Kohta Yoshida

Genetic and Neural Modularity Underlie the Evolution of Schooling Behavior in Threespine Sticklebacks

Although descriptions of striking diversity in animal behavior are plentiful, little is known about the mechanisms by which behaviors change and evolve between groups. To fully understand behavioral evolution, it will be necessary to identify the genetic mechanisms that mediate behavioral change in a natural context. Genetic analysis of behavior can also reveal associations between behavior and morphological or neural phenotypes, providing insight into the proximate mechanisms that control behavior. Relatively few studies to date have successfully identified genes or genomic regions that contribute to behavioral variation among natural populations or species, particularly in vertebrates. Here, we apply genetic approaches to dissect a complex social behavior that has long fascinated biologists, schooling behavior. We performed quantitative trait locus (QTL) analysis of schooling in an F2 intercross between strongly schooling marine and weakly schooling benthic sticklebacks (Gasterosteus aculeatus) and found that distinct genetic modules control different aspects of schooling behavior. Two key components of the behavior, tendency to school and body position when schooling, are uncorrelated in hybrids and map to different genomic regions. Our results further point to a genetic link between one behavioral component, schooling position, and variation in the neurosensory lateral line.

Sex Chromosome Turnover Contributes to Genomic Divergence between Incipient Stickleback Species

Sex chromosomes turn over rapidly in some taxonomic groups, where closely related species have different sex chromosomes. Although there are many examples of sex chromosome turnover, we know little about the functional roles of sex chromosome turnover in phenotypic diversification and genomic evolution. The sympatric pair of Japanese threespine stickleback (Gasterosteus aculeatus) provides an excellent system to address these questions: the Japan Sea species has a neo-sex chromosome system resulting from a fusion between an ancestral Y chromosome and an autosome, while the sympatric Pacific Ocean species has a simple XY sex chromosome system. Furthermore, previous quantitative trait locus (QTL) mapping demonstrated that the Japan Sea neo-X chromosome contributes to phenotypic divergence and reproductive isolation between these sympatric species. To investigate the genomic basis for the accumulation of genes important for speciation on the neo-X chromosome, we conducted whole genome sequencing of males and females of both the Japan Sea and the Pacific Ocean species. No substantial degeneration has yet occurred on the neo-Y chromosome, but the nucleotide sequence of the neo-X and the neo-Y has started to diverge, particularly at regions near the fusion. The neo-sex chromosomes also harbor an excess of genes with sex-biased expression. Furthermore, genes on the neo-X chromosome showed higher non-synonymous substitution rates than autosomal genes in the Japan Sea lineage. Genomic regions of higher sequence divergence between species, genes with divergent expression between species, and QTL for inter-species phenotypic differences were found not only at the regions near the fusion site, but also at other regions along the neo-X chromosome. Neo-sex chromosomes can therefore accumulate substitutions causing species differences even in the absence of substantial neo-Y degeneration.

Heritable Differences in Schooling Behavior among Threespine Stickleback Populations Revealed by a Novel Assay

Identifying the proximate and ultimate mechanisms of social behavior remains a major goal of behavioral biology. In particular, the complex social interactions mediating schooling behavior have long fascinated biologists, leading to theoretical and empirical investigations that have focused on schooling as a group-level phenomenon. However, methods to examine the behavior of individual fish within a school are needed in order to investigate the mechanisms that underlie both the performance and the evolution of schooling behavior. We have developed a technique to quantify the schooling behavior of an individual in standardized but easily manipulated social circumstances. Using our model school assay, we show that threespine sticklebacks (Gasterosteus aculeatus) from alternative habitats differ in behavior when tested in identical social circumstances. Not only do marine sticklebacks show increased association with the model school relative to freshwater benthic sticklebacks, they also display a greater degree of parallel swimming with the models. Taken together, these data indicate that marine sticklebacks exhibit a stronger tendency to school than benthic sticklebacks. We demonstrate that these population-level differences in schooling tendency are heritable and are shared by individuals within a population even when they have experienced mixed-population housing conditions. Finally, we begin to explore the stimuli that elicit schooling behavior in these populations. Our data suggest that the difference in schooling tendency between marine and benthic sticklebacks is accompanied by differential preferences for social vs. non-social and moving vs. stationary shelter options. Our study thus provides novel insights into the evolution of schooling behavior, as well as a new experimental approach to investigate the genetic and neural mechanisms that underlie this complex social behavior.

THE CONTRIBUTION OF FEMALE MEIOTIC DRIVE TO THE EVOLUTION OF NEO-SEX CHROMOSOMES

Sex chromosomes undergo rapid turnover in certain taxonomic groups. One of the mechanisms of sex chromosome turnover involves fusions between sex chromosomes and autosomes. Sexual antagonism, heterozygote advantage, and genetic drift have been proposed as the drivers for the fixation of this evolutionary event. However, all empirical patterns of the prevalence of multiple sex chromosome systems across different taxa cannot be simply explained by these three mechanisms. In this study, we propose that female meiotic drive may contribute to the evolution of neo‐sex chromosomes. The results of this study showed that in mammals, the XY1Y2 sex chromosome system is more prevalent in species with karyotypes of more biarmed chromosomes, whereas the X1X2Y sex chromosome system is more prevalent in species with predominantly acrocentric chromosomes. In species where biarmed chromosomes are favored by female meiotic drive, X‐autosome fusions (XY1Y2 sex chromosome system) will be also favored by female meiotic drive. In contrast, in species with more acrocentric chromosomes, Y‐autosome fusions (X1X2Y sex chromosome system) will be favored just because of the biased mutation rate toward chromosomal fusions. Further consideration should be given to female meiotic drive as a mechanism in the fixation of neo‐sex chromosomes.

Genetic basis for variation in salinity tolerance between stickleback ecotypes.

Adaptation to different salinities can drive and maintain divergence between populations of aquatic organisms. Anadromous and stream ecotypes of threespine stickleback (Gasterosteus aculeatus) are an excellent model to explore the genetic mechanisms underlying osmoregulation divergence. Using a parapatric pair of anadromous and stream stickleback ecotypes, we employed an integrated genomic approach to identify candidate genes important for adaptation to different salinity environments. Quantitative trait loci (QTL) mapping of plasma sodium concentrations under a seawater challenge experiment identified a significant QTL on chromosome 16. To identify candidate genes within this QTL, we first conducted RNA‐seq and microarray analysis on gill tissue to find ecotypic differences in gene expression that were associated with plasma Na+ levels. This resulted in the identification of ten candidate genes. Quantitative PCR analysis on gill tissue of additional Japanese stickleback populations revealed that the majority of the candidate genes showed parallel divergence in expression levels. Second, we conducted whole‐genome sequencing and found five genes that are predicted to have functionally important amino acid substitutions. Finally, we conducted genome scan analysis and found that eight of these candidate genes were located in genomic islands of high differentiation, suggesting that they may be under divergent selection. The candidate genes included those involved in ATP synthesis and hormonal signalling, whose expression or amino acid changes may underlie the variation in salinity tolerance. Further functional molecular analysis of these genes will reveal the causative genetic and genomic changes underlying divergent adaptation.

RNA sequencing reveals small RNAs differentially expressed between incipient Japanese threespine sticklebacks

BackgroundNon-coding small RNAs, ranging from 20 to 30 nucleotides in length, mediate the regulation of gene expression and play important roles in many biological processes. One class of small RNAs, microRNAs (miRNAs), are highly conserved across taxa and mediate the regulation of the chromatin state and the post-transcriptional regulation of messenger RNA (mRNA). Another class of small RNAs is the Piwi-interacting RNAs, which play important roles in the silencing of transposons and other functional genes. Although the biological functions of the different small RNAs have been elucidated in several laboratory animals, little is known regarding naturally occurring variation in small RNA transcriptomes among closely related species.ResultsWe employed next-generation sequencing technology to compare the expression profiles of brain small RNAs between sympatric species of the Japanese threespine stickleback (Gasterosteus aculeatus). We identified several small RNAs that were differentially expressed between sympatric Pacific Ocean and Japan Sea sticklebacks. Potential targets of several small RNAs were identified as repetitive sequences. Female-biased miRNA expression from the old X chromosome was also observed, and it was attributed to the degeneration of the Y chromosome.ConclusionsOur results suggest that expression patterns of small RNA can differ between incipient species and may be a potential mechanism underlying differential mRNA expression and transposon activity.

Different contributions of local‐ and distant‐regulatory changes to transcriptome divergence between stickleback ecotypes

Differential gene expression can play an important role in phenotypic evolution and divergent adaptation. Although differential gene expression can be caused by both local‐ and distant‐regulatory changes, we know little about their relative contribution to transcriptome evolution in natural populations. Here, we conducted expression quantitative trait loci (eQTL) analysis to investigate the genetic architecture underlying transcriptome divergence between marine and stream ecotypes of threespine sticklebacks (Gasterosteus aculeatus). We identified both local and distant eQTLs, some of which constitute hotspots, regions with a disproportionate number of significant eQTLs relative to the genomic background. The majority of local eQTLs including those in the hotspots caused expression changes consistent with the direction of transcriptomic divergence between ecotypes. Genome scan analysis showed that many local eQTLs overlapped with genomic regions of high differentiation. In contrast, nearly half of the distant eQTLs including those in the hotspots caused opposite expression changes, and few overlapped with regions of high differentiation, indicating that distant eQTLs may act as a constraint of transcriptome evolution. Finally, a comparison between two salinity conditions revealed that nearly half of eQTL hotspots were environment specific, suggesting that analysis of genetic architecture in multiple conditions is essential for predicting response to selection.

Ontogenetic stage‐specific quantitative trait loci contribute to divergence in developmental trajectories of sexually dimorphic fins between medaka populations

Sexual dimorphism can evolve when males and females differ in phenotypic optima. Genetic constraints can, however, limit the evolution of sexual dimorphism. One possible constraint is derived from alleles expressed in both sexes. Because males and females share most of their genome, shared alleles with different fitness effects between sexes are faced with intralocus sexual conflict. Another potential constraint is derived from genetic correlations between developmental stages. Sexually dimorphic traits are often favoured at adult stages, but selected against as juvenile, so developmental decoupling of traits between ontogenetic stages may be necessary for the evolution of sexual dimorphism in adults. Resolving intralocus conflicts between sexes and ages is therefore a key to the evolution of age‐specific expression of sexual dimorphism. We investigated the genetic architecture of divergence in the ontogeny of sexual dimorphism between two populations of the Japanese medaka (Oryzias latipes) that differ in the magnitude of dimorphism in anal and dorsal fin length. Quantitative trait loci (QTL) mapping revealed that few QTL had consistent effects throughout ontogenetic stages and the majority of QTL change the sizes and directions of effects on fin growth rates during ontogeny. We also found that most QTL were sex‐specific, suggesting that intralocus sexual conflict is almost resolved. Our results indicate that sex‐ and age‐specific QTL enable the populations to achieve optimal developmental trajectories of sexually dimorphic traits in response to complex natural and sexual selection.

Accumulation of Deleterious Mutations on the Neo-Y Chromosome of Japan Sea Stickleback (Gasterosteus nipponicus)

Degeneration of Y chromosomes is a common evolutionary path of XY sex chromosome systems. Recent genomic studies in flies and plants have revealed that even young neo-sex chromosomes with the age of a few million years show signs of Y degeneration, such as the accumulation of nonsense and frameshift mutations. However, it remains unclear whether neo-Y chromosomes also show rapid degeneration in fishes, which often have homomorphic sex chromosomes. Here, we investigated whether a neo-Y chromosome of Japan Sea stickleback (Gasterosteus nipponicus), which was formed by a Y-autosome fusion within the last 2 million years, accumulates deleterious mutations. Our previous genomic analyses did not detect excess nonsense and frameshift mutations on the Japan Sea stickleback neo-Y. In the present study, we found that the nonrecombining region of the neo-Y near the fusion end has accumulated nonsynonymous mutations altering amino acids of evolutionarily highly conserved residues. Enrichment of gene ontology terms related to protein phosphorylation and cellular protein modification process was found in the genes with potentially deleterious mutations on the neo-Y. These results suggest that the neo-Y of the Japan Sea stickleback has already accumulated mutations that may impair protein functions.

Contribution of gene flow to the evolution of recombination suppression in sex chromosomes

Polymorphism of alleles that benefit one sex but harm the other (sexually antagonistic alleles) generates selective pressures for reduced recombination between themselves and sex-determination loci. Such polymorphism can be maintained within a population when selection coefficients are sufficiently balanced between males and females. However, if regulatory mutations restrict gene expression only to one sex, these alleles become neutral in the other sex and easily fixed within a population, removing the selective pressures for recombination suppression in sex chromosomes. When there is spatial variation in selection regimes, however, alleles that are deleterious in one sex and neutral in the other can be maintained in other neighboring populations and gene flow may continuously supply deleterious alleles. We hypothesized that this maintenance of genetic variation may promote the establishment of recombination suppression in sex chromosomes even in cases where selection is limited to one sex. Using individual-based simulations, we show that spatial variation in male-limited selection and gene flow can promote the establishment of Y-autosome fusions, a special case of recombination suppression in sex chromosomes. This can be explained by the fact that fused Y-chromosomes that capture alleles that are beneficial for local males have a higher mean fitness compared to unfused Y chromosomes in the presence of deleterious gene flow. We also simulated the case of sex-concordant selection and found that gene flow of alleles that are deleterious in both sexes did not substantially increase the establishment rates of Y-autosome fusions across the parameter space examined. This can be accounted for by the fact that foreign alleles that are deleterious in both sexes can be efficiently removed from the population compared to alleles that are neutral in females. These results indicate that how gene flow affects the establishment rates of Y-autosome fusions depends largely on selection regimes. Spatial variation in sex-specific selection and gene flow should be appreciated as a factor affecting sex chromosome evolution.