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.

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.

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.

The evolution and adaptive potential of transcriptional variation in sticklebacks--signatures of selection and widespread heritability.

Evidence implicating differential gene expression as a significant driver of evolutionary novelty continues to accumulate, but our understanding of the underlying sources of variation in expression, both environmental and genetic, is wanting. Heritability in particular may be underestimated when inferred from genetic mapping studies, the predominant "genetical genomics" approach to the study of expression variation. Such uncertainty represents a fundamental limitation to testing for adaptive evolution at the transcriptomic level. By studying the inheritance of expression levels in 10,495 genes (10,527 splice variants) in a threespine stickleback pedigree consisting of 563 individuals, half of which were subjected to a thermal treatment, we show that 74-98% of transcripts exhibit significant additive genetic variance. Dominance variance is also prevalent (41-99% of transcripts), and genetic sources of variation seem to play a more significant role in expression variance in the liver than a key environmental variable, temperature. Among-population comparisons suggest that the majority of differential expression in the liver is likely due to neutral divergence; however, we also show that signatures of directional selection may be more prevalent than those of stabilizing selection. This predominantly aligns with the neutral model of evolution for gene expression but also suggests that natural selection may still act on transcriptional variation in the wild. As genetic variation both within- and among-populations ultimately defines adaptive potential, these results indicate that broad adaptive potential may be found within the transcriptome.

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.

Acclimation of killifish to thermal extremes of hot spring: Transcription of gonadal and liver heat shock genes.

In this study, we explored the hypothesis that killifish acclimate to thermal extremes through regulation of genes involved in stress and metabolism. We examined the liver and gonadal transcription of heat shock proteins (hsp70, hsp90a, hsp90b), glucokinase (gck), and high mobility group b1 (hmgb1) protein in wild killifish species from hot springs and rivers using quantitative real-time PCR. Moreover, we exposed a river killifish species to a long-term thermal regime of hot spring (37-40°C) and examined the liver transcription of the heat shock genes. Our results showed that hot spring killifish showed a significant, strong upregulation of liver hsp90a. Moreover, the testicular transcript levels of hsp90a, hsp90b, and hsp70 were higher in hot spring killifish than the river ones. The results of the common garden experiments showed that the transcripts of hsp70, hsp90b, and hmgb1 were mildly induced (> twofold) at the time when temperature reached to 37-40°C, while the transcripts of hsp90a were strongly induced (17-fold increase). The level of hsp90a was dramatically more upregulated when fish were maintained in thermal extreme (42-fold change higher than in ambient temperature). Moreover, a significant downregulation of gck transcripts was observed at the time when temperature was raised to 37-40°C (80-fold decrease) and during exposure to long-term thermal extreme (56-fold decrease). It can be concluded that the regulation of heat shock genes particularly hsp90a might be a key factor of the acclimation of fish to high temperature environments like hot springs.

Microarray analysis of di-n-butyl phthalate and 17α ethinyl-oestradiol responses in three-spined stickleback testes reveals novel candidate genes for endocrine disruption.

Phthalate esters are plasticizers frequently found in wastewater effluents. Previous studies on phthalates have reported anti-androgenic activity in mammals, causing concerns of their potential effects on the reproduction of aquatic organisms. Another group of environmental endocrine disrupters, steroidal estrogens, are known to inhibit steroid biosynthesis in the gonads, but the effects related to spermatogenesis are not well understood in fish. In this study, three-spined sticklebacks were exposed to di-n-butyl phthalate (DBP) and 17α ethinyl-oestradiol (EE2) at nominal concentrations 35µg/L and 40ng/L, respectively, for four days. The aim of the study was to obtain insight into the acute transcriptional responses putatively associated with endocrine disruption. RNA samples from eight individual male fish per treatment (including controls) were used in microarray analysis, covering the expression of approximately 21,000 genes. In the EE2 treatment the results show transcriptional downregulation of genes associated with steroid biosynthesis pathway and up-regulation of genes involved in pathways related to epidermal growth factor signaling and xenobiotic metabolism. The transcriptional response to DBP was in general weaker than to EE2, but based on enrichment analysis, we suggest adverse effects on retinoid metabolism, creatine kinase activity and cell adhesion. Among the genes showing highest fold changes after DBP treatment compared to control was the teleost fish -specific cytochrome P450 17A2. Overall, this study promotes our understanding on molecular responses to anti-androgens and estrogens in fish testes.

The evolutionary history of Afrocanarian blue tits inferred from genomewide SNPs.

A common challenge in phylogenetic reconstruction is to find enough suitable genomic markers to reliably trace splitting events with short internodes. Here, we present phylogenetic analyses based on genomewide single-nucleotide polymorphisms (SNPs) of an enigmatic avian radiation, the subspecies complex of Afrocanarian blue tits (Cyanistes teneriffae). The two sister species, the Eurasian blue tit (Cyanistes caeruleus) and the azure tit (Cyanistes cyanus), constituted the out-group. We generated a large data set of SNPs for analysis of population structure and phylogeny. We also adapted our protocol to utilize degraded DNA from old museum skins from Libya. We found strong population structuring that largely confirmed subspecies monophyly and constructed a coalescent-based phylogeny with full support at all major nodes. The results are consistent with a recent hypothesis that La Palma and Libya are relic populations of an ancient Afrocanarian blue tit, although a small data set for Libya could not resolve its position relative to La Palma. The birds on the eastern islands of Fuerteventura and Lanzarote are similar to those in Morocco. Together they constitute the sister group to the clade containing the other Canary Islands (except La Palma), in which El Hierro is sister to the three central islands. Hence, extant Canary Islands populations seem to originate from multiple independent colonization events. We also found population divergences in a key reproductive trait, viz. sperm length, which may constitute reproductive barriers between certain populations. We recommend a taxonomic revision of this polytypic species, where several subspecies should qualify for species rank.

Gene expression plasticity evolves in response to colonization of freshwater lakes in threespine stickleback.

Phenotypic plasticity is predicted to facilitate individual survival and/or evolve in response to novel environments. Plasticity that facilitates survival should both permit colonization and act as a buffer against further evolution, with contemporary and derived forms predicted to be similarly plastic for a suite of traits. On the other hand, given the importance of plasticity in maintaining internal homeostasis, derived populations that encounter greater environmental heterogeneity should evolve greater plasticity. We tested the evolutionary significance of phenotypic plasticity in coastal British Columbian postglacial populations of threespine stickleback (Gasterosteus aculeatus) that evolved under greater seasonal extremes in temperature after invading freshwater lakes from the sea. Two ancestral (contemporary marine) and two derived (contemporary freshwater) populations of stickleback were raised near their thermal tolerance extremes, 7 and 22 °C. Gene expression plasticity was estimated for more than 14,000 genes. Over five thousand genes were similarly plastic in marine and freshwater stickleback, but freshwater populations exhibited significantly more genes with plastic expression than marine populations. Furthermore, several of the loci shown to exhibit gene expression plasticity have been previously implicated in the adaptive evolution of freshwater populations, including a gene involved in mitochondrial regulation (PPARAa). Collectively, these data provide molecular evidence that highlights the importance of plasticity in colonization and adaptation to new environments.

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.

Genomic divergence between nine- and three-spined sticklebacks.

BACKGROUND: Comparative genomics approaches help to shed light on evolutionary processes that shape differentiation between lineages. The nine-spined stickleback (Pungitius pungitius) is a closely related species of the ecological 'supermodel' three-spined stickleback (Gasterosteus aculeatus). It is an emerging model system for evolutionary biology research but has garnered less attention and lacks extensive genomic resources. To expand on these resources and aid the study of sticklebacks in a phylogenetic framework, we characterized nine-spined stickleback transcriptomes from brain and liver using deep sequencing. RESULTS: We obtained nearly eight thousand assembled transcripts, of which 3,091 were assigned as putative one-to-one orthologs to genes found in the three-spined stickleback. These sequences were used for evaluating overall differentiation and substitution rates between nine- and three-spined sticklebacks, and to identify genes that are putatively evolving under positive selection. The synonymous substitution rate was estimated to be 7.1 × 10(-9) per site per year between the two species, and a total of 165 genes showed patterns of adaptive evolution in one or both species. A few nine-spined stickleback contigs lacked an obvious ortholog in three-spined sticklebacks but were found to match genes in other fish species, suggesting several gene losses within 13 million years since the divergence of the two stickleback species. We identified 47 SNPs in 25 different genes that differentiate pond and marine ecotypes. We also identified 468 microsatellites that could be further developed as genetic markers in nine-spined sticklebacks. CONCLUSION: With deep sequencing of nine-spined stickleback cDNA libraries, our study provides a significant increase in the number of gene sequences and microsatellite markers for this species, and identifies a number of genes showing patterns of adaptive evolution between nine- and three-spined sticklebacks. We also report several candidate genes that might be involved in differential adaptation between marine and freshwater nine-spined sticklebacks. This study provides a valuable resource for future studies aiming to identify candidate genes underlying ecological adaptation in this and other stickleback species.

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.

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.

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.

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.

Transcriptional responses to hypoxia are enhanced by recurrent hypoxia (hypoxic preconditioning) in the epaulette shark.

All animals require molecular oxygen for aerobic energy production, and oxygen availability has played a particularly important role in the evolution of aquatic animals. This study investigates how previous exposure to hypoxia (preconditioning) primes protective transcriptional responses in a hypoxia-tolerant vertebrate species, the epaulette shark (Hemiscyllium ocellatum). The epaulette shark is a basal cartilaginous fish that in its natural environment experiences cyclic hypoxic periods. We evaluated whether the transcription of a set of crucial prosurvival genes is affected differently by a single short-term (2 h) exposure to sublethal hypoxia compared with eight such successive hypoxia exposures (hypoxia preconditioning). We discovered that hypoxia preconditioning amplifies transcriptional responses compared with animals that experienced a single hypoxic bout. In the heart we observed that hypoxic preconditioning, but not a single hypoxic exposure, resulted in higher transcript levels of genes that regulate oxygen and energy homeostasis, including those of hypoxia-inducible factor-1 alpha, adenosine signaling pathway components, and genes affecting circulation [prostaglandin synthetase 2 (cox-2) and natriuretic peptide C]. This suggests that in a single short-term hypoxic bout, the responses to low oxygen are regulated at the level of pre-existing proteins or translational and posttranslational machinery, whereas transcriptional responses are induced in experiments that parallel the natural environmental cycles of oxygen availability. These findings have general implications for understanding how vertebrates regulate protective gene expression upon physiological stress.

The proteomics of feather development in pied flycatchers (Ficedula hypoleuca) with different plumage coloration.

The genetic theory of morphological evolution postulates that form evolves largely by changing the expression proteins that are functionally conserved. It follows that understanding the function of proteins during different phases of development as well as the mechanisms by which the functions are modified is a prerequisite for understanding evolutionary change. Male pied flycatchers exhibit marked phenotypic variation in their breeding plumage. This variation has repeatedly been shown to have adaptive significance, but the molecular basis of this variation is not known. Here, we characterize the proteome of developing pied flycatcher feathers from differently pigmented males and also introduce a new method for examining the effect sizes of expression differences in protein interaction networks. Approximately 300 proteins were identified in the developing feathers of males. Gene products associated with cellular transport, cell metabolism and protein synthesis formed a large part of the developing feather proteome. Sixty-five proteins associated with the development of the epidermis and/or pigmentation were detected in the data. The examination of expression level differences of protein-protein interaction networks revealed an immunological signalling-related network to exhibit significantly higher expression in black compared to brown males. Additionally, indications of differences in energy balance and oxidative stress related characteristics were detected. Together, these results provide new insight into the molecular mechanisms and evolutionary significance of plumage colour variation.

Female-biased expression on the X chromosome as a key step in sex chromosome evolution in threespine sticklebacks.

Given that the genome of males and females are almost identical with the exception of genes on the Y (or W) chromosome or sex-determining alleles (in organisms without sex chromosomes), it is likely that many downstream processes resulting in sexual dimorphism are produced by changes in regulation. In early stages of sex chromosome evolution, as the Y-chromosome degenerates, gene expression should be significantly impacted for genes residing on the sex chromosome pair as regulatory mutations accumulate. However, this has rarely been examined because most model organisms have clearly diverged sex chromosomes. Fish provide a unique opportunity to examine the evolution of sex chromosomes because genetic sex determination has evolved quite recently in some groups of fish. We compared sex-specific transcription in threespine stickleback (Gasterosteus aculeatus) liver tissue using a long-oligo microarray. Of the 1,268 genes that were differentially expressed between sexes, a highly significant proportion (23%) was concentrated on chromosome 19, corresponding to the recently described nascent sex chromosomes. The sex-biased genes are enriched for different functional categories in males and females, although there is no specific functional enrichment on the sex chromosomes. Female-biased genes are concentrated at one end of the sex chromosome, corresponding to a deletion in the Y, suggesting a lack of global dosage compensation. Prior research on threespine sticklebacks has demonstrated various degrees of dissimilarity in upstream regions of genes on the Y providing a potential mechanism for the observed patterns of female-biased expression. We hypothesize that degeneration of the Y chromosome results in regulatory mutations that create a sex-specific expression pattern and that this physical concentration of sex-biased expression on the nascent sex chromosome may be a key feature characterizing intermediate phases of sex chromosome evolution.

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.

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.