High degree of genetic differentiation in marine three‐spined sticklebacks (Gasterosteus aculeatus)

Populations of widespread marine organisms are typically characterized by a low degree of genetic differentiation in neutral genetic markers, but much less is known about differentiation in genes whose functional roles are associated with specific selection regimes. To uncover possible adaptive population divergence and heterogeneous genomic differentiation in marine three‐spined sticklebacks (Gasterosteus aculeatus), we used a candidate gene‐based genome‐scan approach to analyse variability in 138 microsatellite loci located within/close to (<6 kb) functionally important genes in samples collected from ten geographic locations. The degree of genetic differentiation in markers classified as neutral or under balancing selection—as determined with several outlier detection methods—was low (F_ST = 0.033 or 0.011, respectively), whereas average F_ST for directionally selected markers was significantly higher (F_ST = 0.097). Clustering analyses provided support for genomic and geographic heterogeneity in selection: six genetic clusters were identified based on allele frequency differences in the directionally selected loci, whereas four were identified with the neutral loci. Allelic variation in several loci exhibited significant associations with environmental variables, supporting the conjecture that temperature and salinity, but not optic conditions, are important drivers of adaptive divergence among populations. In general, these results suggest that in spite of the high degree of physical connectivity and gene flow as inferred from neutral marker genes, marine stickleback populations are strongly genetically structured in loci associated with functionally relevant genes.     

Population responses to anthropogenic disturbance: lessons from three‐spined sticklebacks Gasterosteus aculeatus in eutrophic habitats

Human‐induced environmental changes differ from most natural changes in which they happen at a faster rate and require quicker responses from populations. The first response of populations is usually phenotypically plastic alterations of morphology, physiology and behaviour. This plasticity can be favourable and move the population closer to an adaptive peak in the altered environment and, hence, maintain a viable population, or be maladaptive and move the population further from the peak and increase the risk of extinction. The radiation of the three‐spined stickleback Gasterosteus aculeatus from the ocean to different freshwater habitats has provided much information on adaptation to new environmental conditions. Currently, human‐induced eutrophication is changing the breeding areas of these fish, which creates a model system for investigation of responses to rapid environmental disturbance. Results show that a primary reaction is plastic alterations of behaviour, with some adjustments being adaptive while others are not. At the same time, the strength of sexual selection on several traits is relaxed, which could increase the relative importance of survival selection. Whether this will restore population viability depends on the amount of standing genetic variation in the right direction. Human disturbances can be dramatic and resolution of the limit of flexibility and the possibility of genetic adaptation should be important targets of future research.     

Sea‐run migratory behaviour in the Japan Sea form of three‐spined stickleback Gasterosteus aculeatus in the tidal pool of eastern Hokkaido Island,Japan

Juveniles in the Japan Sea form of three‐spined stickleback Gasterosteus aculeatus collected from a tidal pool in eastern Hokkaido Island, Japan, had a unimodal standard length (L_S) frequency distribution in each period, ranging from 11·9 to 31·6 mm, and those of sea‐run migrating individuals collected from the outlet of this tidal pool ranged from 17·0 to 36·0 mm, suggesting juveniles of this form would migrate towards the sea when they reached >17·0 mm. In general linear models (GLM) for testing the best model, there was a relationship between the number of sea‐run migratory individual and precipitation, which suggests that precipitation may trigger migration.     

Predator‐inspection behaviour in female three‐spined sticklebacks Gasterosteus aculeatus is associated with status of gravidity

Differences in predator‐inspection behaviour between gravid and non‐gravid female as well as between male and female three‐spined sticklebacks Gasterosteus aculeatus were investigated. Gravid females confronted with a live rainbow trout Oncorhynchus mykiss showed bolder inspection behaviour than non‐gravid ones. The behaviour of gravid females was comparable with that of males, maybe because both face a high risk of predation. The results indicate that antipredator behaviour in female G. aculeatus is not fixed but adjusted to their reproductive state.     

Evaluation of a non‐invasive tagging system for laboratory studies using three‐spined sticklebacks Gasterosteus aculeatus

A non‐invasive tagging system for individual identification of three‐spined sticklebacks Gasterosteus aculeatus was evaluated. The tags were easily detected via video, and tagged and non‐tagged fish did not differ in terms of growth, activity levels or shoaling behaviour.     

Inbreeding in three‐spined sticklebacks (Gasterosteus aculeatus L.): effects on testis and sperm traits

Mating between relatives often results in inbreeding depression, and is assumed to have a strong effect on fitness traits such as fertility and gonad/gamete quality. However, data concerning this topic are contradictory and particularly scarce in fishes. Three‐spined sticklebacks (Gasterosteus aculeatus L.) show inbreeding depression in fertilization and hatching success, survival rates, body symmetry and behavioural traits. To date, any knowledge of the impact of inbreeding on males' gonads and gametes is lacking in this species. In the present study, testis and sperm traits were quantified in outbred and inbred males. Overall, these traits were not generally impaired by inbreeding, and this result was not changed by a second/third generation of brother–sister matings. However, testes brightness, a potential measure of oxidative stress, was negatively correlated with sperm number. Additionally, inbred males with higher body condition had significantly brighter testes, whereas their sperm number was significantly negatively correlated with sperm quality (as estimated by head volume). Such a trade‐off did not appear in outbred males. The comparatively small impact of inbreeding on testis and sperm traits might be explained by the low number of inbred individuals that reached the reproductive phase. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107 , 510–520.     

A universal and reliable assay for molecular sex identification of three‐spined sticklebacks (Gasterosteus aculeatus)

In heterogametic species, biological differences between the two sexes are ubiquitous, and hence, errors in sex identification can be a significant source of noise and bias in studies where sex‐related sources of variation are of interest or need to be controlled for. We developed and validated a universal multimarker assay for reliable sex identification of three‐spined sticklebacks (Gasterosteus aculeatus). The assay makes use of genotype scores from three sex‐linked loci and utilizes Bayesian probabilistic inference to identify sex of the genotyped individuals. The results, validated with 286 phenotypically sexed individuals from six populations of sticklebacks representing all major genetic lineages (cf. Pacific, Atlantic and Japan Sea), indicate that in contrast to commonly used single‐marker‐based sex identification assays, the developed multimarker assay should be 100% accurate. As the markers in the assay can be scored from agarose gels, it provides a quick and cost‐efficient tool for universal sex identification of three‐spined sticklebacks. The general principle of combining information from multiple markers to improve the reliability of sex identification is transferable and can be utilized to develop and validate similar assays for other species.     

Sexual dimorphism of head morphology in three‐spined stickleback Gasterosteus aculeatus

This study examined sexual dimorphism of head morphology in the ecologically diverse three‐spined stickleback Gasterosteus aculeatus. Male G. aculeatus had longer heads than female G. aculeatus in all 10 anadromous, stream and lake populations examined, and head length growth rates were significantly higher in males in half of the populations sampled, indicating that differences in head size increased with body size in many populations. Despite consistently larger heads in males, there was significant variation in size‐adjusted head length among populations, suggesting that the relationship between head length and body length was flexible. Inter‐population differences in head length were correlated between sexes, thus population‐level factors influenced head length in both sexes despite the sexual dimorphism present. Head shape variation between lake and anadromous populations was greater than that between sexes. The common divergence in head shape between sexes across populations was about twice as important as the sexual dimorphism unique to each population. Finally, much of the sexual dimorphism in head length was due to divergence in the anterior region of the head, where the primary trophic structures were found. It is unclear whether the sexual dimorphism was due to natural selection for niche divergence between sexes or sexual selection. This study improves knowledge of the magnitude, growth rate divergence, inter‐population variation and location of sexual dimorphism in G. aculeatus head morphology.     

Divergence in social foraging among morphs of the three‐spined stickleback,Gasterosteus aculeatus

Foraging in social groups has a number of benefits but can also increase the risk of exploitation. High tendency to shoal may be correlated with groups foraging, although facultatively social fish adjust both shoaling decisions and food resource defence based on intrinsic and extrinsic factors. The main aim of this study was to examine the relationships between shoaling, solitary foraging and aggression, forager tolerance of conspecifics joining at a discovered food patch and forager exploitation of resources discovered by others. We used two intra‐lacustrine three‐spined stickleback morph pairs, lava and mud, and monomorphic morphs from each of lava and mud habitats. The lava morph formed less cohesive shoals, was bolder during solitary foraging, approached and entered an occupied food patch less frequently than the mud morph, suggesting a link between shoaling and the propensity for social foraging. However, shoaling tendency and joiner tolerance were not correlated at a population level. Intralacustrine lava and mud morphs differed more markedly in joiner tolerance than morphs from single habitat lakes, whereas the opposite was true for shoaling tendency. We conclude that, in addition to differentiation in shoaling tendency, the lava and mud morphs differ in social foraging and these variations may act to promote population divergence. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 194–203.     

Regulation of eye and jaw colouration in three‐spined stickleback Gasterosteus aculeatus

Fish can change their skin and eye colour for background matching and signalling. Males of Gasterosteus aculeatus develop ornamental blue eyes and a red jaw during the reproductive season, colours that are further enhanced during courtship. Here, the effects of different hormones on physiological colour changes in the eyes and jaws of male and female G. aculeatus were investigated in vitro. In an in vivo experiment, G. aculeatus were injected with a receptor blocker of a pivotal hormone (noradrenaline) that controls colour change. In males, noradrenaline had aggregating effects on melanophore and erythrophore pigments resulting in blue eyes and a pale jaw, whereas melanocyte‐concentrating hormone (MCH) and melatonin resulted in a pale jaw only. When noradrenalin was combined with melanocyte stimulating hormone (MSH) or prolactin, the jaw became red, while the eyes remained blue. In vivo injection of yohimbine, an alpha‐2 adrenoreceptor blocker, resulted in dispersion of melanophore pigment in the eyes and inhibited the blue colouration. Altogether, the data suggest that noradrenalin has a pivotal role in the short‐term enhancement of the ornamental colouration of male G. aculeatus, potentially together with MSH or prolactin. This study also found a sex difference in the response to MCH, prolactin and melatonin, which may result from different appearance strategies in males, versus the more cryptic females.     

Cain and Abel reloaded? Kin recognition and male–male aggression in three‐spined sticklebacks Gasterosteus aculeatus L.

The influence of relatedness on male–male aggression was tested in three‐spined sticklebacks Gasterosteus aculeatus. The intensity of aggression against brothers and non‐kin males did not differ significantly, indicating that kin recognition plays at most a minor role in aggressive interactions between male G. aculeatus.     

Effects of VIE tagging and partial tissue sampling on the immune response of three‐spined stickleback Gasterosteus aculeatus

A 14 day experiment on effects of visible implant elastomer (VIE) tagging and spine‐clipping of three‐spined stickleback Gasterosteus aculeatus showed significant increases in immune response, particularly in the granulocyte:lymphocyte ratio, in both treatments and the sham control. A minimum two‐week recovery after handling, anaesthesia, tagging and spine‐clipping is recommended to minimize effect of manipulation on the immune system.     

Computational identification of miRNAs,their targets and functions in three‐spined stickleback (Gasterosteus aculeatus)

An intriguing question in biology is how the evolution of gene regulation is shaped by natural selection in natural populations. Among the many known regulatory mechanisms, regulation of gene expression by microRNAs (miRNAs) is of critical importance. However, our understanding of their evolution in natural populations is limited. Studying the role of miRNAs in three‐spined stickleback, an important natural model for speciation research, may provide new insights into adaptive polymorphisms. However, lack of annotation of miRNA genes in its genome is a bottleneck. To fill this research gap, we used the genome of three‐spined stickleback to predict miRNAs and their targets. We predicted 1486 mature miRNAs using the homology‐based miRNA prediction approach. We then performed functional annotation and enrichment analysis of these targets, which identified over‐represented motifs. Further, a database resource (GAmiRdb) has been developed for dynamically searching miRNAs and their targets exclusively in three‐spined stickleback. Finally, the database was used in two case studies focusing on freshwater adaptation in natural populations. In the first study, we found 44 genomic regions overlapping with predicted miRNA targets. In the second study, we identified two SNPs altering the MRE seed site of sperm‐specific glyceraldehyde‐3‐phosphate gene. These findings highlight the importance of the GAmiRdb knowledge base in understanding adaptive evolution.     

Further refinement of the non‐invasive procedure for measuring steroid production in the male three‐spined stickleback Gasterosteus aculeatus

Measurement of steroids that are released into the water via the gills has previously been shown to be an effective way of studying the reproductive endocrinology of the male three‐spined stickleback Gasterosteus aculeatus without having to kill the fish. In the present paper, a previous observation on the existence of a compound other than 11‐ketotestosterone (11‐KT) in water, which cross‐reacted in the 11‐KT radioimmunoassay was repeated. The amounts of this compound, however, were not sufficient to warrant a separation step prior to carrying out assay. The lack of association between androstenedione levels in water and those in plasma was also confirmed. For the first time, the amounts of testosterone released into the water were shown to be positively correlated with the amounts in plasma, the sampling procedure (placing the fish for 30 min in 50 ml water) had no effect on the rate of release of cortisol but caused a rapid drop in the rate of release of 11‐KT (which means that the fish should not be sampled twice in short succession), physical interaction between two nesting males (which was accompanied by aggression) significantly increased the rate of release of 11‐KT, androstenedione and testosterone (but not of cortisol) and the rate of release of 11‐KT was at its maximum between 2 and 4 h after exposure.     

Latitudinal variation in photoperiodic response of the three‐spined stickleback Gasterosteus aculeatus in western North America

Reproductive maturation in both male and female three‐spined stickleback Gasterosteus aculeatus was strongly photoperiodic in a northern population (Alaska, 61° N) but not in a southern population (Oregon, 43° N) from western North America. Increasing reliance on photoperiod with increasing latitude is a general phenomenon among vertebrates, and is probably due to the anticipation of a narrower window of opportunity for reproduction and development at higher latitudes.     

A field reciprocal transplant experiment reveals asymmetric costs of migration between lake and river ecotypes of three‐spined sticklebacks (Gasterosteus aculeatus)

Theory of local adaptation predicts that nonadapted migrants will suffer increased costs compared to local residents. Ultimately this process can result in the reduction of gene flow and culminate in speciation. Here, we experimentally investigated the relative fitness of migrants in foreign habitats, focusing on diverging lake and river ecotypes of three‐spined sticklebacks. A reciprocal transplant experiment performed in the field revealed asymmetric costs of migration: whereas mortality of river fish was increased under lake conditions, lake migrants suffered from reduced growth relative to river residents. Selection against migrants thus involved different traits in each habitat but generally contributed to bidirectional reduction in gene flow. Focusing particularly on the parasitic environments, migrant fish differed from resident fish in the parasite community they harboured. This pattern correlated with both cellular phenotypes of innate immunity as well as with allelic variation at the genes of the major histocompatibility complex. In addition to showing the costs of migration in three‐spined sticklebacks, this study highlights the role of asymmetric selection particularly from parasitism in genotype sorting and in the emergence of local adaptation.     

Effects of perceived predation risk and social environment on the development of three‐spined stickleback (Gasterosteus aculeatus) morphology

Phenotypically plastic changes in response to variation in perceived predation risk are widespread, but little is known about if and how social environment modulates induced responses to predation risk. We investigated the influence of perceived predation risk (i.e. chemical cues from a predator) and social environment (i.e. one, two or 20 individuals reared together) on three‐spined stickleback (Gasterosteus aculeatus) morphology in a factorial common garden experiment. We found that exposure to chemical cues from potential predators did not influence growth or body condition or induce more robust morphological defences (i.e. lateral plate numbers and dorsal spine lengths). However, sticklebacks exposed to predator cues developed longer caudal peduncles and larger eyes as compared with fish from the control treatment. As these responses may improve sticklebacks’ ability to avoid piscine predation, they might be adaptive. Social environment/density also influenced expression of some traits, but these effects were independent of predation‐risk treatment effects. In general, these results suggest that apart from the classic morphological defence structures, which appear mostly constitutive, three‐spined sticklebacks are capable of expressing potentially adaptive morphological responses to chemical cues from potential predators.     

Spectral composition and visual foraging in the three‐spined stickleback (Gasterosteidae: Gasterosteus aculeatus L.): elucidating the role of ultraviolet wavelengths

Visual signalling can be affected by both the intensity and spectral distribution of environmental light. In shallow aquatic habitats, the spectral range available for visually mediated behaviour, such as foraging, can reach from ultraviolet (UV) to long wavelengths in the human visible range. However, the relative importance of different wavebands in foraging behaviour is generally unknown. Here, we test how the spectral composition of ambient light influences the behaviour of three‐spined sticklebacks (Gasterosteus aculeatus) when foraging for live cladoceran Daphnia magna. Although paying particular attention to the UV waveband, we measured the foraging preferences of sticklebacks for prey presented under four different spectral conditions. These conditions selectively removed UV (UV–), short‐wave (SW–), mid‐wave (MW–) or long‐wave (LW–) light from the entire spectrum. The absence of UV and long wavelengths strongly reduced prey attractiveness for G. aculeatus compared with conditions without short‐wave and mid‐wave light. To control for potential light habitat preferences in the main experiment, we conducted a further choice experiment without prey stimuli. Fish in these trials did not discriminate significantly between the different spectral conditions. When comparing both experiments, it was observed that, although filter preferences for MW– and LW– conditions were virtually consistent, they differed at shorter wavelengths, with a reduced preference for UV– conditions and, at the same time, an increased preference for SW– conditions in the presence of prey. Thus, prey choice seems to be strongly affected by visual information at the short‐wave end of the spectrum. The foraging preferences were also mirrored by the chromatic contrast values between prey and the experimental background, as calculated for each lighting condition using a series of physiological models on stickleback perception. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 359–368.     

A benthic predatory fish does not cause selection on armour traits in three‐spined stickleback Gasterosteus aculeatus (Gasterosteiformes: Gasterosteidae)

Predation can promote divergence between prey populations and contribute to ecological speciation. In theory, predators can also constrain prey population divergence. In coastal British Columbia, Canada, Gasterosteus aculeatus (three‐spined stickleback) species pairs only occur in lakes with a single species of predatory fish: Oncorhynchus clarkii (the cutthroat trout). Similar lakes containing additional predatory fish species (Cottus asper, prickly sculpins; Oncorhynchus mykiss, rainbow trout) contain only single species of morphologically intermediate stickleback, suggesting that these predators prevent the coexistence of stickleback species pairs. We conducted a mesocosm experiment to investigate how prickly sculpins might constrain divergence, by quantifying their impact on survival and natural selection on antipredator (armour) traits in F₂ stickleback from a cross between ecologically divergent populations. We tested three hypotheses: (1) sculpin predation on sticklebacks reduces survival in a way that could result in their exclusion from certain niches; (2) sculpins compete with stickleback; (3) sculpins respond to prey vulnerabilities in similar ways to cutthroat trout, tending to constrain rather than to enhance divergence. We found that sculpins significantly reduce stickleback survival, that their presence per se does not reduce growth in stickleback, and that predation did not result in selection on any of the armour traits measured, or on gill raker length, which is an important trophic trait. These results tend to refute hypotheses (2) and (3), while supporting hypothesis (1). © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 877–885.     

Adaptive evolution of lateral plates in three‐spined stickleback Gasterosteus aculeatus: a case study in functional analysis of natural variation

The three‐spined stickleback Gasterosteus aculeatus is a model species for studying questions in ecology and evolution. The rapid diversification of G. aculeatus in post‐glacial freshwater environments, combined with recently developed molecular tools, provides a unique opportunity to study the functional basis of fitness variation in natural populations. In derived freshwater populations, a number of morphological traits have diverged in parallel from the marine ancestral state, including the number of lateral armour plates. Evolution of reduced armour in freshwater populations is due to positive selection from both abiotic and biotic mechanisms. The major effect gene (ectodysplasin‐A or Eda), along with several minor effect genetic regions, has recently been shown to control lateral plate variation. Field experiments have further determined the fitness consequences of allelic variation at the major effect locus. This work helps elucidate the mechanisms connecting genetic variation with phenotypic variation and fitness in the wild, a synthesis that should be applicable to many other phenotypic traits and species of fishes.