Ultrastructural analysis of throat dermal tissue and chromatophore components in the threespine stickleback (Gasterosteus aculeatus).

The threespine stickleback (Gasterosteus aculeatus) is an important model for studying the evolution of nuptial coloration, but histological analyses of color are largely lacking. Previous analyses of one nuptial coloration trait, orange-red coloration along the body, have indicated carotenoids are the main pigment producing this color. In addition, recent gene expression studies found variation in the correlates of throat coloration between the sexes and between populations, raising the possibility of variation in the mechanisms underlying superficially similar coloration. We used transmission electron microscopy (TEM) to investigate the histological correlates of color in the throat dermal tissue of threespine stickleback from Western North America, within and between sexes, populations, and ecotypes. Ultrastructural analysis revealed carotenoid-containing erythrophores to be the main chromatophore component associated with orange-red coloration in both males and females across populations. In individuals where some darkening of the throat tissue was present, with no obvious orange-red coloration, erythrophores were not detected. Melanophore presence was more population-specific in expression, including being the only chromatophore component detected in a population of darker fish. We found no dermal chromatophore units within colorless throat tissue. This work confirms the importance of carotenoids and the erythrophore in producing orange-red coloration across sexes, as well as melanin within the melanophore in producing darkened coloration, but does not reveal broad histological differences among populations with similar coloration.

Gene expression in male and female stickleback from populations with convergent and divergent throat coloration.

Understanding of genetic mechanisms underlying variation in sexual dichromatism remains limited, especially for carotenoid-based colors. We addressed this knowledge gap in a gene expression study with threespine stickleback. We compared male and female throat tissues across five populations, including two in which female red coloration has evolved convergently. We found that the expression of individual genes, gene ontologies, and coexpression networks associated with red female color within a population differed between California and British Columbia populations, suggesting differences in underlying mechanisms. Comparing females from each of these populations to females from populations dominated by dull females, we again found extensive expression differences. For each population, genes and networks associated with female red color showed the same patterns for males only inconsistently. The functional roles of genes showing correlated expression with female color are unclear within populations, whereas genes highlighted through inter-population comparisons include some previously suggested to function in carotenoid pathways. Among these, the most consistent patterns involved TTC39B (Tetratricopeptide Repeat Domain 39B), which is within a known red coloration QTL in stickleback and implicated in red coloration in other taxa.

Evolution and assessment of colour patterns in stream-resident and anadromous male threespine stickleback Gasterosteus aculeatus from three regions.

We compared the colour patterns of free swimming, reproductively active male threespine stickleback Gasterosteus aculeatus of the anadromous and stream ecotypes from three geographically distinct regions. Consistent with the hypothesis of environmentally mediated selection, our results indicate ecologically replicated differences in G. aculeatus coloration between anadromous and stream-resident populations, and that G. aculeatus probably have the visual acuity to discriminate colour pattern differences between anadromous and stream-resident fish.

Variation in female aggression in 2 three-spined stickleback populations with female throat and spine coloration.

Despite growing interest in female ornament evolution, we still have a rudimentary understanding of female display traits relative to similar traits in males. Under one popular adaptive scenario, female ornaments are hypothesized to function in female-female competition and serve as badges of status, such that their expression is linked with elevated aggression in some cases. In this study, we investigated the relationship between 2 female ornaments-male-like red throat color and red spine coloration-and female aggression in 2 independently derived stream-resident populations of three-spined stickleback Gasterosteus aculeatus. Using simulated intrusions, we tested whether females with redder ornaments were generally more aggressive, and for variation in aggressive and social behaviors between the 2 populations. We found that the red intensity of the throat and spine did not predict aggression levels in either population, suggesting a limited role for both female ornaments during female-female interaction. The 2 populations exhibited different levels of aggressive behaviors, unrelated to the color patches. Our results suggest that variation in selective pressures between populations may promote interpopulation variance in aggressive behavior but not the correlation between female ornamentation and aggression, and raise the possibility that red coloration may have evolved through different mechanisms or processes in the 2 populations.

Genetic Architecture of Conspicuous Red Ornaments in Female Threespine Stickleback.

Explaining the presence of conspicuous female ornaments that take the form of male-typical traits has been a longstanding challenge in evolutionary biology. Such female ornaments have been proposed to evolve via both adaptive and nonadaptive evolutionary processes. Determining the genetic underpinnings of female ornaments is important for elucidating the mechanisms by which such female traits arise and persist in natural populations, but detailed information about their genetic basis is still scarce. In this study, we investigated the genetic architecture of two ornaments, the orange-red throat and pelvic spine, in the threespine stickleback (Gasterosteus aculeatus). Throat coloration is male-specific in ancestral marine populations but has evolved in females in some derived stream populations, whereas sexual dimorphism in pelvic spine coloration is variable among populations. We find that ornaments share a common genetic architecture between the sexes. At least three independent genomic regions contribute to red throat coloration, and harbor candidate genes related to pigment production and pigment cell differentiation. One of these regions is also associated with spine coloration, indicating that both ornaments might be mediated partly via pleiotropic genetic mechanisms.

Conspicuous female ornamentation and tests of male mate preference in threespine sticklebacks (Gasterosteus aculeatus).

Sexual selection drives the evolution of exaggerated male ornaments in many animal species. Female ornamentation is now acknowledged also to be common but is generally less well understood. One example is the recently documented red female throat coloration in some threespine stickleback (Gasterosteus aculeatus) populations. Although female sticklebacks often exhibit a preference for red male throat coloration, the possibility of sexual selection on female coloration has been little studied. Using sequential and simultaneous mate choice trials, we examined male mate preferences for female throat color, as well as pelvic spine color and standard length, using wild-captured threespine sticklebacks from the Little Campbell River, British Columbia. In a multivariate analysis, we found no evidence for a population-level mate preference in males, suggesting the absence of directional sexual selection on these traits arising from male mate choice. Significant variation was detected among males in their preference functions, but this appeared to arise from differences in their mean responsiveness across mating trials and not from variation in the strength (i.e., slope) of their preference, suggesting the absence of individual-level preferences as well. When presented with conspecific intruder males, male response decreased as intruder red throat coloration increased, suggesting that males can discriminate color and other aspects of phenotype in our experiment and that males may use these traits in intrasexual interactions. The results presented here are the first to explicitly address male preference for female throat color in threespine sticklebacks.

Male choice in the stream-anadromous stickleback complex.

Studies of mating preferences and pre-mating reproductive isolation have often focused on females, but the potential importance of male preferences is increasingly appreciated. We investigated male behavior in the context of reproductive isolation between divergent anadromous and stream-resident populations of threespine stickleback, Gasterosteus aculeatus, using size-manipulated females of both ecotypes. Specifically, we asked if male courtship preferences are present, and if they are based on relative body size, non-size aspects of ecotype, or other traits. Because male behaviors were correlated with each other, we conducted a principal components analysis on the correlations and ran subsequent analyses on the principal components. The two male ecotypes differed in overall behavioral frequencies, with stream-resident males exhibiting consistently more vigorous and positive courtship than anadromous males, and an otherwise aggressive behavior playing a more positive role in anadromous than stream-resident courtship. We observed more vigorous courtship toward smaller females by (relatively small) stream-resident males and the reverse pattern for (relatively large) anadromous males. Thus size-assortative male courtship preferences may contribute to reproductive isolation in this system, although preferences are far from absolute. We found little indication of males responding preferentially to females of their own ecotype independent of body size.

Colour polymorphism and correlated characters: genetic mechanisms and evolution.

Colour polymorphisms (CP's) continue to be of interest to evolutionary biologists because of their general tractability, importance in studies of selection and potential role in speciation. Since some of the earliest studies of CP, it has been evident that alternative colour morphs often differ in features other than colour. Here we review the rapidly accumulating evidence concerning the genetic mechanisms underlying correlations between CP and other traits in animals. We find that evidence for genetic correlations is now available for taxonomically diverse systems and that physical linkage and regulatory mechanisms including transcription factors, cis-regulatory elements, and hormone systems provide pathways for the ready accumulation or modification of these correlations. Moreover, physical linkage and regulatory mechanisms may both contribute to genetic correlation in some of the best-studied systems. These results raise the possibility that negative frequency-dependent selection and disruptive selection might often be acting on suites of traits and that the cumulative effects of such selection, as well as correlational selection, may be important to CP persistence and evolution. We consider additional evolutionary implications. We recommend continued efforts to elucidate the mechanisms underlying CP-correlated characters and the more frequent application of comparative approaches, looking at related species that vary in character correlations and patterns of selection. We also recommend efforts to elucidate how frequency-dependent selection may act on suites of characters.

Environment-contingent sexual selection in a colour polymorphic fish.

Sexual selection could be a driving force in the maintenance of intraspecific variation, but supporting observations from nature are limited. Here, we test the hypothesis that spatial heterogeneity of the visual environment can influence sexual selection on colourful male secondary traits such that selective advantage is environment contingent. Using a small fish endemic to Sulawesi, Indonesia (Telmatherina sarasinorum) that has five male colour morphs varying in frequency between two visually distinct mating habitats, we used direct behavioural observations to test the environment-contingent selection hypothesis. These observations were combined with measurements of the visual environment, fish coloration and the sensitivity of visual photopigments to determine whether differential morph conspicuousness was associated with reproductive success across habitats. We found that blue and yellow males are most conspicuous in different habitats, where they also have the highest reproductive fitness. A less conspicuous grey morph also gained high reproductive success in both habitats, raising the possibility that alternative behaviours may also contribute to reproductive success. In a comprehensive analysis, conspicuousness was strongly correlated with reproductive success across morphs and environments. Our results suggest an important role for spatially heterogeneous environments in the maintenance of male colour polymorphism.

Microhabitat variation and sexual selection can maintain male color polymorphisms.

Male color polymorphism may be an important precursor to sympatric speciation by sexual selection, but the processes maintaining such polymorphisms are not well understood. Here, we develop a formal model of the hypothesis that male color polymorphisms may be maintained by variation in the sensory environment resulting in microhabitat-specific selection pressures. We analyze the evolution of two male color morphs when color perception (by females and predators) is dependent on the microhabitat in which natural and sexual selection occur. We find that an environment of heterogeneous microhabitats can lead to the maintenance of color polymorphism despite asymmetries in the strengths of natural and sexual selection and in microhabitat proportions. We show that sexual selection alone is sufficient for polymorphism maintenance over a wide range of parameter space, even when female preferences are weak. Polymorphisms can also be maintained by natural selection acting alone, but the conditions for polymorphism maintenance by natural selection will usually be unrealistic for the case of microhabitat variation. Microhabitat variation and sexual selection for conspicuous males may thus provide a situation particularly favorable to the maintenance of male color polymorphisms. These results are important both because of the general insight they provide into a little appreciated mechanism for the maintenance of variation in natural populations and because such variation is an important prerequisite for sympatric speciation.

Cuckoldry incites cannibalism: male fish turn to cannibalism when perceived certainty of paternity decreases.

Perceived certainty of paternity is expected to influence a male's behavior toward his offspring: if he is uncertain of his reproductive success with a current brood due to the presence of cuckolders, it may benefit him to invest instead in future reproduction. A decrease in perceived certainty of paternity incites filial cannibalism (the eating of one's own offspring) in some teleost fishes that provide parental care; however, no work has demonstrated that cannibalism increases proportionately with increased levels of cuckoldry. Here we show for the first time in a fish with no parental care that as the number of cuckolders at a spawning event increases, so does the probability that a male will cannibalize eggs. In field observations of Telmatherina sarasinorum, a small fish endemic to Sulawesi, Indonesia, males increased filial cannibalism behavior threefold in the presence of one cuckolder and nearly sixfold in the presence of two or more cuckolders. This suggests that males may use detection of cuckolders as an indication that the paternity of current offspring has been compromised.

Linking color polymorphism maintenance and speciation.

Here, we review the recently burgeoning literature on color polymorphisms, seeking to integrate studies of the maintenance of genetic variation and the evolution of reproductive isolation. Our survey reveals that several mechanisms, some operating between populations and others within them, can contribute to both color polymorphism persistence and speciation. As expected, divergent selection clearly can couple with gene flow to maintain color polymorphism and mediate speciation. More surprisingly, recent evidence suggests that diverse forms of within-population sexual selection can generate negative frequency dependence and initiate reproductive isolation. These findings deserve additional study, particularly concerning the roles of heterogeneous visual environments and correlational selection. Finally, comparative studies and more comprehensive approaches are required to elucidate when color polymorphism evolves, persists, or leads to speciation.

Parallel evolution and inheritance of quantitative traits.

Parallel phenotypic evolution, the independent evolution of the same trait in closely related lineages, is interesting because it tells us about the contribution of natural selection to phenotypic evolution. Haldane and others have proposed that parallel evolution also results from a second process, the similarly biased production of genetic variation in close relatives, an idea that has received few tests. We suggest that influence of shared genetic biases should be detectable by the disproportionate use of the same genes in independent instances of parallel phenotypic evolution. We show how progress in testing this prediction can be made through simple tests of parallel inheritance of genetic differences: similar additive, dominance, and epistasis components in analysis of line means and similar effective numbers of loci. We demonstrate parallel inheritance in two traits, lateral plate number and body shape, in two lineages of threespine stickleback that have adapted independently to freshwater streams on opposite sides of the Pacific Ocean. Notably, reduction of plate number in freshwater involves a substitution at the same major locus in both lineages. Our results represent only a first step in the study of the genetics of parallel phenotypic evolution in sticklebacks. Nevertheless, we have shown how such studies can be employed to test the genetic hypothesis of parallel evolution and how study of parallel evolution might yield insights into the roles of both selection and genetic constraint in phenotypic evolution.

Evidence for ecology's role in speciation.

A principal challenge in testing the role of natural selection in speciation is to connect the build-up of reproductive isolation between populations to divergence of ecologically important traits. Demonstrations of 'parallel speciation', or assortative mating by selective environment, link ecology and isolation, but the phenotypic traits mediating isolation have not been confirmed. Here we show that the parallel build-up of mating incompatibilities between stickleback populations can be largely accounted for by assortative mating based on one trait, body size, which evolves predictably according to environment. In addition to documenting the influence of body size on reproductive isolation for stickleback populations spread across the Northern Hemisphere, we have confirmed its importance through a new experimental manipulation. Together, these results suggest that speciation may arise largely as a by-product of ecological differences and divergent selection on a small number of phenotypic traits.

Directional sexual selection on chroma and within-pattern colour contrast in Labeotropheus fuelleborni.

Speciation via intersexual selection on male nuptial colour pattern is thought to have been a major force in promoting the explosive speciation of African haplochromine cichlids, yet there is very little direct empirical evidence of directional preferences within populations. In this study, we used objective spectrophotometry and analyses based on visual physiology to determine whether females of the Katale population of Labeotropheus fuelleborni, a Lake Malawi haplochromine, prefer males that have higher chroma and more within-pattern colour contrast. In paired male preference tests, female Katale L. fuelleborni showed increasing preferences for males with more relatively saturated colours on their flanks. They also showed increasing preferences for males with relatively higher contrast levels among flank elements. This is the first empirical evidence, to our knowledge, for male colour as a directionally sexually selected trait within a haplochromine cichlid population.