Genetic relationships among marine and freshwater populations of the European three-spined stickleback (Gasterosteus aculeatus) revealed by microsatellites

To assess the population genetic structure of the three-spined stickleback, Gasterosteus aculeatus, variability at 18 microsatellite loci was examined in 1724 individuals from 74 locations covering most of the species distribution range in Europe. The results revealed high overall degree of differentiation (F(ST) = 0.21) but contrasting level of divergence and genetic variability between habitat types. Marine populations were genetically relatively uniform even across great geographical distances as compared to substantial differentiation among freshwater populations. Analysis of molecular variance indicated low but significant (2.7%) variation in allele frequencies between geographical regions, but a negligible effect of habitat type (0.2%). The phylogenetic pattern was not explained by habitat type, but a weak signal of populations clustering according to geographical or water system origin was found. The results support the view that three-spined stickleback marine ancestors colonized northern European fresh waters during the postglacial marine submergence c. 10,000 years ago, whereas in the Mediterranean region colonization probably dates back to the Pleistocene. The independent origins of river and lake populations indicate that they originate from multiple colonizations rather than sharing common ancestry. In the continuous marine environment, the low degree of differentiation among populations can be explained by gene flow among subpopulations and large effective population size buffering divergence in neutral markers. In contrast, among postglacially established freshwater populations differentiation appears to be driven by genetic drift and isolation. The stepwise mutations appear to have contributed to the population differentiation in the southern part of the three-spined stickleback distribution range.     

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 genetic dynamics of three-spined sticklebacks (Gasterosteus aculeatus) in anthropogenic altered habitats

In industrialized and/or agriculturally used landscapes, inhabiting species are exposed to a variety of anthropogenic changes in their environments. Genetic diversity may be reduced if populations encounter founder events, bottlenecks, or isolation. Conversely, genetic diversity may increase if populations adapt to changes in selective regimes in newly created habitats. With the present study, genetic variability of 918 sticklebacks from 43 samplings (21.3 ± 3.8 per sample) at 36 locations from cultivated landscapes in Northwest Germany was analyzed at nine neutral microsatellite loci. To test if differentiation is influenced by habitat alterations, sticklebacks were collected from ancient running waters and adjacent artificial stagnant waters, from brooks with salt water inflow of anthropogenic and natural origin and adjacent freshwater sites. Overall population structure was dominated by isolation by distance (IBD), which was significant across all populations, and analysis of molecular variance (AMOVA) revealed that 10.6% of the variation was explained by river catchment area. Populations in anthropogenic modified habitats deviated from the general IBD structure and in the AMOVA, grouping by habitat type running/stagnant water explained 4.9% of variation and 1.4% of the variation was explained by salt-/freshwater habitat. Sticklebacks in salt-polluted water systems seem to exhibit elevated migratory activity between fresh- and saltwater habitats, reducing IBD. In other situations, populations showed distinct signs of genetic isolation, which in some locations was attributed to mechanical migration barriers, but in others to potential anthropogenic induced bottleneck or founder effects. The present study shows that anthropogenic habitat alterations may have diverse effects on the population genetic structure of inhabiting species. Depending on the type of habitat change, increased genetic differentiation, diversification, or isolation are possible consequences.     

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.     

Sex differences in pectoral muscles but not in pectoral fins in the three‐spined stickleback Gasterosteus aculeatus

The pectoral muscle index ( I _PM)( I _PM = 100 M _PM M⁻¹, where M _PM and M are the pectoral muscle and body masses, respectively) fin‐area and fin ray length were studied over a year in male and female three‐spined stickleback Gasterosteus aculeatus from a marine population (Öresund, Sweden) kept under simulated natural light and temperature conditions. A castration‐replacement experiment was used to test androgen effects on the I _PM, fin‐area and fin ray length. Non‐breeding males were castrated or sham‐operated in winter ( i.e . the fish had low levels of androgens). Castrated control and sham‐operated fish were implanted with empty Silastic capsules and castrated groups with capsules containing the androgens testosterone or 11‐ketoandrostenedione into the abdominal cavity. The experiment was terminated after 41 days, when the controls had matured. No morphological differences were found in pectoral fins between sexes during the year, except during the peak breeding season (May), where females showed larger fin‐area and longer fin ray in length compared to males. No effects of androgens treatment or of castration on pectoral fin‐area or fin ray length was observed. Breeding and non‐breeding males showed higher I _PM compared to females. The lower I _PM in females than in males could not be explained by the larger gonads in the former alone, as a sex difference in I _PM was still present after deduction of the ovaries from the female body mass. The I _PM was higher in sham‐operated compared to castrated fish. No effects of androgens treatment on I _PM was observed.     

The role of calcium and predation on plate morph evolution in the three‐spined stickleback (Gasterosteus aculeatus)

While the genetic basis to plate morph evolution of the three‐spined stickleback (Gasterosteus aculeatus) is well described, the environmental variables that select for different plate and spine morphs are incompletely understood. Using replicate populations of three‐spined sticklebacks on North Uist, Scotland, we previously investigated the role of predation pressure and calcium limitation on the adaptive evolution of stickleback morphology and behavior. While dissolved calcium proved a significant predictor of plate and spine morph, predator abundance did not. Ecol. Evol., xxx, 2014 and xxx performed a comparable analysis to our own to address the same question. They failed to detect a significant effect of dissolved calcium on morphological evolution, but did establish a significant effect of predation; albeit in the opposite direction to their prediction.     

Heritability of DNA methylation in threespine stickleback (Gasterosteus aculeatus)

Epigenetic mechanisms underlying phenotypic change are hypothesized to contribute to population persistence and adaptation in the face of environmental change. To date, few studies have explored the heritability of intergenerationally stable methylation levels in natural populations, and little is known about the relative contribution of cis- and trans-regulatory changes to methylation variation. Here, we explore the heritability of DNA methylation, and conduct methylation quantitative trait loci (meQTLs) analysis to investigate the genetic architecture underlying methylation variation between marine and freshwater ecotypes of threespine stickleback (Gasterosteus aculeatus). We quantitatively measured genome-wide DNA methylation in fin tissue using reduced representation bisulfite sequencing of F1 and F2 crosses, and their marine and freshwater source populations. We identified cytosines (CpG sites) that exhibited stable methylation levels across generations. We found that additive genetic variance explained an average of 24–35% of the methylation variance, with a number of CpG sites possibly autonomous from genetic control. We also detected both cis- and trans-meQTLs, with only trans-meQTLs overlapping with previously identified genomic regions of high differentiation between marine and freshwater ecotypes. Finally, we identified the genetic architecture underlying two key CpG sites that were differentially methylated between ecotypes. These findings demonstrate a potential role for DNA methylation in facilitating adaptation to divergent environments and improve our understanding of the heritable basis of population epigenomic variation.     

Glacial refuges for three‐spined stickleback in the Iberian Peninsula: mitochondrial DNA phylogeography

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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.     

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.     

Identification of novel microRNA genes in freshwater and marine ecotypes of the three‐spined stickleback (Gasterosteus aculeatus)

The three‐spined stickleback (Gasterosteus aculeatus L.) is an important model organism for studying the molecular mechanisms of speciation and adaptation to salinity. Despite increased interest to microRNA discovery and recent publication on microRNA prediction in the three‐spined stickleback using bioinformatics approaches, there is still a lack of experimental support for these data. In this paper, high‐throughput sequencing technology was applied to identify microRNA genes in gills of the three‐spined stickleback. In total, 595 miRNA genes were discovered; half of them were predicted in previous computational studies and were confirmed here as microRNAs expressed in gill tissue. Moreover, 298 novel microRNA genes were identified. The presence of miRNA genes in selected ‘divergence islands’ was analysed and 10 miRNA genes were identified as not randomly located in ‘divergence islands’. Regulatory regions of miRNA genes were found enriched with selective SNPs that may play a role in freshwater adaptation.     

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.     

Reproductive and histopathological effects of long-term experimental exposure to bis(tributyltin)oxide (TBTO) on the three-spined stickleback, Gasterosteus aculeatus Linnaeus

Three-spined sticklebacks, Gasterosteus aculeatus Linnaeus, were exposed to 0, 0.1, 1.0, 2.5 and 10 μg bis(tributyltin)oxide (TBTO) 1⁻¹ for up to 7.5 months in a flow-through system of sea water. As early as 4 weeks after the start of the experiment, fish from the 10 μg TBTO 1⁻¹ group showed diminishing appetite, increasing lethargy and opaque eyes. The mortality in this group after 2 months of exposure was 80%. No significant differences between the control and the exposed groups were noted concerning fecundity, hatched fry and frequencies of malformed fry. The gonad somatic index (GSI) was determined before and during the spawning period. GSI in control fish increased significantly, while that in the exposed groups remained unchanged between the two sampling occasions. Structural examination of the gills from fish exposed for 2 months to 10 μg TBTO I⁻¹ revealed fusion between secondary lamellae and gradual cell degeneration. The chloride cells were characterized by degenerating mitochondria. Hepatocytes in fish exposed to 2.5 and 10 μg TBTO I⁻¹ contained an increased number of lipid-containing vacuoles and the nucleoli were disorganized and fragmented.     

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.     

Intraspecific variation of spectral sensitivity in threespine stickleback (Gasterosteus aculeatus) from different photic regimes

To examine the influence of the spectral characteristics of underwater light on spectral sensitivity of the ON and OFF visual pathways, compound action potential recordings were made from retinal ganglion cells of threespine stickleback from different photic regimes. In fish from a red-shifted photic regime (P₅₀ 680 nm for downwelling light at 1m), peak sensitivity of both the ON and OFF pathways was limited to long wavelength light (_max 600–620). In contrast, the ON pathway of fish from a comparatively blue-shifted (P₅₀ 566 nm) photic regime exhibited sensitivity to medium (_max 540–560) and long (_max 600 nm) wavelengths, while the OFF pathway exhibited peak sensitivity to only medium (_max 540 nm) wavelength light. In a third population, where the the ambient light is moderately red-shifted (P₅₀ 629 nm), the ON pathway once again exhibited only a long wavelength sensitivity peak at 620 nm, while the OFF pathway exhibited sensitivity to both medium (_max 560 nm) and long (_max 600–620 nm) wavelength light. These findings suggest that the photic environment plays an integral role in shaping spectral sensitivity of the ON and OFF pathways.     

Rapid genetic divergence in postglacial populations of threespine stickleback (Gasterosteus aculeatus): the role of habitat type, drainage and geographical proximity

The goal of this study was to evaluate the role of common ancestry, and of geographical or reproductive isolation, in genetic divergence in populations of threespine sticklebacks (Gasterosteus aculeatus). Using seven DNA microsatellite loci we compared the effects of habitat type, drainage system and geographical proximity on genetic distance among 16 populations situated in an area in Schleswig-Holstein (Germany) that became deglaciated approximately 12 000 years ago. Stickleback population structure correlated only weakly with drainage system, whereas the primary divergence was among habitat types. Phylogenetic analysis revealed that lake (n = 7) and river (n = 5) populations formed two distinct clades (Cavalli-Sforza's and Edwards' chord distance, 82-100% bootstrap support) at approximately equal genetic distances to a third clade, comprising putative estuarine (n = 4) ancestors. Allele frequencies in lake and river populations represented different subsets of the genetically more diverse estuarine populations. In nested amovas approximately twice the genetic variance was distributed among lake vs. river vs. estuarine populations as compared with the combined effects of drainage system and geographical distance. Limited gene flow between habitat types must have been established after postglacial colonization, suggesting ecological hybrid inferiority or behavioural mating barriers between ecotypes. Within estuarine and lake populations, population differentiation followed an isolation-by-distance model. Given the high observed heterozygosities within the 16 study populations (HO = 0.65-0.87), the mean divergence between lake and river population pairs (FST = 0.18 +/- 0.007) would be reached after 300-6000 generations in a stepwise mutation model, depending on the size of N(e). This demonstrates both the utility of hypervariable microsatellites for detecting recent population divergences and the danger of operating at temporal or spatial scales which are beyond their resolution.     

Effects of Schistocephalus solidus (Cestoda) on the food intake and diet of the three-spined stickleback, Gasterosteus aculeatus

Seasonal differences were detected between uninfected sticklebacks and those infected with Schistocephalus solidus in their stomach fullness and diet composition.     

Fecundity compensation in the three‐spined stickleback Gasterosteus aculeatus infected by the diphyllobothriidean cestode Schistocephalus solidus

Causal explanations for host reproductive phenotypes influenced by parasitism fit into three broad evolutionary models: (1) non‐adaptive side effect; (2) adaptive parasitic manipulation; and (3) adaptive host defence. This study demonstrates fecundity compensation, an adaptive non‐immunological host defence, in the three‐spined stickleback fish (Gasterosteus aculeatus) infected by the diphyllobothriidean cestode Schistocephalus solidus. Both infected and uninfected female sticklebacks produced egg clutches at the same age and size. The reproductive capacity of infected females decreased rapidly with increased parasite : host body mass ratio. Body condition was lower in infected females than uninfected females and decreased with increasing parasite : host mass ratio. Females with clutches had greater body condition than those without clutches. A point biserial correlation showed that there was a body condition threshold necessary for clutch production to occur. Host females apparently had the capacity to produce egg clutches until the prolonged effects of nutrient theft by the parasite and the drain on resources from reproduction precluded clutch formation. Clutch mass, adjusted for female body mass, did not differ significantly between infected and uninfected females. Infected females apparently maintained the same level of reproductive allotment (egg mass as proportion of body mass) as uninfected females. Infected females produced larger clutches of smaller eggs than uninfected females, revealing a trade‐off between egg mass and egg number, consistent with the fecundity compensation hypothesis. The rapid loss of reproductive capacity with severity of infection probably reflects the influence of the parasite combined with a trade‐off between current and future reproduction in the host. Inter‐annual differences in reproductive performance may have reflected ecological influences on host pathology and/or intra‐annual seasonal changes. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

A method for the automated long‐term monitoring of three‐spined stickleback Gasterosteus aculeatus shoal dynamics

This paper describes and evaluates a flexible, non‐invasive tagging system for the automated identification and long‐term monitoring of individual three‐spined sticklebacks Gasterosteus aculeatus. The system is based on barcoded tags, which can be reliably and robustly detected and decoded to provide information on an individual's identity and location. Because large numbers of fish can be individually tagged, it can be used to monitor individual‐ and group‐level dynamics within fish shoals.