De Novo Sequencing, Assembly, and Annotation of Four Threespine Stickleback Genomes Based on Microfluidic Partitioned DNA Libraries.

: The threespine stickleback is a geographically widespread and ecologically highly diverse fish that has emerged as a powerful model system for evolutionary genomics and developmental biology. Investigations in this species currently rely on a single high-quality reference genome, but would benefit from the availability of additional, independently sequenced and assembled genomes. We present here the assembly of four new stickleback genomes, based on the sequencing of microfluidic partitioned DNA libraries. The base pair lengths of the four genomes reach 92-101% of the standard reference genome length. Together with their de novo gene annotation, these assemblies offer a resource enhancing genomic investigations in stickleback. The genomes and their annotations are available from the Dryad Digital Repository (https://doi.org/10.5061/dryad.113j3h7).

Variation in sexual brain size dimorphism over the breeding cycle in the three-spined stickleback.

Snapshot analyses have demonstrated dramatic intraspecific variation in the degree of brain sexual size dimorphism (SSD). Although brain SSD is believed to be generated by the sex-specific cognitive demands of reproduction, the relative roles of developmental and population-specific contributions to variation in brain SSD remain little studied. Using a common garden experiment, we tested for sex-specific changes in brain anatomy over the breeding cycle in three-spined stickleback (Gasterosteus aculeatus) sampled from four locations in northern Europe. We found that the male brain increased in size (ca. 24%) significantly more than the female brain towards breeding, and that the resulting brain SSD was similar (ca. 20%) for all populations over the breeding cycle. Our findings support the notion that the stickleback brain is highly plastic and changes over the breeding cycle, especially in males, likely as an adaptive response to the cognitive demands of reproduction (e.g. nest construction and parental care). The results also provide evidence to suggest that breeding-related changes in brain size may be the reason for the widely varying estimates of brain SSD across studies of this species, cautioning against interpreting brain size measurements from a single time point as fixed/static.

Heterochronic development of lateral plates in the three-spined stickleback induced by thyroid hormone level alterations.

The three-spined stickleback Gasterosteus aculeatus is an important model for studying microevolution and parallel adaptation to freshwater environments. Marine and freshwater forms differ markedly in their phenotype, especially in the number of lateral plates, which are serially repeated elements of the exoskeleton. In fishes, thyroid hormones are involved in adaptation to salinity, as well as the developmental regulation of serially repeated elements. To study how thyroid hormones influence lateral plate development, we manipulated levels of triiodothyronine and thiourea during early ontogeny in a marine and freshwater population with complete and low plate phenotypes, respectively. The development of lateral plates along the body and keel was heterochronic among experimental groups. Fish with a low dosage of exogenous triiodothyronine and those treated with thiourea exhibited retarded development of bony plates compared to both control fish and those treated with higher a triiodothyronine dosage. Several triiodothyronine-treated individuals of the marine form expressed the partial lateral plate phenotype. Some individuals with delayed development of lateral plates manifested 1-2 extra bony plates located above the main row of lateral plates.

Sticklebacks adapted to divergent osmotic environments show differences in plasticity for kidney morphology and candidate gene expression.

Novel physiological challenges in different environments can promote the evolution of divergent phenotypes, either through plastic or genetic changes. Environmental salinity serves as a key barrier to the distribution of nearly all aquatic organisms, and species diversification is likely to be enabled by adaptation to alternative osmotic environments. The threespine stickleback (Gasterosteus aculeatus) is a euryhaline species with populations found both in marine and freshwater environments. It has evolved both highly plastic and locally adapted phenotypes due to salinity-derived selection, but the physiological and genetic basis of adaptation to salinity is not fully understood. We integrated comparative cellular morphology of the kidney, a key organ for osmoregulation, and candidate gene expression to explore the underpinnings of evolved variation in osmotic plasticity within two populations of sticklebacks from distinct salinity zones in the Baltic Sea: the high salinity Kattegat, representative of the ancestral marine habitat; and the low salinity Bay of Bothnia. A common-garden experiment revealed that kidney morphology in the ancestral high-salinity population had a highly plastic response to salinity conditions whereas this plastic response was reduced in the low-salinity population. Candidate gene expression in kidney tissue revealed a similar pattern of population-specific differences, with a higher degree of plasticity in the native high-salinity population. Together these results suggest that renal cellular morphology has become canalized to low salinity, and that these structural differences may have functional implications for osmoregulation.

Environmental enrichment, sexual dimorphism, and brain size in sticklebacks.

Evidence for phenotypic plasticity in brain size and the size of different brain parts is widespread, but experimental investigations into this effect remain scarce and are usually conducted using individuals from a single population. As the costs and benefits of plasticity may differ among populations, the extent of brain plasticity may also differ from one population to another. In a common garden experiment conducted with three-spined sticklebacks (Gasterosteus aculeatus) originating from four different populations, we investigated whether environmental enrichment (aquaria provided with structural complexity) caused an increase in the brain size or size of different brain parts compared to controls (bare aquaria). We found no evidence for a positive effect of environmental enrichment on brain size or size of different brain parts in either of the sexes in any of the populations. However, in all populations, males had larger brains than females, and the degree of sexual size dimorphism (SSD) in relative brain size ranged from 5.1 to 11.6% across the populations. Evidence was also found for genetically based differences in relative brain size among populations, as well as for plasticity in the size of different brain parts, as evidenced by consistent size differences among replicate blocks that differed in their temperature.

Comparison of catch per unit effort among four minnow trap models in the three-spined stickleback (Gasterosteus aculeatus) fishery.

Minnow traps are commonly used in the stickleback (Gasterostidae) fishery, but the potential differences in catch per unit effort (CPUE) among different minnow trap models are little studied. We compared the CPUE of four different minnow trap models in field experiments conducted with three-spined sticklebacks (Gasterosteus aculeatus). Marked (up to 26 fold) differences in median CPUE among different trap models were observed. Metallic uncoated traps yielded the largest CPUE (2.8 fish/h), followed by metallic black nylon-coated traps (1.3 fish/h). Collapsible canvas traps yielded substantially lower CPUEs (black: 0.7 fish/h; red: 0.1 fish/h) than the metallic traps. Laboratory trials further revealed significant differences in escape probabilities among the different trap models. While the differences in escape probability can explain at least part of the differences in CPUE among the trap models (e.g. high escape rate and low CPUE in red canvas traps), discrepancies between model-specific CPUEs and escape rates suggests that variation in entrance rate also contributes to the differences in CPUE. In general, and in accordance with earlier data on nine-spined stickleback (Pungitius pungitius) trapping, the results suggest that uncoated metallic (Gee-type) traps are superior to the other commonly used minnow trap models in stickleback fisheries.

Population genomic evidence for adaptive differentiation in Baltic Sea three-spined sticklebacks.

BACKGROUND: The degree of genetic differentiation among populations experiencing high levels of gene flow is expected to be low for neutral genomic sites, but substantial divergence can occur in sites subject to directional selection. Studies of highly mobile marine fish populations provide an opportunity to investigate this kind of heterogeneous genomic differentiation, but most studies to this effect have focused on a relatively low number of genetic markers and/or few populations. Hence, the patterns and extent of genomic divergence in high-gene flow marine fish populations remain poorly understood. RESULTS: We here investigated genome-wide patterns of genetic variability and differentiation in 10 marine populations of three-spined stickleback (Gasterosteus aculeatus) distributed across a steep salinity and temperature gradient in the Baltic Sea, by utilizing > 30,000 single nucleotide polymorphisms obtained with a pooled RAD-seq approach. We found that genetic diversity and differentiation varied widely across the genome, and identified numerous fairly narrow genomic regions exhibiting signatures of both divergent and balancing selection. Evidence was uncovered for substantial genetic differentiation associated with both salinity and temperature gradients, and many candidate genes associated with local adaptation in the Baltic Sea were identified. CONCLUSIONS: The patterns of genetic diversity and differentiation, as well as candidate genes associated with adaptation in Baltic Sea sticklebacks were similar to those observed in earlier comparisons between marine and freshwater populations, suggesting that similar processes may be driving adaptation to brackish and freshwater environments. Taken together, our results provide strong evidence for heterogenic genomic divergence driven by local adaptation in the face of gene flow along an environmental gradient in the post-glacially formed Baltic Sea.

Temporal stability of genetic variability and differentiation in the three-spined stickleback (Gasterosteus aculeatus).

Temporal variation in allele frequencies, whether caused by deterministic or stochastic forces, can inform us about interesting demographic and evolutionary phenomena occurring in wild populations. In spite of the continued surge of interest in the genetics of three-spined stickleback (Gasterosteus aculeatus) populations, little attention has been paid towards the temporal stability of allele frequency distributions, and whether there are consistent differences in effective size (Ne) of local populations. We investigated temporal stability of genetic variability and differentiation in 15 microsatellite loci within and among eight collection sites of varying habitat type, surveyed twice over a six-year time period. In addition, Nes were estimated with the expectation that they would be lowest in isolated ponds, intermediate in larger lakes and largest in open marine sites. In spite of the marked differences in genetic variability and differentiation among the study sites, the temporal differences in allele frequencies, as well as measures of genetic diversity and differentiation, were negligible. Accordingly, the Ne estimates were temporally stable, but tended to be lower in ponds than in lake or marine habitats. Hence, we conclude that allele frequencies in putatively neutral markers in three-spined sticklebacks seem to be temporally stable - at least over periods of few generations - across a wide range of habitat types differing markedly in levels of genetic variability, effective population size and gene flow.

Contrasting patterns of genetic and phenotypic differentiation in two invasive salmonids in the southern hemisphere.

Invasion success may be expected to increase with residence time (i.e., time since first introduction) and secondary releases (i.e., those that follow the original introduction), but this has rarely been tested in natural fish populations. We compared genetic and phenotypic divergence in rainbow trout and brown trout in Chile and the Falkland Islands to test the prediction that adaptive divergence, measured as P ST/F ST, would increase with residence time and secondary releases. We also explored whether interspecific competition between invaders could drive phenotypic divergence. Residence time had no significant effect on genetic diversity, phenotypic divergence, effective population size, or signatures of expansion of invasive trout. In contrast, secondary releases had a major effect on trout invasions, and rainbow trout populations mostly affected by aquaculture escapees showed significant divergence from less affected populations. Coexistence with brown trout had a positive effect on phenotypic divergence of rainbow trout. Our results highlight an important role of secondary releases in shaping fish invasions, but do not support the contention that older invaders are more differentiated than younger ones. They also suggest that exotic trout may not have yet developed local adaptations in these recently invaded habitats, at least with respect to growth-related traits.

Geographic variation in age structure and longevity in the nine-spined stickleback (Pungitius pungitius).

Variation in age and size of mature nine-spined sticklebacks (Pungitius pungitius) within and among 16 Fennoscandian populations were assessed using skeletochronology. The average age of individuals in a given population varied from 1.7 to 4.7 years. Fish from pond populations were on average older than those from lake and marine populations, and females tended to be older than males. Reproduction in marine and lake populations commenced typically at an age of two years, whereas that in ponds at an age of three years. The maximum life span of the fish varied from 3 to 7 years. Mean body size within and among populations increased with increasing age, but the habitat and population differences in body size persisted even after accounting for variation in population age (and sex) structure. Hence, the population differences in mean body size are not explainable by age differences alone. As such, much of the pronounced intraspecific variation in population age structure can be attributed to delayed maturation and extended longevity of the pond fish. The results are contrasted and discussed in the context of similar data from the three-spined stickleback (Gasterosteus aculeatus) occupying the same geographic area.

Variation in age and size in Fennoscandian three-spined sticklebacks (Gasterosteus aculeatus).

Average age and maximum life span of breeding adult three-spined sticklebacks (Gasterosteus aculeatus) were determined in eight Fennoscandian localities with the aid of skeletochronology. The average age varied from 1.8 to 3.6 years, and maximum life span from three to six years depending on the locality. On average, fish from marine populations were significantly older than those from freshwater populations, but variation within habitat types was large. We also found significant differences in mean body size among different habitat types and populations, but only the population differences remained significant after accounting for variation due to age effects. These results show that generation length and longevity in three-spined sticklebacks can vary significantly from one locality to another, and that population differences in mean body size cannot be explained as a simple consequence of differences in population age structure. We also describe a nanistic population from northern Finland exhibiting long life span and small body size.

Heterogeneous Genomic Differentiation in marine threespine sticklebacks: adaptation along an environmental gradient.

Evolutionary divergence among populations occupying ecologically distinct environments can occur even in the face of on-going gene flow. However, the genetic underpinnings, as well as the scale and magnitude at which this differentiation occurs in marine habitats are not well understood. We investigated the patterns and degree of genomic heterogeneity in threespine sticklebacks (Gasterosteus aculeatus) by assessing genetic variability in 20 nongenic and 20 genic (associated with genes important for freshwater adaptation) microsatellite loci in samples collected from 38 locations spanning the entire Baltic Sea coast to the North Sea boundary. Population divergence (F(ST) ≈ 0.026) and structuring (five genetic clusters) was significantly more pronounced in the genic as compared to nongenic markers (F(ST) ≈ 0.008; no genetic clusters). Patterns of divergence in the genic markers--45% of which were identified as outliers--correlated with local differences in salinity. Yet, a strong positive correlation between divergence in genic and nongenic markers, and their association with environmental factors suggests that adaptive divergence is reducing gene flow across the genome. Apart from providing a clear demonstration of heterogeneous genomic patterns of differentiation in a marine species, the results are indicative of adaptive population structuring across the relatively young Baltic Sea in spite of ample opportunities for gene flow.

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 FST 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.

Characterizing genic and nongenic molecular markers: comparison of microsatellites and SNPs.

The implications of transitioning to single nucleotide polymorphism (SNPs) from microsatellite markers (MSs) have been investigated in a number of population genetics studies, but the effect of genomic location on the amount of information each type of marker reveals has not been explored in detail. We developed novel SNP markers flanking 1 kb regions of 13 genic (within gene or <1 kb away from gene) and 13 nongenic (>10 kb from annotated gene) MSs in the threespine stickleback genome to obtain comparable data for both types of markers. We analysed patterns of genetic diversity and divergence on various geographic scales after converting the SNP loci within each genomic region into haplotypes. Marker type (SNP haplotype or MS) and location (genic or nongenic) significantly affected most estimates of population diversity and divergence. Between-lineage divergence was significantly higher in SNP haplotypes (genic and nongenic), however, within-lineage divergence was similar between marker types. Most divergence and diversity measures were uncorrelated between markers, except for population differentiation which was correlated between MSs and SNP haplotypes (both genic and nongenic). Broad-scale population structure and assignment were similarly resolved by both marker types, however, only the MSs were able to delimit fine-scale population structuring, particularly when genic and nongenic markers were combined. These results demonstrate that estimates of genetic variability and differentiation among populations can be strongly influenced by marker type, their genomic location in relation to genes and by the interaction of these two factors. This highlights the importance of having an awareness of the inherent strengths and limitations associated with different molecular tools to select the most appropriate methods for accurately addressing various ecological and evolutionary questions.

Global analysis of genes involved in freshwater adaptation in threespine sticklebacks (Gasterosteus aculeatus).

Examples of parallel evolution of phenotypic traits have been repeatedly demonstrated in threespine sticklebacks (Gasterosteus aculeatus) across their global distribution. Using these as a model, we performed a targeted genome scan--focusing on physiologically important genes potentially related to freshwater adaptation--to identify genetic signatures of parallel physiological evolution on a global scale. To this end, 50 microsatellite loci, including 26 loci within or close to (<6 kb) physiologically important genes, were screened in paired marine and freshwater populations from six locations across the Northern Hemisphere. Signatures of directional selection were detected in 24 loci, including 17 physiologically important genes, in at least one location. Although no loci showed consistent signatures of selection in all divergent population pairs, several outliers were common in multiple locations. In particular, seven physiologically important genes, as well as reference ectodysplasin gene (EDA), showed signatures of selection in three or more locations. Hence, although these results give some evidence for consistent parallel molecular evolution in response to freshwater colonization, they suggest that different evolutionary pathways may underlie physiological adaptation to freshwater habitats within the global distribution of the threespine stickleback.