Morphological asymmetry of insular freshwater populations of threespine stickleback

Fluctuating asymmetry (FA), random deviations from perfect symmetry in a bilateral organism, has been widely used as a proxy for developmental instability in stressed populations. In order to test the utility of FA of resident freshwater threespine stickleback (Gasterosteus aculeatus) as a biomonitoring tool for contaminated sites, we compared levels of asymmetry of seven morphological traits of threespine stickleback collected from lakes at three islands with a history of military contamination and three islands with no military history, in the Aleutian Archipelago, Alaska. Traits examined include eye diameter, operculum width, pectoral fin ray number, pectoral fin width, lateral plate number, lateral plate length, and pelvic spine length. All morphometric traits demonstrated some degree of FA or directional asymmetry (DA), but the military history of a lake was not a predictor of the degree or type of asymmetry. Overall, the patterns of asymmetry were similar for all traits, irrespective of military contamination at a lake. Our results demonstrate that the suite of threespine stickleback traits measured are not suitable for examination of FA as a proxy for aquatic pollution in this region. DA also does not appear to be suitable as an indicator of aquatic pollution, but may instead be driven by local ecological factors such as predation.     

Genetic and morphometric divergence in threespine stickleback in the Chignik catchment,Alaska

Divergent selection pressures induced by different environmental conditions typically lead to variation in life history, behavior, and morphology. When populations are locally adapted to their current environment, selection may limit movement into novel sites, leading to neutral and adaptive genetic divergence in allopatric populations. Subsequently, divergence can be reinforced by development of pre‐ or postzygotic barriers to gene flow. The threespine stickleback, Gasterosteus aculeatus, is a primarily marine fish that has invaded freshwater repeatedly in postglacial times. After invasion, the established freshwater populations typically show rapid diversification of several traits as they become reproductively isolated from their ancestral marine population. In this study, we examine the genetic and morphometric differentiation between sticklebacks living in an open system comprising a brackish water lagoon, two freshwater lakes, and connecting rivers. By applying a set of microsatellite markers, we disentangled the genetic relationship of the individuals across the diverse environments and identified two genetic populations: one associated with brackish and the other with the freshwater environments. The “brackish” sticklebacks were larger and had a different body shape than those in freshwater. However, we found evidence for upstream migration from the brackish lagoon into the freshwater environments, as fish that were genetically and morphometrically similar to the lagoon fish were found in all freshwater sampling sites. Regardless, few F1‐hybrids were identified, and it therefore appears that some pre‐ and/or postzygotic barriers to gene flow rather than geographic distance are causing the divergence in this system.     

Family origin and the response of threespine stickleback, Gasterosteus aculeatus, to thermal acclimation

To establish whether family origin affects the response of the threespine stickleback (Gasterosteus aculeatus) to thermal acclimation, we examined the rates of feeding, growth, and food conversion, relative tissue and organ masses and activities of a mitochondrial and a glycolytic enzyme in pectoral and axial muscle of individually housed fish from six families during acclimation to 8 degrees C and 23 degrees C. Feeding rates differed among families but were consistently higher in warm-acclimated than cold-acclimated fish. Growth rates differed among families. In four families growth was greater at 8 degrees C; these families generally had higher conversion efficiencies at 8 degrees C than 23 degrees C. For two families, growth was greater at 23 degrees C than 8 degrees C and conversion efficiencies did not differ between 8 degrees C and 23 degrees C. Relative tissue and organ masses (percent axial muscle, hepatosomatic, gut and kidney indices) differed with gender and among families (hepatosomatic, gut and kidney indices) but little with acclimation status. In all families and in both muscles, activities of the mitochondrial enzyme, citrate synthase (CS), were increased by cold acclimation. Axial muscle levels of the glycolytic enzyme, lactate dehydrogenase (LDH), were not affected by thermal acclimation or family origin, but were strongly correlated with the hepatosomatic index and axial muscle protein content. Pectoral muscle levels of LDH were affected by family origin which also influenced the response to thermal acclimation. Similar patterns were observed for specific activities and total muscle contents of these enzymes. Stickleback family origin influenced rates of feeding and growth and the thermal sensitivity of growth rates but not the compensatory increase in muscle CS levels with cold acclimation. The differing thermal sensitivities of growth could reflect distinct strategies for the timing of juvenile growth.     

Genetic structure of threespine stickleback,Gasterosteus aculeatus,in Lake Harutori,Japan, with reference to coexisting anadromous and freshwater forms

Both anadromous and freshwater forms of threespine sticklebacks (Gasterosteus aculeatus) inhabit Lake Harutori on the pacific coast of eastern Hokkaido, Japan. Since the two groups of threespine sticklebacks, Pacific Ocean and Japan Sea groups, which showed high genetic differentiation between them, were sympatrically distributed on the Pacific Ocean coast of Hokkaido Island, the genetic structures of the two forms were examined in Lake Harutori. Allozyme analyses of the two forms showed that most specimens from Lake Harutori belonged to the Pacific Ocean group, with a few fish belonging to the Japan Sea group or representing hybrids between the two groups. Both anadromous and freshwater sticklebacks were detected in the Pacific Ocean group. There were no significant differences in allelic frequencies at 17 presumptive loci between the two forms. Analyses of genetic relationships among 5 populations showed that the freshwater population from Lake Harutori was genetically more closely related to the sympatric anadromous population than to the freshwater population from a neighboring river. These results suggested that anadromous and freshwater sticklebacks in Lake Harutori form a single breeding population, and that the two forms might represent a life-history polymorphism within a single population.     

Genetic basis of sexual dimorphism in the threespine stickleback Gasterosteus aculeatus

Sexual dimorphism (SD) in morphological, behavioural and physiological features is common, but the genetics of SD in the wild has seldom been studied in detail. We investigated the genetic basis of SD in morphological traits of threespine stickleback (Gasterosteus aculeatus) by conducting a large breeding experiment with fish from an ancestral marine population that acts as a source of morphological variation. We also examined the patterns of SD in a set of 38 wild populations from different habitats to investigate the relationship between the genetic architecture of SD of the marine ancestral population in relation to variation within and among natural populations. The results show that genetic architecture in terms of heritabilities, additive genetic variances and covariances (as well as correlations) is very similar in the two sexes in spite of the fact that many of the traits express significant SD. Furthermore, population differences in threespine stickleback body shape and armour SD appear to have evolved despite constraints imposed by genetic architecture. This implies that constraints for the evolution of SD imposed by strong genetic correlations are not as severe and absolute as commonly thought.     

Individuals' diet diversity influences gut microbial diversity in two freshwater fish (threespine stickleback and Eurasian perch)

Vertebrates' diets profoundly influence the composition of symbiotic gut microbial communities. Studies documenting diet‐microbiota associations typically focus on univariate or categorical diet variables. However, in nature individuals often consume diverse combinations of foods. If diet components act independently, each providing distinct microbial colonists or nutrients, we expect a positive relationship between diet diversity and microbial diversity. We tested this prediction within each of two fish species (stickleback and perch), in which individuals vary in their propensity to eat littoral or pelagic invertebrates or mixtures of both prey. Unexpectedly, in most cases individuals with more generalised diets had less diverse microbiota than dietary specialists, in both natural and laboratory populations. This negative association between diet diversity and microbial diversity was small but significant, and most apparent after accounting for complex interactions between sex, size and diet. Our results suggest that multiple diet components can interact non‐additively to influence gut microbial diversity.     

Allometric relationships in morphological traits associated with foraging,swimming ability,and predator defense reveal adaptations toward brackish and freshwater environments in the threespine stickleback

Freshwater colonization by threespine stickleback has led to divergence in morphology between ancestral marine and derived freshwater populations, making them ideal for studying natural selection on phenotypes. In an open brackish–freshwater system, we previously discovered two genetically distinct stickleback populations that also differ in geometric shape: one mainly found in the brackish water lagoon and one throughout the freshwater system. As shape and size are not perfectly correlated, the aim of this study was to identify the morphological trait(s) that separated the populations in geometric shape. We measured 23 phenotypes likely to be important for foraging, swimming capacity, and defense against predation. The lateral plate morphs in freshwater displayed few significant changes in trait sizes, but the low plated expressed feeding traits more associated with benthic habitats. When comparing the completely plated genetically assigned populations, the freshwater, the hybrids, the migrants and the lagoon fish, many of the linear traits had different slopes and intercepts in trait‐size regressions, precluding our ability to directly compare all traits simultaneously, which most likely results from low variation in body length for the lagoon and migrant population. We found the lagoon stickleback population to be more specialized toward the littoral zone, displaying benthic traits such as large, deep bodies with smaller eyes compared to the freshwater completely plated morph. Further, the lagoon and migrant fish had an overall higher body coverage of lateral plates compared to freshwater fish, and the dorsal and pelvic spines were longer. Evolutionary constraints due to allometric scaling relationships could explain the observed, overall restricted, differences in morphology between the sticklebacks in this study, as most traits have diversified in common allometric trajectories. The observed differences in foraging and antipredation traits between the fish with a lagoon and freshwater genetic signature are likely a result of genetic or plastic adaptations toward brackish and freshwater environments.     

Reductions in prolonged swimming capacity following freshwater colonization in multiple threespine stickleback populations

We compared ancestral anadromous-marine and nonmigratory, stream-resident threespine stickleback (Gasterosteus aculeatus) populations to examine the outcome of relaxed selection on prolonged swimming performance. We reared marine and stream-resident fish from two locations in a common environment and found that both stream-resident populations had lower critical swimming speeds (U(crits) ) than marine populations. F1 hybrids from the two locations displayed significant differences in dominance, suggesting that the genetic basis for variation in U(crit) differs between locations. To determine which traits evolved in conjunction with, and may underlie, differences in performance capacity we measured a suite of traits known to affect prolonged swimming performance in fish. Although some candidate traits did not evolve (standard metabolic rate and two body shape traits), multiple morphological (pectoral fin size, shape, and four body shape measures) and physiological (maximum metabolic rate; MMR) traits evolved in the predicted direction in both stream-resident populations. However, data from F1 hybrids suggested that only one of these traits (MMR) had dominance effects similar to those of U(crit) in both locations. Overall, our data suggest that reductions in prolonged swimming performance were selected for in nonmigratory populations of threespine stickleback, and that decreases in MMR may mediate these reductions in performance.     

Locomotory capacity of Baltic Sea and freshwater populations of the threespine stickleback (Gasterosteus aculeatus)

Two freshwater populations and one marine population (Baltic Sea) of threespine stickeback (Gasterosteus aculeatus) from Northeastern Germany were studied with regard to locomotory capacity: sustained swimming performance, activities of key enzymes in axial muscle, pectoral fin muscle and heart, and morphology. We postulated that life history differences between migratory Baltic Sea and resident freshwater populations could have led to a divergence in their locomotory capacity. The activity of citrate synthase (CS) in pectoral muscle correlated with critical swimming speed. Critical swimming speed, aerobic and anaerobic capacity of the pectoral fin muscle were population-specific. The Baltic Sea sticklebacks had a higher locomotory capacity (activity of CS in pectoral muscle, critical swimming speed) than sticklebacks of one freshwater population. However, another freshwater population expressed a similar locomotory capacity as the Baltic Sea population. In addition, Baltic Sea sticklebacks had a greater mass and lower anaerobic capacity of the pectoral fin muscle than the freshwater sticklebacks. The results are interpreted as an indication of a proceeding divergence between marine and resident freshwater populations and between freshwater populations of G. aculeatus originating from marine ancestors. The migratory Baltic Sea sticklebacks had better morphological prerequisites for sustained swimming than both freshwater populations, but there was no general difference in the locomotory capacity between marine and freshwater sticklebacks. However, their morphology could favour a more effective locomotion in the Baltic Sea sticklebacks.     

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

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

Classification of threespine stickleback along the benthic-limnetic axis

Many species of fish display morphological divergence between individuals feeding on macroinvertebrates associated with littoral habitats (benthic morphotypes) and individuals feeding on zooplankton in the limnetic zone (limnetic morphotypes). Threespine stickleback (Gasterosteus aculeatus L.) have diverged along the benthic-limnetic axis into allopatric morphotypes in thousands of populations and into sympatric species pairs in several lakes. However, only a few well known populations have been studied because identifying additional populations as either benthic or limnetic requires detailed dietary or observational studies. Here we develop a Fisher's linear discriminant function based on the skull morphology of known benthic and limnetic stickleback populations from the Cook Inlet Basin of Alaska and test the feasibility of using this function to identify other morphologically divergent populations. Benthic and limnetic morphotypes were separable using this technique and of 45 populations classified, three were identified as morphologically extreme (two benthic and one limnetic), nine as moderately divergent (three benthic and six limnetic) and the remaining 33 populations as morphologically intermediate. Classification scores were found to correlate with eye size, the depth profile of lakes, and the presence of invasive northern pike (Esox lucius). This type of classification function provides a means of integrating the complex morphological differences between morphotypes into a single score that reflects the position of a population along the benthic-limnetic axis and can be used to relate that position to other aspects of stickleback biology.     

Migratory history of the threespine stickleback Gasterosteus aculeatus

 The migratory history of two highly divergent forms (the Japan Sea and Pacific Ocean forms) of the threespine stickleback Gasterosteus aculeatus collected from Japanese brackish water (seawater) and freshwater was studied by examining strontium (Sr) and calcium (Ca) concentrations in their otoliths using wavelength dispersive X-ray spectrometry on an electron microprobe. The Sr : Ca ratios in the otoliths changed with salinity of the habitat. The otolith Sr : Ca ratios of the freshwater resident-type samples of the Pacific Ocean form showed consistently low Sr : Ca ratios, averaging 0.85–0.96 × 10⁻³ from the core to the edge. In contrast, the otolith Sr : Ca ratios of the anadromous type of both the Japan Sea and Pacific Ocean forms fluctuated strongly along the life history transects in accordance with their migration patterns from seawater to freshwater. The higher ratios in the anadromous type, averaging 5.4 × 10⁻³, in the otolith region from the core to 200 μm, corresponded to the seagoing period, suggesting that otolith Sr : Ca ratios are affected by ambient water salinity. These findings clearly indicate that otolith Sr : Ca ratios reflect individual life histories, and that these two highly divergent forms of stickleback have a flexible migration strategy. Received: May 23, 2002 / Revised: July 29, 2002 / Accepted: August 19, 2002 Acknowledgments We are grateful to Dr. S. Mori of Gifu Keizai University, and Miss M. Yamada and Messrs. M. Kume and T. Kitamura of Hokkaido University, for their assistance in sampling. This work was supported in part by Grant-in-Aid No. 13760138 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Correspondence to:Takaomi Arai     

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.     

Lateral plate asymmetry,diet and parasitism in threespine stickleback

Individuals with random left–right departures from bilateral symmetry are predicted to exhibit fitness reduction including increased parasitism. In an insular lake population of stickleback (Gasterosteus aculeatus) from the Queen Charlotte Islands, Western Canada, phenotypes with high or low number of lateral bony plates exhibited increased plate asymmetry relative to modal phenotypes. Asymmetric lateral plate phenotypes had increased prevalence and to a lesser extent intensity of parasitism relative to symmetric individuals, suggesting that differences in genetic resistance to pathogens contributed to unequal parasitism. The effect occurred mainly in the larger adults and during the warmest season, which may be due to the high metabolic costs incurred during the summer breeding season. Dietary differences between symmetric and asymmetric phenotypes were also detected and could contribute to unequal infection rates by mediating exposure to infected prey items. Our study, which is one of the first long‐term field assessments of asymmetry and parasitism, yields results that are consistent with studies linking asymmetry to reduced fitness and indicate that lateral plate asymmetry can be an indicator of poor individual quality, despite its apparent directionality.     

Natural selection and the genetics of adaptation in threespine stickleback

Growing knowledge of the molecular basis of adaptation in wild populations is expanding the study of natural selection. We summarize ongoing efforts to infer three aspects of natural selection—mechanism, form and history—from the genetics of adaptive evolution in threespine stickleback that colonized freshwater after the last ice age. We tested a mechanism of selection for reduced bony armour in freshwater by tracking genotype and allele frequency changes at an underlying major locus (Ectodysplasin) in transplanted stickleback populations. We inferred disruptive selection on genotypes at the same locus in a population polymorphic for bony armour. Finally, we compared the distribution of phenotypic effect sizes of genes underlying changes in body shape with that predicted by models of adaptive peak shifts following colonization of freshwater. Studies of the effects of selection on genes complement efforts to identify the molecular basis of adaptive differences, and improve our understanding of phenotypic evolution.     

Reverse evolution of armor plates in the threespine stickleback

Faced with sudden environmental changes, animals must either adapt to novel environments or go extinct. Thus, study of the mechanisms underlying rapid adaptation is crucial not only for the understanding of natural evolutionary processes but also for the understanding of human-induced evolutionary change, which is an increasingly important problem [1-8]. In the present study, we demonstrate that the frequency of completely plated threespine stickleback fish (Gasterosteus aculeatus) has increased in an urban freshwater lake (Lake Washington, Seattle, Washington) within the last 40 years. This is a dramatic example of "reverse evolution,"[9] because the general evolutionary trajectory is toward armor-plate reduction in freshwater sticklebacks [10]. On the basis of our genetic studies and simulations, we propose that the most likely cause of reverse evolution is increased selection for the completely plated morph, which we suggest could result from higher levels of trout predation after a sudden increase in water transparency during the early 1970s. Rapid evolution was facilitated by the existence of standing allelic variation in Ectodysplasin (Eda), the gene that underlies the major plate-morph locus [11]. The Lake Washington stickleback thus provides a novel example of reverse evolution, which is probably caused by a change in allele frequency at the major plate locus in response to a changing predation regime.     

Armor morphology and reproductive output in threespine stickleback,Gasterosteus aculeatus

Synopsis The threespine stickleback, Gasterosteus aculeatus, is an extensively armored fish inhabiting both marine and fresh waters across its holarctic distribution. Marine fish nearly always possess a full complement of bony lateral plates running from just behind the head to the tail, and a robust pelvic girdle complex. These armor features appear to constrain lateral and ventral abdominal distention, and therefore clutch volume. Freshwater populations in many areas exhibit variable reduction in lateral plate number, and in some regions the pelvic girdle is also reduced or lost. Freshwater populations also vary in the degree of abdominal distention exhibited by gravid females. We tested whether reduction in armoring might be correlated with increased clutch volume using five populations from the Cook Inlet area of Alaska. The hypothesis that populations having reduced pelvic girdle complexes would have greater size-adjusted clutch volumes was not supported. In fact, our two full-pelvic populations as a group had larger volumes. Similarly, size-adjusted clutch volumes were not related to pelvic phenotype within either of our two pelvic-reduced populations, nor to lateral plate morph within a fifth population. Other factors that may explain the interpopulation differences in clutch volume in threespine stickleback include body shape, food quantity and quality, intensity of predation, and even behavior. Except for a preliminary analysis of body shape, these possibilities remain unexplored. The concept of phenotypic integration suggests that these factors should be analyzed as a suite rather than individually.