics.

In species with alternative reproductive tactics, there is much empirical support that parasitically spawning males have larger testes and greater sperm numbers as an evolved response to a higher degree of sperm competition, but support for higher sperm performance (motility, longevity and speed) by such males is inconsistent. We used the sand goby (Pomatoschistus minutus) to test whether sperm performance differed between breeding-coloured males (small testes, large mucus-filled sperm-duct glands; build nests lined with sperm-containing mucus, provide care) and parasitic sneaker-morph males (no breeding colouration, large testes, rudimentary sperm-duct glands; no nest, no care). We compared motility (per cent motile sperm), velocity, longevity of sperm, gene expression of testes and sperm morphometrics between the two morphs. We also tested if sperm-duct gland contents affected sperm performance. We found a clear difference in gene expression of testes between the male morphs with 109 transcripts differentially expressed between the morphs. Notably, several mucin genes were upregulated in breeding-coloured males and two ATP-related genes were upregulated in sneaker-morph males. There was a partial evidence of higher sperm velocity in sneaker-morph males, but no difference in sperm motility. Presence of sperm-duct gland contents significantly increased sperm velocity, and nonsignificantly tended to increase sperm motility, but equally so for the two morphs. The sand goby has remarkably long-lived sperm, with only small or no decline in motility and velocity over time (5 min vs. 22 h), but again, this was equally true for both morphs. Sperm length (head, flagella, total and flagella-to-head ratio) did not differ between morphs and did not correlate with sperm velocity for either morph. Thus, other than a clear difference in testes gene expression, we found only modest differences between the two male morphs, confirming previous findings that increased sperm performance as an adaptation to sperm competition is not a primary target of evolution.

Invader at the edge - Genomic origins and physiological differences of round gobies across a steep urban salinity gradient.

Species invasions are a global problem of increasing concern, especially in highly connected aquatic environments. Despite this, salinity conditions can pose physiological barriers to their spread, and understanding them is important for management. In Scandinavia's largest cargo port, the invasive round goby (Neogobius melanostomus) is established across a steep salinity gradient. We used 12,937 SNPs to identify the genetic origin and diversity of three sites along the salinity gradient and round goby from western, central and northern Baltic Sea, as well as north European rivers. Fish from two sites from the extreme ends of the gradient were also acclimated to freshwater and seawater, and tested for respiratory and osmoregulatory physiology. Fish from the high-salinity environment in the outer port showed higher genetic diversity, and closer relatedness to the other regions, compared to fish from lower salinity upstream the river. Fish from the high-salinity site also had higher maximum metabolic rate, fewer blood cells and lower blood Ca2+. Despite these genotypic and phenotypic differences, salinity acclimation affected fish from both sites in the same way: seawater increased the blood osmolality and Na+ levels, and freshwater increased the levels of the stress hormone cortisol. Our results show genotypic and phenotypic differences over short spatial scales across this steep salinity gradient. These patterns of the physiologically robust round goby are likely driven by multiple introductions into the high-salinity site, and a process of sorting, likely based on behaviour or selection, along the gradient. This euryhaline fish risks spreading from this area, and seascape genomics and phenotypic characterization can inform management strategies even within an area as small as a coastal harbour inlet.

Ancestral Sperm Ecotypes Reveal Multiple Invasions of a Non-Native Fish in Northern Europe.

For externally fertilising organisms in the aquatic environment, the abiotic fertilisation medium can be a strong selecting force. Among bony fishes, sperm are adapted to function in a narrow salinity range. A notable exception is the family Gobiidae, where several species reproduce across a wide salinity range. The family also contains several wide-spread invasive species. To better understand how these fishes tolerate such varying conditions, we measured sperm performance in relation to salinity from a freshwater and a brackish population within their ancestral Ponto-Caspian region of the round goby, Neogobius melanostomus. These two ancestral populations were then compared to nine additional invaded sites across northern Europe, both in terms of their sperm traits and by using genomic SNP markers. Our results show clear patterns of ancestral adaptations to freshwater and brackish salinities in their sperm performance. Population genomic analyses show that the ancestral ecotypes have generally established themselves in environments that fit their sperm adaptations. Sites close to ports with intense shipping show that both outbreeding and admixture can affect the sperm performance of a population in a given salinity. Rapid adaptation to local conditions is also supported at some sites. Historical and contemporary evolution in the traits of the round goby sperm cells is tightly linked to the population and seascape genomics as well as biogeographic processes in these invasive fishes. Since the risk of a population establishing in an area is related to the genotype by environment match, port connectivity and the ancestry of the round goby population can likely be useful for predicting the species spread.

Sperm adaptation in relation to salinity in three goby species.

In externally fertilizing species, the gametes of both males and females are exposed to the influences of the environment into which they are released. Sperm are sensitive to abiotic factors such as salinity, but they are also affected by biotic factors such as sperm competition. In this study, the authors compared the performance of sperm of three goby species, the painted goby, Pomatoschistus pictus, the two-spotted goby, Pomatoschistus flavescens, and the sand goby, Pomatoschistus minutus. These species differ in their distributions, with painted goby having the narrowest salinity range and sand goby the widest. Moreover, data from paternity show that the two-spotted goby experiences the least sperm competition, whereas in the sand goby sperm competition is ubiquitous. The authors took sperm samples from dissected males and exposed them to high salinity water (31 PSU) representing the North Sea and low salinity water (6 PSU) representing the brackish Baltic Sea Proper. They then used computer-assisted sperm analysis to measure the proportion of motile sperm and sperm swimming speed 10 min and 20 h after sperm activation. The authors found that sperm performance depended on salinity, but there seemed to be no relationship to the species' geographical distribution in relation to salinity range. The species differed in the proportion of motile sperm, but there was no significant decrease in sperm motility during 20 h. The sand goby was the only species with motile sperm after 72 h.

Cytosine methylation patterns suggest a role of methylation in plastic and adaptive responses to temperature in European grayling (Thymallus thymallus) populations.

Temperature is a key environmental parameter affecting both the phenotypes and distributions of organisms, particularly ectotherms. Rapid organismal responses to thermal environmental changes have been described for several ectotherms; however, the underlying molecular mechanisms often remain unclear. Here, we studied whole genome cytosine methylation patterns of European grayling (Thymallus thymallus) embryos from five populations with contemporary adaptations of early life history traits at either 'colder' or 'warmer' spawning grounds. We reared fish embryos in a common garden experiment using two temperatures that resembled the 'colder' and 'warmer' conditions of the natal natural environments. Genome-wide methylation patterns were similar in populations originating from colder thermal origin subpopulations, whereas single nucleotide polymorphisms uncovered from the same data identified strong population structure among isolated populations, but limited structure among interconnected populations. This was surprising because the previously studied gene expression response among populations was mostly plastic, and mainly influenced by the developmental temperature. These findings support the hypothesis of the magnified role of epigenetic mechanisms in modulating plasticity. The abundance of consistently changing methylation loci between two warmer-to-colder thermal origin population pairs suggests that local adaptation has shaped the observed methylation patterns. The dynamic nature of the methylomes was further highlighted by genome-wide and site-specific plastic responses. Our findings support both the presence of a plastic response in a subset of CpG loci, and the evolutionary role of methylation divergence between populations adapting to contrasting thermal environments.

Post-glacial establishment of locally adapted fish populations over a steep salinity gradient.

Studies of colonization of new habitats that appear from rapidly changing environments are interesting and highly relevant to our understanding of divergence and speciation. Here, we analyse phenotypic and genetic variation involved in the successful establishment of a marine fish (sand goby, Pomatoschistus minutus) over a steep salinity drop from 35 PSU in the North Sea (NE Atlantic) to two PSU in the inner parts of the post-glacial Baltic Sea. We first show that populations are adapted to local salinity in a key reproductive trait, the proportion of motile sperm. Thereafter, we show that genome variation at 22,190 single nucleotide polymorphisms (SNPs) shows strong differentiation among populations along the gradient. Sequences containing outlier SNPs and transcriptome sequences, mapped to a draft genome, reveal associations with genes with relevant functions for adaptation in this environment but without overall evidence of functional enrichment. The many contigs involved suggest polygenic differentiation. We trace the origin of this differentiation using demographic modelling and find the most likely scenario is that at least part of the genetic differentiation is older than the Baltic Sea and is a result of isolation of two lineages prior to the current contact over the North Sea-Baltic Sea transition zone.

Endless forms of sexual selection.

In recent years, the field of sexual selection has exploded, with advances in theoretical and empirical research complementing each other in exciting ways. This perspective piece is the product of a "stock-taking" workshop on sexual selection and sexual conflict. Our aim is to identify and deliberate on outstanding questions and to stimulate discussion rather than provide a comprehensive overview of the entire field. These questions are organized into four thematic sections we deem essential to the field. First we focus on the evolution of mate choice and mating systems. Variation in mate quality can generate both competition and choice in the opposite sex, with implications for the evolution of mating systems. Limitations on mate choice may dictate the importance of direct vs. indirect benefits in mating decisions and consequently, mating systems, especially with regard to polyandry. Second, we focus on how sender and receiver mechanisms shape signal design. Mediation of honest signal content likely depends on integration of temporally variable social and physiological costs that are challenging to measure. We view the neuroethology of sensory and cognitive receiver biases as the main key to signal form and the 'aesthetic sense' proposed by Darwin. Since a receiver bias is sufficient to both initiate and drive ornament or armament exaggeration, without a genetically correlated or even coevolving receiver, this may be the appropriate 'null model' of sexual selection. Thirdly, we focus on the genetic architecture of sexually selected traits. Despite advances in modern molecular techniques, the number and identity of genes underlying performance, display and secondary sexual traits remains largely unknown. In-depth investigations into the genetic basis of sexual dimorphism in the context of long-term field studies will reveal constraints and trajectories of sexually selected trait evolution. Finally, we focus on sexual selection and conflict as drivers of speciation. Population divergence and speciation are often influenced by an interplay between sexual and natural selection. The extent to which sexual selection promotes or counteracts population divergence may vary depending on the genetic architecture of traits as well as the covariance between mating competition and local adaptation. Additionally, post-copulatory processes, such as selection against heterospecific sperm, may influence the importance of sexual selection in speciation. We propose that efforts to resolve these four themes can catalyze conceptual progress in the field of sexual selection, and we offer potential avenues of research to advance this progress.

QTLs Associated with Resistance to Cardiomyopathy Syndrome in Atlantic Salmon.

Cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus is a major disease affecting the Norwegian Atlantic salmon industry. Three different populations of Atlantic salmon from the Mowi breeding program were used in this study. The first 2 populations (population 1 and 2) were naturally infected in a field outbreak, while the third population (population 3) went through a controlled challenged test. The aim of the study was to estimate the heritability, the genetic correlation between populations and perform genome-wide association analysis for resistance to this disease. Survival data from population 1 and 2 and heart atrium histology score data from population 3 was analyzed. A total of 571, 4312, and 901 fish from population 1, 2, and 3, respectively were genotyped with a noncommercial 55,735 Affymetrix marker panel. Genomic heritability ranged from 0.12 to 0.46 and the highest estimate was obtained from the challenge test dataset. The genetic correlation between populations was moderate (0.51-0.61). Two chromosomal regions (SSA27 and SSA12) contained single nucleotide polymorphisms associated with resistance to CMS. The highest association signal (P = 6.9751 × 10-27) was found on chromosome 27. Four genes with functional roles affecting viral resistance (magi1, pi4kb, bnip2, and ha1f) were found to map closely to the identified quantitative trait loci (QTLs). In conclusion, genetic variation for resistance to CMS was observed in all 3 populations. Two important quantitative trait loci were detected which together explain half of the total genetic variance, suggesting strong potential application for marker-assisted selection and genomic predictions to improve CMS resistance.

Modularity Facilitates Flexible Tuning of Plastic and Evolutionary Gene Expression Responses during Early Divergence.

Gene expression changes have been recognized as important drivers of adaptation to changing environmental conditions. Little is known about the relative roles of plastic and evolutionary responses in complex gene expression networks during the early stages of divergence. Large gene expression data sets coupled with in silico methods for identifying coexpressed modules now enable systems genetics approaches also in nonmodel species for better understanding of gene expression responses during early divergence. Here, we combined gene coexpression analyses with population genetics to separate plastic and population (evolutionary) effects in expression networks using small salmonid populations as a model system. We show that plastic and population effects were highly variable among the six identified modules and that the plastic effects explained larger proportion of the total eigengene expression than population effects. A more detailed analysis of the population effects using a QST - FST comparison across 16,622 annotated transcripts revealed that gene expression followed neutral expectations within modules and at the global level. Furthermore, two modules showed enrichment for genes coding for early developmental traits that have been previously identified as important phenotypic traits in thermal responses in the same model system indicating that coexpression analysis can capture expression patterns underlying ecologically important traits. We suggest that module-specific responses may facilitate the flexible tuning of expression levels to local thermal conditions. Overall, our study indicates that plasticity and neutral evolution are the main drivers of gene expression variance in the early stages of thermal adaptation in this system.

Cold temperature represses daily rhythms in the liver transcriptome of a stenothermal teleost under decreasing day length.

The climate-change-driven increase in temperature is occurring rapidly and decreasing the predictability of seasonal rhythms at high latitudes. It is therefore urgent to understand how a change in the relationship between photoperiod and temperature can affect ectotherms in these environments. We tested whether temperature affects daily rhythms of transcription in a cold-adapted salmonid using high-throughput RNA sequencing. Arctic char (Salvelinus alpinus) from a subarctic population were reared at a high and a low temperature (15 and 8°C) for 1 month under natural, decreasing day length during late summer. Liver transcriptomes were compared between samples collected in the middle and towards the end of the light period and in the middle of the dark period. Daily variation in transcription was lower in fish from the low temperature compared with strong daily variation in warm-acclimated fish, suggesting that cold temperatures dampen the cycling of transcriptional rhythms under a simultaneously decreasing day length. Different circadian clock genes had divergent expression patterns, responding either by decreased expression or by increased rhythmicity at 15°C compared with 8°C. The results point out mechanisms that can affect the ability of fish to adapt to increasing temperatures caused by climate change.

Immigrant reproductive dysfunction facilitates ecological speciation.

The distributions of species are not only determined by where they can survive - they must also be able to reproduce. Although immigrant inviability is a well-established concept, the fact that immigrants also need to be able to effectively reproduce in foreign environments has not been fully appreciated in the study of adaptive divergence and speciation. Fertilization and reproduction are sensitive life-history stages that could be detrimentally affected for immigrants in non-native habitats. We propose that "immigrant reproductive dysfunction" is a hitherto overlooked aspect of reproductive isolation caused by natural selection on immigrants. This idea is supported by results from experiments on an externally fertilizing fish (sand goby, Pomatoschistus minutus). Growth and condition of adults were not affected by non-native salinity whereas males spawning as immigrants had lower sperm motility and hatching success than residents. We interpret these results as evidence for local adaptation or acclimation of sperm, and possibly also components of paternal care. The resulting loss in fitness, which we call "immigrant reproductive dysfunction," has the potential to reduce gene flow between populations with locally adapted reproduction, and it may play a role in species distributions and speciation.

Rapid, broad-scale gene expression evolution in experimentally harvested fish populations.

Gene expression changes potentially play an important role in adaptive evolution under human-induced selection pressures, but this has been challenging to demonstrate in natural populations. Fishing exhibits strong selection pressure against large body size, thus potentially inducing evolutionary changes in life history and other traits that may be slowly reversible once fishing ceases. However, there is a lack of convincing examples regarding the speed and magnitude of fisheries-induced evolution, and thus, the relevant underlying molecular-level effects remain elusive. We use wild-origin zebrafish (Danio rerio) as a model for harvest-induced evolution. We experimentally demonstrate broad-scale gene expression changes induced by just five generations of size-selective harvesting, and limited genetic convergence following the cessation of harvesting. We also demonstrate significant allele frequency changes in genes that were differentially expressed after five generations of size-selective harvesting. We further show that nine generations of captive breeding induced substantial gene expression changes in control stocks likely due to inadvertent selection in the captive environment. The large extent and rapid pace of the gene expression changes caused by both harvest-induced selection and captive breeding emphasizes the need for evolutionary enlightened management towards sustainable fisheries.

Regulatory Architecture of Gene Expression Variation in the Threespine Stickleback Gasterosteus aculeatus.

Much adaptive evolutionary change is underlain by mutational variation in regions of the genome that regulate gene expression rather than in the coding regions of the genes themselves. An understanding of the role of gene expression variation in facilitating local adaptation will be aided by an understanding of underlying regulatory networks. Here, we characterize the genetic architecture of gene expression variation in the threespine stickleback (Gasterosteus aculeatus), an important model in the study of adaptive evolution. We collected transcriptomic and genomic data from 60 half-sib families using an expression microarray and genotyping-by-sequencing, and located expression quantitative trait loci (eQTL) underlying the variation in gene expression in liver tissue using an interval mapping approach. We identified eQTL for several thousand expression traits. Expression was influenced by polymorphism in both cis- and trans-regulatory regions. Trans-eQTL clustered into hotspots. We did not identify master transcriptional regulators in hotspot locations: rather, the presence of hotspots may be driven by complex interactions between multiple transcription factors. One observed hotspot colocated with a QTL recently found to underlie salinity tolerance in the threespine stickleback. However, most other observed hotspots did not colocate with regions of the genome known to be involved in adaptive divergence between marine and freshwater habitats.

Plastic and Evolutionary Gene Expression Responses Are Correlated in European Grayling (Thymallus thymallus) Subpopulations Adapted to Different Thermal Environments.

Understanding how populations adapt to changing environmental conditions is a long-standing theme in evolutionary biology. Gene expression changes have been recognized as an important driver of local adaptation, but relatively little is known regarding the direction of change and in particular, about the interplay between plastic and evolutionary gene expression. We have previously shown that the gene expression profiles of European grayling (Thymallus thymallus) populations inhabiting different thermal environments include both plastic and evolutionary components. However, whether the plastic and evolutionary responses were in the same direction was not investigated in detail, nor was the identity of the specific genes involved. In this study, we show that the plastic changes in protein expression in response to different temperatures are highly correlated with the evolutionary response in grayling subpopulations adapted to different thermal environments. This finding provides preliminary evidence that the plastic response most likely facilitates adaptation during the early phases of colonization of thermal environments. The proteins that showed significant changes in expression level between warm and cold temperature treatments were mostly related to muscle development, which is consistent with earlier findings demonstrating muscle mass differentiation between cold and warm grayling populations.