Genome-wide analysis of histone modifications can contribute to the identification of candidate cis-regulatory regions in the threespine stickleback fish.

BACKGROUND: Cis-regulatory mutations often underlie phenotypic evolution. However, because identifying the locations of promoters and enhancers in non-coding regions is challenging, we have fewer examples of identified causative cis-regulatory mutations that underlie naturally occurring phenotypic variations than of causative amino acid-altering mutations. Because cis-regulatory elements have epigenetic marks of specific histone modifications, we can detect cis-regulatory elements by mapping and analyzing them. Here, we investigated histone modifications and chromatin accessibility with cleavage under targets and tagmentation (CUT&Tag) and assay for transposase-accessible chromatin-sequencing (ATAC-seq). RESULTS: Using the threespine stickleback (Gasterosteus aculeatus) as a model, we confirmed that the genes for which nearby regions showed active marks, such as H3K4me1, H3K4me3, and high chromatin accessibility, were highly expressed. In contrast, the expression levels of genes for which nearby regions showed repressive marks, such as H3K27me3, were reduced, suggesting that our chromatin analysis protocols overall worked well. Genomic regions with peaks of histone modifications showed higher nucleotide diversity within and between populations. By comparing gene expression in the gills of the marine and stream ecotypes, we identified several insertions and deletions (indels) with transposable element fragments in the candidate cis-regulatory regions. CONCLUSIONS: Thus, mapping and analyzing histone modifications can help identify cis-regulatory elements and accelerate the identification of causative mutations in the non-coding regions underlying naturally occurring phenotypic variations.

Tempo and mode in karyotype evolution revealed by a probabilistic model incorporating both chromosome number and morphology.

Karyotype, including the chromosome and arm numbers, is a fundamental genetic characteristic of all organisms and has long been used as a species-diagnostic character. Additionally, karyotype evolution plays an important role in divergent adaptation and speciation. Centric fusion and fission change chromosome numbers, whereas the intra-chromosomal movement of the centromere, such as pericentric inversion, changes arm numbers. A probabilistic model simultaneously incorporating both chromosome and arm numbers has not been established. Here, we built a probabilistic model of karyotype evolution based on the "karyograph", which treats karyotype evolution as a walk on the two-dimensional space representing the chromosome and arm numbers. This model enables analysis of the stationary distribution with a stable karyotype for any given parameter. After evaluating their performance using simulated data, we applied our model to two large taxonomic groups of fish, Eurypterygii and series Otophysi, to perform maximum likelihood estimation of the transition rates and reconstruct the evolutionary history of karyotypes. The two taxa significantly differed in the evolution of arm number. The inclusion of speciation and extinction rates demonstrated possibly high extinction rates in species with karyotypes other than the most typical karyotype in both groups. Finally, we made a model including polyploidization rates and applied it to a small plant group. Thus, the use of this probabilistic model can contribute to a better understanding of tempo and mode in karyotype evolution and its possible role in speciation and extinction.

A Cryptic Sex-Linked Locus Revealed by the Elimination of a Master Sex-Determining Locus in Medaka Fish.

AbstractSex chromosomes rapidly turn over in several taxonomic groups. Sex chromosome turnover is generally thought to start with the appearance of a new sex-determining gene on an autosome while an old sex-determining gene still exists, followed by the fixation of the new one. However, we do not know how prevalent the transient state is, where multiple sex-determining loci coexist within natural populations. Here, we removed a Y chromosome with a master male-determining gene DMY from medaka fish using high temperature-induced sex-reversed males. After four generations, the genomic characteristics of a sex chromosome were found on one chromosome, which was an autosome in the original population. Thus, the elimination of a master sex-determining locus can reveal a cryptic locus with a possible sex-determining effect, which can be the seed for sex chromosome turnover. Our results suggest that populations that seem to have a single-locus XY system may have other chromosomal regions with sex-determining effects. In conclusion, the coexistence of multiple sex-determining genes in a natural population may be more prevalent than previously thought. Experimental elimination of a master sex-determining locus may serve as a promising method for finding a locus that can be a protosex chromosome.

Repositioning of centromere-associated repeats during karyotype evolution in Oryzias fishes.

The karyotype, which is the number and shape of chromosomes, is a fundamental characteristic of all eukaryotes. Karyotypic changes play an important role in many aspects of evolutionary processes, including speciation. In organisms with monocentric chromosomes, it was previously thought that chromosome number changes were mainly caused by centric fusions and fissions, whereas chromosome shape changes, that is, changes in arm numbers, were mainly due to pericentric inversions. However, recent genomic and cytogenetic studies have revealed examples of alternative cases, such as tandem fusions and centromere repositioning, found in the karyotypic changes within and between species. Here, we employed comparative genomic approaches to investigate whether centromere repositioning occurred during karyotype evolution in medaka fishes. In the medaka family (Adrianichthyidae), the three phylogenetic groups differed substantially in their karyotypes. The Oryzias latipes species group has larger numbers of chromosome arms than the other groups, with most chromosomes being metacentric. The O. javanicus species group has similar numbers of chromosomes to the O. latipes species group, but smaller arm numbers, with most chromosomes being acrocentric. The O. celebensis species group has fewer chromosomes than the other two groups and several large metacentric chromosomes that were likely formed by chromosomal fusions. By comparing the genome assemblies of O. latipes, O. javanicus, and O. celebensis, we found that repositioning of centromere-associated repeats might be more common than simple pericentric inversion. Our results demonstrated that centromere repositioning may play a more important role in karyotype evolution than previously appreciated.

Multiple colonizations and hybridization of a freshwater fish group on a satellite island of Sulawesi.

Repeated colonizations and resultant hybridization may increase lineage diversity on an island if introgression occurs only in a portion of the indigenous island lineage. Therefore, to precisely understand how island biodiversity was shaped, it is essential to reconstruct the history of secondary colonization and resultant hybridization both in time and space. In this study, we reconstructed the history of multiple colonizations of the Oryzias woworae species group, a freshwater fish group of the family Adrianichthyidae, from Sulawesi Island to its southeast satellite island, Muna Island. Phylogenetic and species tree analyses using genome-wide single-nucleotide polymorphisms revealed that all local populations on Muna Island were monophyletic, but that there were several genetically distinct lineages within the island. Population structure and phylogenetic network analyses demonstrated that colonization of this island occurred more than once, and that secondary colonization and resultant introgressive hybridization occurred only in one local population on the island. The spatially heterogeneous introgression induced by the multiple colonizations were also supported by differential admixture analyses. In addition, the differential admixture analyses detected reverse colonization from Muna Island to the Sulawesi mainland. Coalescence-based demographic inference estimated that these mutual colonizations occurred during the middle to late Quaternary period, during which sea level repeatedly declined; this indicates that the colonizations occurred via land bridges. We conclude that these mutual colonizations between Muna Island and the Sulawesi mainland, and the resultant spatially heterogeneous introgression shaped the current biodiversity of this species group in this area.

Genetic basis for the evolution of pelvic-fin brooding, a new mode of reproduction, in a Sulawesian fish.

Modes of reproduction in animals are diverse, with different modes having evolved independently in multiple lineages across a variety of taxa. However, an understanding of the genomic change driving the transition between different modes of reproduction is limited. Several ricefishes (Adrianichthyidae) on the island of Sulawesi have a unique mode of reproduction called "pelvic-fin brooding," wherein females carry externally fertilized eggs until hatching using their pelvic fins. Phylogenomic analysis demonstrated pelvic-fin brooders to have evolved at least twice in two distant clades of the Adrianichthyidae. We investigated the genetic architecture of the evolution of this unique mode of reproduction. Morphological analyses and laboratory observations revealed that females of pelvic-fin brooders have longer pelvic fins and a deeper abdominal concavity, and that they can carry an egg clutch for longer than nonbrooding adrianichthyids, suggesting that these traits play important roles in this reproductive mode. Quantitative trait locus mapping using a cross between a pelvic-fin brooder Oryzias eversi and a nonbrooding O. dopingdopingensis reveals different traits involved in pelvic-fin brooding to be controlled by different loci on different chromosomes. Genomic analyses of admixture detected no signatures of introgression between two lineages with pelvic-fin brooders, indicating that introgression is unlikely to be responsible for repeated evolution of pelvic-fin brooding. These findings suggest that multiple independent mutations may have contributed to the convergent evolution of this novel mode of reproduction.

Diversity of sex chromosomes in Sulawesian medaka fishes.

Recent genetic and genomic studies have revealed tremendous diversity in sex chromosomes across diverse taxa. Closely related species with different sex chromosomes provide us excellent opportunities to investigate the driving forces and the consequences of sex chromosome turnover. In the present study, we investigated the diversity of sex chromosomes of 13 Oryzias species from Sulawesi, Indonesia, which diversified during the last 4.86 million years. Using pooled sequencing, we found sex chromosomes in nine species that all had XY systems, with a species being possibly modified by multiple loci. Seven species (O. woworae, O. asinua, O. wolasi, O. matanensis, O. celebensis, O. hadiatyae, and O. dopingdopingensis) share linkage group (LG) 24 as sex chromosomes; however, they differed in the length and magnitude of sequence divergence between the X and Y chromosomes. The sex chromosome of O. eversi was LG4, which has not been reported as a sex chromosome in any other medaka species. In O. sarasinorum, LG16 and LG22 are associated with sex. Although LG16 was found to be sex-linked in another medaka species previously examined, the sex-determining regions did not overlap. No significant signatures for sex chromosomes were identified in the other four species (O. marmoratus, O. nigrimas, O. nebulosus, and O. orthognathus). Frequent turnovers and the great diversity of the sex chromosomes will make Sulawesian medaka species a model system for investigating the driving forces and consequences of sex chromosome turnover.

Ancient Admixture in Freshwater Halfbeaks of the Genus Nomorhamphus in Southeast Sulawesi.

Freshwater halfbeaks of the genus Nomorhamphus (Zenarchopteridae) uniquely diversified on Sulawesi Island, where tectonic movements have been very active since the Pliocene. Most species of this genus have quite limited distributions, which indicates that geographic isolations have contributed to their diversification. In this study, we demonstrated that secondary contacts and resultant admixtures between long-isolated species/populations may have also been important. We found that the mitochondrial phylogeny of a group of Nomorhamphus in Southeast Sulawesi was discordant with the nuclear phylogeny. Most notably, individuals in the upper and lower streams of the Moramo River, a small river in this region, clustered with each other in the mitochondrial phylogeny but not in the nuclear phylogeny; in the latter, the lower-stream individuals formed a clade with individuals in the Anduna River, a different river with no present water connection to the Moramo River. Phylogenetic network and population structure analyses using genomic data obtained from RNA-seq revealed that the lower-stream Moramo population admixed with the upper-stream Moramo lineage in ancient times. These findings indicate that the observed mito-nuclear discordance was caused by mitochondrial introgression and not incomplete lineage sorting. The phylogenetic network also revealed several other admixtures between ancient lineages. Repeated admixtures were also evidenced by topological incongruence in population trees estimated using the RNA-seq data. We propose that activities of many fault systems dissecting Southeast Sulawesi caused repeated secondary contact.

The evolutionary ecology of fatty-acid variation: Implications for consumer adaptation and diversification.

The nutritional diversity of resources can affect the adaptive evolution of consumer metabolism and consumer diversification. The omega-3 long-chain polyunsaturated fatty acids eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) have a high potential to affect consumer fitness, through their widespread effects on reproduction, growth and survival. However, few studies consider the evolution of fatty acid metabolism within an ecological context. In this review, we first document the extensive diversity in both primary producer and consumer fatty acid distributions amongst major ecosystems, between habitats and amongst species within habitats. We highlight some of the key nutritional contrasts that can shape behavioural and/or metabolic adaptation in consumers, discussing how consumers can evolve in response to the spatial, seasonal and community-level variation of resource quality. We propose a hierarchical trait-based approach for studying the evolution of consumers' metabolic networks and review the evolutionary genetic mechanisms underpinning consumer adaptation to EPA and DHA distributions. In doing so, we consider how the metabolic traits of consumers are hierarchically structured, from cell membrane function to maternal investment, and have strongly environment-dependent expression. Finally, we conclude with an outlook on how studying the metabolic adaptation of consumers within the context of nutritional landscapes can open up new opportunities for understanding evolutionary diversification.

Multiple paths to the same destination: Influence of gene flow on convergent evolution.

Elucidation of the genetic mechanisms of convergent evolution, the evolution of similar or the same phenotypes in phylogenetically independent lineages, helps predict how populations will respond to the same selective pressures. Convergent evolution can be caused by either the fixation of identical-by-descent alleles, independent mutations at the same gene, or mutations in different genes controlling the same trait. To what extent does the fixation of identical-by-descent alleles lead to convergent evolution in isolated populations where inflow of adaptive alleles from other populations is limited? In a From the Cover article in this issue of Molecular Ecology, Kemppainen et al. (2021) compared the genetic basis for the reduction of pelvic structures in three isolated freshwater populations of nine-spined stickleback (Pungitius pungitius) from Northern Europe. The authors used quantitative trait loci (QTL) mapping to reveal that the pelvic reduction in these three populations was caused by mutations at different genetic loci. In contrast to studies in three-spined stickleback (Gasterosteus aculeatus), where independently derived Pitx1 mutations were shown to be responsible for plate reduction across multiple freshwater populations, Kemppainen et al. (2021) found Pitx1 to be the candidate causative gene for only one population of P. pungitius. This study highlights the importance of genetic studies of convergent evolution, not only in the presence of gene flow but also in its absence for a better understanding of the genetic architecture of convergent evolution.

Patterns of genomic divergence and introgression between Japanese stickleback species with overlapping breeding habitats.

With only a few absolute geographic barriers in marine environments, the factors maintaining reproductive isolation among marine organisms remain elusive. However, spatial structuring in breeding habitat can contribute to reproductive isolation. This is particularly important for marine organisms that migrate to use fresh- or brackish water environments to breed. The Japanese Gasterosteus stickleback species, the Pacific Ocean three-spined stickleback (G. aculeatus) and the Japan Sea stickleback (G. nipponicus) overwinter in the sea, but migrate to rivers for spawning. Although they co-occur at several locations across the Japanese islands, they are reproductively isolated. Our previous studies in Bekanbeushi River showed that the Japan Sea stickleback spawns in the estuary, while the Pacific Ocean stickleback mainly spawns further upstream in freshwater. Overall genomic divergence was very high with many interspersed regions of introgression. Here, we investigated genomic divergence and introgression between the sympatric species in the much shorter Tokotan River, where they share spawning sites. The levels of genome-wide divergence were reduced and introgression was increased, suggesting that habitat isolation substantially contributes to a reduction in gene flow. We also found that genomic regions of introgression were largely shared between the two systems. Furthermore, some regions of introgression were located near loci with a heterozygote advantage for juvenile survival. Taken together, introgression may be partially driven by adaptation in this system. Although, the two species remain clearly genetically differentiated. Regions with low recombination rates showed especially low introgression. Speciation reversal is therefore likely prevented by barriers other than habitat isolation.

Species divergence and repeated ancient hybridization in a Sulawesian lake system.

An increasing volume of empirical studies demonstrated that hybridization between distant lineages may have promoted speciation in various taxa. However, the timing, extent and direction of introgressive hybridization remain unknown in many cases. Here, we report a possible case in which repeated hybridization promoted divergence of Oryzias ricefishes (Adrianichthyidae) on Sulawesi, an island of Wallacea. Four Oryzias species are endemic to the Malili Lake system in central Sulawesi, which is composed of five tectonic lakes; of these, one lake is inhabited by two species. Morphological and population genomic analyses of genome-wide single-nucleotide polymorphisms revealed that these two sympatric species are phylogenetically sister to but substantially reproductively isolated from each other. Analyses of admixture and comparison of demographic models revealed that the two sympatric species experienced several substantial introgressions from outgroup populations that probably occurred soon after they had secondary contact with each other in the lake. However, the ratio of migrants from the outgroups was estimated to be different between the two species, which is consistent with the hypothesis that these introgressions aided their divergence or prevented them from forming a hybrid swarm. Repeated lake fragmentations and fusions may have promoted diversification of this freshwater fish species complex that is endemic to this ancient lake system.

Copy number variation of a fatty acid desaturase gene Fads2 associated with ecological divergence in freshwater stickleback populations.

Fitness of aquatic animals can be limited by the scarcity of nutrients such as long-chain polyunsaturated fatty acids, especially docosahexaenoic acid (DHA). DHA availability from diet varies among aquatic habitats, imposing different selective pressures on resident animals to optimize DHA acquisition and synthesis. For example, DHA is generally poor in freshwater ecosystems compared to marine ecosystems. Our previous work revealed that, relative to marine fishes, several freshwater fishes evolved higher copy numbers of the fatty acid desaturase2 (Fads2) gene, which encodes essential enzymes for DHA biosynthesis, likely compensating for the limited availability of DHA in freshwater. Here, we demonstrate that Fads2 copy number also varies between freshwater sticklebacks inhabiting lakes and streams with stream fish having higher Fads2 copy number. Additionally, populations with benthic-like morphology possessed higher Fads2 copy number than those with planktivore-like morphology. This may be because benthic-like fish mainly feed on DHA-deficient prey such as macroinvertebrates whereas planktivore-like fish forage more regularly on DHA-rich prey, like copepods. Our results suggest that Fads2 copy number variation arises from ecological divergence not only between organisms exploiting marine and freshwater habitats but also between freshwater organisms exploiting divergent resources.

Mesozoic origin and 'out-of-India' radiation of ricefishes (Adrianichthyidae).

The Indian subcontinent has an origin geologically different from Eurasia, but many terrestrial animal and plant species on it have congeneric or sister species in other parts of Asia, especially in the Southeast. This faunal and floral similarity between India and Southeast Asia is explained by either of the two biogeographic scenarios, 'into-India' or 'out-of-India'. Phylogenies based on complete mitochondrial genomes and five nuclear genes were undertaken for ricefishes (Adrianichthyidae) to examine which of these two biogeographic scenarios fits better. We found that Oryzias setnai, the only adrianichthyid distributed in and endemic to the Western Ghats, a mountain range running parallel to the western coast of the Indian subcontinent, is sister to all other adrianichthyids from eastern India and Southeast-East Asia. Divergence time estimates and ancestral area reconstructions reveal that this western Indian species diverged in the late Mesozoic during the northward drift of the Indian subcontinent. These findings indicate that adrianichthyids dispersed eastward 'out-of-India' after the collision of the Indian subcontinent with Eurasia, and subsequently diversified in Southeast-East Asia. A review of geographic distributions of 'out-of-India' taxa reveals that they may have largely fuelled or modified the biodiversity of Eurasia.

The genomic landscape at a late stage of stickleback speciation: High genomic divergence interspersed by small localized regions of introgression.

Speciation is a continuous process and analysis of species pairs at different stages of divergence provides insight into how it unfolds. Previous genomic studies on young species pairs have revealed peaks of divergence and heterogeneous genomic differentiation. Yet less known is how localised peaks of differentiation progress to genome-wide divergence during the later stages of speciation in the presence of persistent gene flow. Spanning the speciation continuum, stickleback species pairs are ideal for investigating how genomic divergence builds up during speciation. However, attention has largely focused on young postglacial species pairs, with little knowledge of the genomic signatures of divergence and introgression in older stickleback systems. The Japanese stickleback species pair, composed of the Pacific Ocean three-spined stickleback (Gasterosteus aculeatus) and the Japan Sea stickleback (G. nipponicus), which co-occur in the Japanese islands, is at a late stage of speciation. Divergence likely started well before the end of the last glacial period and crosses between Japan Sea females and Pacific Ocean males result in hybrid male sterility. Here we use coalescent analyses and Approximate Bayesian Computation to show that the two species split approximately 0.68-1 million years ago but that they have continued to exchange genes at a low rate throughout divergence. Population genomic data revealed that, despite gene flow, a high level of genomic differentiation is maintained across the majority of the genome. However, we identified multiple, small regions of introgression, occurring mainly in areas of low recombination rate. Our results demonstrate that a high level of genome-wide divergence can establish in the face of persistent introgression and that gene flow can be localized to small genomic regions at the later stages of speciation with gene flow.

Multiple waves of freshwater colonization of the three-spined stickleback in the Japanese Archipelago.

BACKGROUND: The three-spined stickleback (Gasterosteus aculeatus) is a remarkable system to study the genetic mechanisms underlying parallel evolution during the transition from marine to freshwater habitats. Although the majority of previous studies on the parallel evolution of sticklebacks have mainly focused on postglacial freshwater populations in the Pacific Northwest of North America and northern Europe, we recently use Japanese stickleback populations for investigating shared and unique features of adaptation and speciation between geographically distant populations. However, we currently lack a comprehensive phylogeny of the Japanese three-spined sticklebacks, despite the fact that a good phylogeny is essential for any evolutionary and ecological studies. Here, we conducted a phylogenomic analysis of the three-spined stickleback in the Japanese Archipelago. RESULTS: We found that freshwater colonization occurred in multiple waves, each of which may reflect different interglacial isolations. Some of the oldest freshwater populations from the central regions of the mainland of Japan (hariyo populations) were estimated to colonize freshwater approximately 170,000 years ago. The next wave of colonization likely occurred approximately 100,000 years ago. The inferred origins of several human-introduced populations showed that introduction occurred mainly from nearby habitats. We also found a new habitat of the three-spined stickleback sympatric with the Japan Sea stickleback (Gasterosteus nipponicus). CONCLUSIONS: These Japanese stickleback systems differ from those in the Pacific Northwest of North America and northern Europe in terms of divergence time and history. Stickleback populations in the Japanese Archipelago offer valuable opportunities to study diverse evolutionary processes in historical and contemporary timescales.

The Origin of a New Sex Chromosome by Introgression between Two Stickleback Fishes.

Introgression is increasingly recognized as a source of genetic diversity that fuels adaptation. Its role in the evolution of sex chromosomes, however, is not well known. Here, we confirm the hypothesis that the Y chromosome in the ninespine stickleback, Pungitius pungitius, was established by introgression from the Amur stickleback, P. sinensis. Using whole genome resequencing, we identified a large region of Chr 12 in P. pungitius that is diverged between males and females. Within but not outside of this region, several lines of evidence show that the Y chromosome of P. pungitius shares a most recent common ancestor not with the X chromosome, but with the homologous chromosome in P. sinensis. Accumulation of repetitive elements and gene expression changes on the new Y are consistent with a young sex chromosome in early stages of degeneration, but other hallmarks of Y chromosomes have not yet appeared. Our findings indicate that porous species boundaries can trigger rapid sex chromosome evolution.

Differences in the contributions of sex linkage and androgen regulation to sex-biased gene expression in juvenile and adult sticklebacks.

Different evolutionary interests between males and females can lead to the evolution of sexual dimorphism. However, intersex genetic correlations due to the shared genome can constrain the evolution of sexual dimorphism, resulting in intra-locus sexual conflict. One of the mechanisms resolving this conflict is sex linkage, which allows males and females to carry different alleles on sex chromosomes. Another is a regulatory mutation causing sex-biased gene expression, which is often mediated by gonadal steroids in vertebrates. How do these two mechanisms differ in the contributions to the resolution of intra-locus sexual conflict? The magnitude of sexual conflict often varies between the juvenile and adult stages. Because gonadal steroids change in titre during development, we hypothesized that gonadal steroids play a role in sexual dimorphism expression only at certain developmental stages, whereas sex linkage is more important for sexual dimorphism expressed throughout life. Our brain transcriptome analysis of juvenile and adult threespine sticklebacks showed that the majority of genes that were sex-biased in both stages were sex-linked. The relative contribution of androgen-dependent regulation to the sex-biased transcriptome increased and that of sex linkage declined in adults compared to juveniles. The magnitude of the sex differences was greater in sex-linked genes than androgen-responsive genes, suggesting that sex linkage is more effective than androgen regulation in the production of large sex differences in gene expression. Overall, our data are consistent with the hypothesis that sex linkage is effective in resolving sexual conflict throughout life, whereas androgen-dependent regulation can contribute to temporary resolution of sexual conflict.

Diversity in reproductive seasonality in the three-spined stickleback, Gasterosteus aculeatus.

The annual timing of reproduction is a key life history trait with a large effect on fitness. Populations often vary in the timing and duration of reproduction to adapt to different seasonality of ecological and environmental variables between habitats. However, little is known about the molecular genetic mechanisms underlying interpopulation variation in reproductive seasonality. Here, we demonstrate that the three-spined stickleback (Gasterosteus aculeatus) is a good model for molecular genetic analysis of variations in reproductive seasonality. We first compiled data on reproductive seasons of diverse ecotypes, covering marine-anadromous, lake and stream ecotypes, of three-spined stickleback inhabiting a wide range of latitudes. Our analysis showed that both ecotype and latitude significantly contribute to variation in reproductive seasons. Stream ecotypes tend to start breeding earlier and end later than other ecotypes. Populations from lower latitudes tend to start breeding earlier than those from higher latitudes in all three ecotypes. Additionally, stream ecotypes tend to have extended breeding seasons at lower latitudes than at higher latitudes, leading to nearly year-round reproduction in the most southern stream populations. A review of recent progress in our understanding of the physiological mechanisms underlying seasonal reproduction in the three-spined stickleback indicates that photoperiod is an important external cue that stimulates and/or suppresses reproduction in this species. Taking advantage of genomic tools available for this species, the three-spined stickleback will be a good model to investigate what kinds of genes and mutations underlie variations in the physiological signalling pathways that regulate reproduction in response to photoperiod.

Functional divergence of a heterochromatin-binding protein during stickleback speciation.

Intragenomic conflict, the conflict of interest between different genomic regions within an individual, is proposed as a mechanism driving both the rapid evolution of heterochromatin-related proteins and the establishment of intrinsic genomic incompatibility between species. Although molecular studies of laboratory model organisms have demonstrated the link between heterochromatin evolution and hybrid abnormalities, we know little about their link in natural systems. Previously, we showed that F1 hybrids between the Japan Sea stickleback and the Pacific Ocean stickleback show hybrid male sterility and found a region responsible for hybrid male sterility on the X chromosome, but did not identify any candidate genes. In this study, we first screened for genes rapidly evolving under positive selection during the speciation of Japanese sticklebacks to find genes possibly involved in intragenomic conflict. We found that the region responsible for hybrid male sterility contains a rapidly evolving gene encoding a heterochromatin-binding protein TRIM24B. We conducted biochemical experiments and showed that the binding affinity of TRIM24B to a heterochromatin mark found at centromeres and transposons, histone H4 lysine 20 trimethylation (H4K20me3), is reduced in the Japan Sea stickleback. In addition, mRNA expression levels of Trim24b were different between the Japan Sea and the Pacific Ocean testes. Further expression analysis of genes possibly in the TRIM24B-regulated pathway showed that some gypsy retrotransposons are overexpressed in the F1 hybrid testes. We, therefore, demonstrate that a heterochromatin-binding protein can evolve rapidly under positive selection and functionally diverge during stickleback speciation.