Strontium isotope analysis of otoliths reveals differences in the habitat salinity among three sympatric stickleback species of the genus Pungitius.

The analysis of otolith Sr isotope ratios (87Sr/86Sr) is a powerful method to study fish migration in freshwater areas. However, few studies have applied this method to study fish movement in brackish-water environments. Furthermore, despite the fact that habitat differentiation has been shown to drive genetic differentiation and reproductive isolation among stickleback fish, no studies have used the otolith 87Sr/86Sr ratios to analyze habitat differentiation between stickleback ecotypes and species. In this study, we analyzed the otolith 87Sr/86Sr ratios of three sympatric stickleback species of the genus Pungitius in the Shiomi River on Hokkaido Island, Japan: P. tymensis, the brackish-water type of the P. pungitius-P. sinensis complex, and the freshwater type of the P. pungitius-P. sinensis complex. First, we created a mixing equation to depict the relationship between habitat salinity and the 87Sr/86Sr ratios of river water. We found that the otolith 87Sr/86Sr ratios differed significantly among the three species, indicating that the three species utilize habitats with different salinities: P. tymensis and the brackish-water type inhabit freshwater and brackish-water environments, respectively, with the freshwater type using intermediate habitats. In addition, we found that some freshwater individuals moved to habitats with higher salinities as they grew. Our study demonstrates that the analysis of otolith 87Sr/86Sr ratios is a useful method for studying the habitat use of fish in brackish-water environments and habitat differentiation among closely related sympatric and parapatric species.

Genetic basis of speciation and adaptation: from loci to causative mutations.

Does evolution proceed in small steps or large leaps? How repeatable is evolution? How constrained is the evolutionary process? Answering these long-standing questions in evolutionary biology is indispensable for both understanding how extant biodiversity has evolved and predicting how organisms and ecosystems will respond to changing environments in the future. Understanding the genetic basis of phenotypic diversification and speciation in natural populations is key to properly answering these questions. The leap forward in genome sequencing technologies has made it increasingly easier to not only investigate the genetic architecture but also identify the variant sites underlying adaptation and speciation in natural populations. Furthermore, recent advances in genome editing technologies are making it possible to investigate the functions of each candidate gene in organisms from natural populations. In this article, we discuss how these recent technological advances enable the analysis of causative genes and mutations and how such analysis can help answer long-standing evolutionary biology questions. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

Convergent copy number increase of genes associated with freshwater colonization in fishes.

Copy number variation (CNV) can cause phenotypic changes. However, in contrast to amino acid substitutions and cis-regulatory changes, little is known about the functional categories of genes in which CNV is important for adaptation to novel environments. It is also unclear whether the same genes repeatedly change the copy numbers for adapting to similar environments. Here, we investigate CNV associated with freshwater colonization in fishes, which was observed multiple times across different lineages. Using 48 ray-finned fishes across diverse orders, we identified 23 genes whose copy number increases were associated with freshwater colonization. These genes showed enrichment for peptide receptor activity, hexosyltransferase activity and unsaturated fatty acid metabolism. We further revealed that three of the genes showed copy number increases in freshwater populations compared to marine ancestral populations of the stickleback genus Gasterosteus. These results indicate that copy number increases of genes involved in fatty acid metabolism (FADS2), immune function (PSMB8a) and thyroid hormone metabolism (UGT2) may be important for freshwater colonization at both the inter-order macroevolutionary scale and at the intra-genus microevolutionary scale. Further analysis across diverse taxa will help to understand the role of CNV in the adaptation to novel environments. This article is part of the theme issue 'Genetic basis of adaptation and speciation: from loci to causative mutations'.

Construction of a chromosome-level Japanese stickleback species genome using ultra-dense linkage analysis with single-cell sperm sequencing.

It is still difficult to construct the genomes of higher organisms as their genome sequences must be extended to the length of the chromosome by linkage analysis. In this study, we attempted to provide an innovative alternative to conventional linkage analysis by devising a method to genotype sperm using 10× Genomics single-cell genome sequencing libraries to generate a linkage map without interbreeding individuals. A genome was assembled using sperm from the Japanese stickleback Gasterosteus nipponicus, with single-cell genotyping yielding 1 864 430 very dense hetero-SNPs and an average coverage per sperm cell of 0.13×. In total, 1665 sperm were used, which is an order of magnitude higher than the number of recombinations used for conventional linkage analysis. We then improved the linkage analysis tool scaffold extender with low depth linkage analysis (SELDLA) to analyze the data according to the characteristics of the single-cell genotyping data. Finally, we were able to determine the chromosomal location (97.1%) and orientation (64.4%) of the contigs in the 456 Mb genome of G. nipponicus, sequenced using nanopores. This method promises to be a useful tool for determining the genomes of non-model organisms for which breeding systems have not yet been established by linkage analysis.

Copy number variation of a fatty acid desaturase gene Fads2 associated with ecological divergence in freshwater stickleback populations.

Fitness of aquatic animals can be limited by the scarcity of nutrients such as long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). DHA availability from diet varies among aquatic habitats, imposing different selective pressures on resident animals to optimize DHA acquisition and synthesis. For example, DHA is generally poor in freshwater ecosystems compared to marine ecosystems. Our previous work revealed that, relative to marine fishes, several freshwater fishes evolved higher copy numbers of the fatty acid desaturase2 (Fads2) gene, which encodes essential enzymes for DHA biosynthesis, likely compensating for the limited availability of DHA in freshwater. Here, we demonstrate that Fads2 copy number also varies between freshwater sticklebacks inhabiting lakes and streams with stream fish having higher Fads2 copy number. Additionally, populations with benthic-like morphology possessed higher Fads2 copy number than those with planktivore-like morphology. This may be because benthic-like fish mainly feed on DHA-deficient prey such as macroinvertebrates whereas planktivore-like fish forage more regularly on DHA-rich prey, like copepods. Our results suggest that Fads2 copy number variation arises from ecological divergence not only between organisms exploiting marine and freshwater habitats but also between freshwater organisms exploiting divergent resources.

The evolutionary ecology of fatty-acid variation: Implications for consumer adaptation and diversification.

The nutritional diversity of resources can affect the adaptive evolution of consumer metabolism and consumer diversification. The omega-3 long-chain polyunsaturated fatty acids eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) have a high potential to affect consumer fitness, through their widespread effects on reproduction, growth and survival. However, few studies consider the evolution of fatty acid metabolism within an ecological context. In this review, we first document the extensive diversity in both primary producer and consumer fatty acid distributions amongst major ecosystems, between habitats and amongst species within habitats. We highlight some of the key nutritional contrasts that can shape behavioural and/or metabolic adaptation in consumers, discussing how consumers can evolve in response to the spatial, seasonal and community-level variation of resource quality. We propose a hierarchical trait-based approach for studying the evolution of consumers' metabolic networks and review the evolutionary genetic mechanisms underpinning consumer adaptation to EPA and DHA distributions. In doing so, we consider how the metabolic traits of consumers are hierarchically structured, from cell membrane function to maternal investment, and have strongly environment-dependent expression. Finally, we conclude with an outlook on how studying the metabolic adaptation of consumers within the context of nutritional landscapes can open up new opportunities for understanding evolutionary diversification.

Patterns of genomic divergence and introgression between Japanese stickleback species with overlapping breeding habitats.

With only a few absolute geographic barriers in marine environments, the factors maintaining reproductive isolation among marine organisms remain elusive. However, spatial structuring in breeding habitat can contribute to reproductive isolation. This is particularly important for marine organisms that migrate to use fresh- or brackish water environments to breed. The Japanese Gasterosteus stickleback species, the Pacific Ocean three-spined stickleback (G. aculeatus) and the Japan Sea stickleback (G. nipponicus) overwinter in the sea, but migrate to rivers for spawning. Although they co-occur at several locations across the Japanese islands, they are reproductively isolated. Our previous studies in Bekanbeushi River showed that the Japan Sea stickleback spawns in the estuary, while the Pacific Ocean stickleback mainly spawns further upstream in freshwater. Overall genomic divergence was very high with many interspersed regions of introgression. Here, we investigated genomic divergence and introgression between the sympatric species in the much shorter Tokotan River, where they share spawning sites. The levels of genome-wide divergence were reduced and introgression was increased, suggesting that habitat isolation substantially contributes to a reduction in gene flow. We also found that genomic regions of introgression were largely shared between the two systems. Furthermore, some regions of introgression were located near loci with a heterozygote advantage for juvenile survival. Taken together, introgression may be partially driven by adaptation in this system. Although, the two species remain clearly genetically differentiated. Regions with low recombination rates showed especially low introgression. Speciation reversal is therefore likely prevented by barriers other than habitat isolation.

Multiple waves of freshwater colonization of the three-spined stickleback in the Japanese Archipelago.

BACKGROUND: The three-spined stickleback (Gasterosteus aculeatus) is a remarkable system to study the genetic mechanisms underlying parallel evolution during the transition from marine to freshwater habitats. Although the majority of previous studies on the parallel evolution of sticklebacks have mainly focused on postglacial freshwater populations in the Pacific Northwest of North America and northern Europe, we recently use Japanese stickleback populations for investigating shared and unique features of adaptation and speciation between geographically distant populations. However, we currently lack a comprehensive phylogeny of the Japanese three-spined sticklebacks, despite the fact that a good phylogeny is essential for any evolutionary and ecological studies. Here, we conducted a phylogenomic analysis of the three-spined stickleback in the Japanese Archipelago. RESULTS: We found that freshwater colonization occurred in multiple waves, each of which may reflect different interglacial isolations. Some of the oldest freshwater populations from the central regions of the mainland of Japan (hariyo populations) were estimated to colonize freshwater approximately 170,000 years ago. The next wave of colonization likely occurred approximately 100,000 years ago. The inferred origins of several human-introduced populations showed that introduction occurred mainly from nearby habitats. We also found a new habitat of the three-spined stickleback sympatric with the Japan Sea stickleback (Gasterosteus nipponicus). CONCLUSIONS: These Japanese stickleback systems differ from those in the Pacific Northwest of North America and northern Europe in terms of divergence time and history. Stickleback populations in the Japanese Archipelago offer valuable opportunities to study diverse evolutionary processes in historical and contemporary timescales.

Differences in the contributions of sex linkage and androgen regulation to sex-biased gene expression in juvenile and adult sticklebacks.

Different evolutionary interests between males and females can lead to the evolution of sexual dimorphism. However, intersex genetic correlations due to the shared genome can constrain the evolution of sexual dimorphism, resulting in intra-locus sexual conflict. One of the mechanisms resolving this conflict is sex linkage, which allows males and females to carry different alleles on sex chromosomes. Another is a regulatory mutation causing sex-biased gene expression, which is often mediated by gonadal steroids in vertebrates. How do these two mechanisms differ in the contributions to the resolution of intra-locus sexual conflict? The magnitude of sexual conflict often varies between the juvenile and adult stages. Because gonadal steroids change in titre during development, we hypothesized that gonadal steroids play a role in sexual dimorphism expression only at certain developmental stages, whereas sex linkage is more important for sexual dimorphism expressed throughout life. Our brain transcriptome analysis of juvenile and adult threespine sticklebacks showed that the majority of genes that were sex-biased in both stages were sex-linked. The relative contribution of androgen-dependent regulation to the sex-biased transcriptome increased and that of sex linkage declined in adults compared to juveniles. The magnitude of the sex differences was greater in sex-linked genes than androgen-responsive genes, suggesting that sex linkage is more effective than androgen regulation in the production of large sex differences in gene expression. Overall, our data are consistent with the hypothesis that sex linkage is effective in resolving sexual conflict throughout life, whereas androgen-dependent regulation can contribute to temporary resolution of sexual conflict.

Diversity in reproductive seasonality in the three-spined stickleback, Gasterosteus aculeatus.

The annual timing of reproduction is a key life history trait with a large effect on fitness. Populations often vary in the timing and duration of reproduction to adapt to different seasonality of ecological and environmental variables between habitats. However, little is known about the molecular genetic mechanisms underlying interpopulation variation in reproductive seasonality. Here, we demonstrate that the three-spined stickleback (Gasterosteus aculeatus) is a good model for molecular genetic analysis of variations in reproductive seasonality. We first compiled data on reproductive seasons of diverse ecotypes, covering marine-anadromous, lake and stream ecotypes, of three-spined stickleback inhabiting a wide range of latitudes. Our analysis showed that both ecotype and latitude significantly contribute to variation in reproductive seasons. Stream ecotypes tend to start breeding earlier and end later than other ecotypes. Populations from lower latitudes tend to start breeding earlier than those from higher latitudes in all three ecotypes. Additionally, stream ecotypes tend to have extended breeding seasons at lower latitudes than at higher latitudes, leading to nearly year-round reproduction in the most southern stream populations. A review of recent progress in our understanding of the physiological mechanisms underlying seasonal reproduction in the three-spined stickleback indicates that photoperiod is an important external cue that stimulates and/or suppresses reproduction in this species. Taking advantage of genomic tools available for this species, the three-spined stickleback will be a good model to investigate what kinds of genes and mutations underlie variations in the physiological signalling pathways that regulate reproduction in response to photoperiod.

A key metabolic gene for recurrent freshwater colonization and radiation in fishes.

Colonization of new ecological niches has triggered large adaptive radiations. Although some lineages have made use of such opportunities, not all do so. The factors causing this variation among lineages are largely unknown. Here, we show that deficiency in docosahexaenoic acid (DHA), an essential ω-3 fatty acid, can constrain freshwater colonization by marine fishes. Our genomic analyses revealed multiple independent duplications of the fatty acid desaturase gene Fads2 in stickleback lineages that subsequently colonized and radiated in freshwater habitats, but not in close relatives that failed to colonize. Transgenic manipulation of Fads2 in marine stickleback increased their ability to synthesize DHA and survive on DHA-deficient diets. Multiple freshwater ray-finned fishes also show a convergent increase in Fads2 copies, indicating its key role in freshwater colonization.

Functional divergence of a heterochromatin-binding protein during stickleback speciation.

Intragenomic conflict, the conflict of interest between different genomic regions within an individual, is proposed as a mechanism driving both the rapid evolution of heterochromatin-related proteins and the establishment of intrinsic genomic incompatibility between species. Although molecular studies of laboratory model organisms have demonstrated the link between heterochromatin evolution and hybrid abnormalities, we know little about their link in natural systems. Previously, we showed that F1 hybrids between the Japan Sea stickleback and the Pacific Ocean stickleback show hybrid male sterility and found a region responsible for hybrid male sterility on the X chromosome, but did not identify any candidate genes. In this study, we first screened for genes rapidly evolving under positive selection during the speciation of Japanese sticklebacks to find genes possibly involved in intragenomic conflict. We found that the region responsible for hybrid male sterility contains a rapidly evolving gene encoding a heterochromatin-binding protein TRIM24B. We conducted biochemical experiments and showed that the binding affinity of TRIM24B to a heterochromatin mark found at centromeres and transposons, histone H4 lysine 20 trimethylation (H4K20me3), is reduced in the Japan Sea stickleback. In addition, mRNA expression levels of Trim24b were different between the Japan Sea and the Pacific Ocean testes. Further expression analysis of genes possibly in the TRIM24B-regulated pathway showed that some gypsy retrotransposons are overexpressed in the F1 hybrid testes. We, therefore, demonstrate that a heterochromatin-binding protein can evolve rapidly under positive selection and functionally diverge during stickleback speciation.

Parallel transcriptome evolution in stream threespine sticklebacks.

Natural selection can cause similar phenotypic evolution in phylogenetically independent lineages inhabiting similar environments. Compared to morphological, behavioral, and physiological traits, little is known about the parallel evolution of transcriptome. Furthermore, the relative contribution of cis- and trans-regulatory changes to parallel transcriptome evolution largely remains unclear. The threespine stickleback fish (Gasterosteus aculeatus) is a great model for studying parallel evolution because its ancestral marine populations independently colonized freshwater habitats in multiple geographical regions, resulting in independent pairs of marine and freshwater ecotypes in each region. Here, we investigated transcriptomic parallelism among the marine and stream ecotypes of Japanese and Canadian threespine sticklebacks by conducting common garden experiments and microarray analysis of the brain, which controls several physiological and behavioral traits differing between these ecotypes. We found parallel expression differences in 103 genes, including those encoding the enzymes involved in taurine synthesis and glycoprotein hydrolysis. The number of genes differentially expressed in parallel was significantly larger than the number of genes showing an antiparallel pattern (71 genes). To investigate the genetic architecture underlying transcriptome divergence, we re-analyzed the previous expression quantitative trait locus (eQTL) data and found that most eQTLs were located on the same chromosome as the transcripts, possibly in cis-regulatory regions. Furthermore, the effect sizes of the eQTLs on the same chromosomes were larger than those on different chromosomes. Thus, we found that divergence in the brain transcriptome between the ecotypes shows parallelism and is mainly caused by genetic changes occurring on the same chromosome as the target genes.

Lateralized expression of left-right axis formation genes is shared by adult brains of lefty and righty scale-eating cichlids.

Variation in the laterality often exists within species and can be maintained by frequency-dependent selection. Although the molecular developmental mechanisms underlying the left-right axis formation have been investigated, the genomic mechanisms underlying variation in laterality remain largely unknown. The scale-eating cichlid Perissodus microlepis in Lake Tanganyika exhibit lateralized predation; lefty individuals with the mouth opening toward the right preferentially attack on the prey's left trunk, while righty individuals with the opposite opening attacks on the right trunk. Here, we performed RNA-sequencing and subsequent confirmation with quantitative-PCR in the telencephalon, optic tectum, and hindbrain of the cichlid and identified five genes (pkd1b, ntn1b, ansn, pde6g, and rbp4l1) that were differentially expressed between the hemispheres regardless of the laterality. Surprisingly, pkd1b and ntn1b are involved in nodal and netrin signalling, respectively, which are important for left-right asymmetry formation during early embryogenesis. This result indicates that nodal- and netrin-related signals may also play important roles in the maintenance of asymmetry in adult brain. By contrast, no genes showed reversal of lateral differences between lefty and righty individuals in any brain regions examined, suggesting that laterality in the scale-eating cichlid does not simply result from inversion of the left-right asymmetry of gene expression.

Sex Differences in Recombination in Sticklebacks.

Recombination often differs markedly between males and females. Here we present the first analysis of sex-specific recombination in Gasterosteus sticklebacks. Using whole-genome sequencing of 15 crosses between G. aculeatus and G. nipponicus, we localized 698 crossovers with a median resolution of 2.3 kb. We also used a bioinformatic approach to infer historical sex-averaged recombination patterns for both species. Recombination is greater in females than males on all chromosomes, and overall map length is 1.64 times longer in females. The locations of crossovers differ strikingly between sexes. Crossovers cluster toward chromosome ends in males, but are distributed more evenly across chromosomes in females. Suppression of recombination near the centromeres in males causes crossovers to cluster at the ends of long arms in acrocentric chromosomes, and greatly reduces crossing over on short arms. The effect of centromeres on recombination is much weaker in females. Genomic differentiation between G. aculeatus and G. nipponicus is strongly correlated with recombination rate, and patterns of differentiation along chromosomes are strongly influenced by male-specific telomere and centromere effects. We found no evidence for fine-scale correlations between recombination and local gene content in either sex. We discuss hypotheses for the origin of sexual dimorphism in recombination and its consequences for sexually antagonistic selection and sex chromosome evolution.

Phylogenomics reveals habitat-associated body shape divergence in Oryzias woworae species group (Teleostei: Adrianichthyidae).

The Oryzias woworae species group, composed of O. asinua, O. wolasi, and O. woworae, is widely distributed in southeastern Sulawesi, an island in the Indo-Australian Archipelago. Deep-elongated body shape divergence is evident among these three species to the extent that it is used as a species-diagnostic character. These fishes inhabit a variety of habitats, ranging from upper streams to ponds, suggesting that the body shape divergence among the three species may reflect adaptation to local environments. First, our geometric morphometrics among eight local populations of this species group revealed that the three species cannot be separated by body shape and that riverine populations had more elongated bodies and longer caudal parts than lacustrine populations. Second, their phylogenetic relationships did not support the presence of three species; phylogenies using mitochondrial DNA and genomic data obtained from RNA-Seq revealed that the eight populations could not be sorted into three different clades representing three described species. Third, phylogenetic corrections of body shape variations and ancestral state reconstruction of body shapes demonstrated that body shape divergence between riverine and lacustrine populations persisted even if the phylogenies were considered and that body shape evolved rapidly irrespective of phylogeny. Sexual dimorphism in body shape was also evident, but the degree of dimorphism did not significantly differ between riverine and lacustrine populations after phylogenetic corrections, suggesting that sexual selection may not substantially contribute to geographical variations in body shape. Overall, these results indicate that the deep-elongated body shape divergence of the O. woworae species group evolved locally in response to habitat environments, such as water currents, and that a thorough taxonomic reexamination of the O. woworae species group may be necessary.

Different contributions of local- and distant-regulatory changes to transcriptome divergence between stickleback ecotypes.

Differential gene expression can play an important role in phenotypic evolution and divergent adaptation. Although differential gene expression can be caused by both local- and distant-regulatory changes, we know little about their relative contribution to transcriptome evolution in natural populations. Here, we conducted expression quantitative trait loci (eQTL) analysis to investigate the genetic architecture underlying transcriptome divergence between marine and stream ecotypes of threespine sticklebacks (Gasterosteus aculeatus). We identified both local and distant eQTLs, some of which constitute hotspots, regions with a disproportionate number of significant eQTLs relative to the genomic background. The majority of local eQTLs including those in the hotspots caused expression changes consistent with the direction of transcriptomic divergence between ecotypes. Genome scan analysis showed that many local eQTLs overlapped with genomic regions of high differentiation. In contrast, nearly half of the distant eQTLs including those in the hotspots caused opposite expression changes, and few overlapped with regions of high differentiation, indicating that distant eQTLs may act as a constraint of transcriptome evolution. Finally, a comparison between two salinity conditions revealed that nearly half of eQTL hotspots were environment specific, suggesting that analysis of genetic architecture in multiple conditions is essential for predicting response to selection.

The function of appendage patterning genes in mandible development of the sexually dimorphic stag beetle.

One of the defining features of the evolutionary success of insects is the morphological diversification of their appendages, especially mouthparts. Although most insects share a common mouthpart ground plan, there is remarkable diversity in the relative size and shapes of these appendages among different insect lineages. One of the most prominent examples of mouthpart modification can be found in the enlargement of mandibles in stag beetles (Coleoptera, Insecta). In order to understand the proximate mechanisms of mouthpart modification, we investigated the function of appendage-patterning genes in mandibular enlargement during extreme growth of the sexually dimorphic mandibles of the stag beetle Cyclommatus metallifer. Based on knowledge from Drosophila and Tribolium studies, we focused on seven appendage patterning genes (Distal-less (Dll), aristaless (al), dachshund (dac), homothorax (hth), Epidermal growth factor receptor (Egfr), escargot (esg), and Keren (Krn). In order to characterize the developmental function of these genes, we performed functional analyses by using RNA interference (RNAi). Importantly, we found that RNAi knockdown of dac resulted in a significant mandible size reduction in males but not in female mandibles. In addition to reducing the size of mandibles, dac knockdown also resulted in a loss of the serrate teeth structures on the mandibles of males and females. We found that al and hth play a significant role during morphogenesis of the large male-specific inner mandibular tooth. On the other hand, knockdown of the distal selector gene Dll did not affect mandible development, supporting the hypothesis that mandibles likely do not contain the distal-most region of the ancestral appendage and therefore co-option of Dll expression is unlikely to be involved in mandible enlargement in stag beetles. In addition to mandible development, we explored possible roles of these genes in controlling the divergent antennal morphology of Coleoptera.

Genetic basis for variation in salinity tolerance between stickleback ecotypes.

Adaptation to different salinities can drive and maintain divergence between populations of aquatic organisms. Anadromous and stream ecotypes of threespine stickleback (Gasterosteus aculeatus) are an excellent model to explore the genetic mechanisms underlying osmoregulation divergence. Using a parapatric pair of anadromous and stream stickleback ecotypes, we employed an integrated genomic approach to identify candidate genes important for adaptation to different salinity environments. Quantitative trait loci (QTL) mapping of plasma sodium concentrations under a seawater challenge experiment identified a significant QTL on chromosome 16. To identify candidate genes within this QTL, we first conducted RNA-seq and microarray analysis on gill tissue to find ecotypic differences in gene expression that were associated with plasma Na+ levels. This resulted in the identification of ten candidate genes. Quantitative PCR analysis on gill tissue of additional Japanese stickleback populations revealed that the majority of the candidate genes showed parallel divergence in expression levels. Second, we conducted whole-genome sequencing and found five genes that are predicted to have functionally important amino acid substitutions. Finally, we conducted genome scan analysis and found that eight of these candidate genes were located in genomic islands of high differentiation, suggesting that they may be under divergent selection. The candidate genes included those involved in ATP synthesis and hormonal signalling, whose expression or amino acid changes may underlie the variation in salinity tolerance. Further functional molecular analysis of these genes will reveal the causative genetic and genomic changes underlying divergent adaptation.

Relaxin-related gene expression differs between anadromous and stream-resident stickleback (Gasterosteus aculeatus) following seawater transfer.

Relaxin (RLN) is a hormone that was originally identified as a regulator of pregnancy and reproduction. However, recent mammalian studies have demonstrated that relaxins also have potent osmoregulatory actions. In mammals, six relaxin family peptides have been identified: RLN1/2, RLN3, insulin-like peptide (INSL) 3, INSL4, INSL5, and INSL6. Previous genome database searches have revealed that teleosts also possess multiple relaxin family genes. However, the functions of these relaxin family peptides in teleosts remain unclear. In order to gain insight into the osmoregulatory functions of teleost relaxins, we studied the relaxin family peptides in euryhaline three-spined sticklebacks (Gasterosteus aculeatus), which have diversified into a variety of ecotypes. Rln3a, rln3b, and rln transcripts were abundant in the stickleback brain, whereas insl5b transcript levels were highest in the intestine among tissues. Seawater challenge experiments showed that transcript levels of rln3a, rln3b, and rln in the brain changed significantly after seawater transfer. Particularly, rln3b showed different patterns of temporal changes between anadromous and stream-resident morphs. The transcript levels of relaxin family peptide receptors, rxfp1, rxfp2b, rxfp3-2a, and rxfp3-2b, did not exhibit substantial changes in the brain, although these were constantly higher in the anadromous morph than the stream-resident morph. These results suggest that stickleback relaxin systems are differentially regulated by salinity signals, at least at the transcriptional level, and anadromous and stream-resident morphs differ in relaxin signaling pathways. The differences in the expression of relaxin-related genes between these two morphs provide a foundation for further exploration of the osmoregulatory function of relaxins in teleosts.