Plate reduction in southern Japanese freshwater populations of threespine stickleback (Gasterosteus aculeatus).

Adaptation to similar environments can lead to the evolution of similar phenotypes in phylogenetically independent lineages. However, the extent of parallel evolution often varies. Because such variations can be due to environmental heterogeneity among seemingly similar habitats, identification of the environmental factors that cause non-parallel patterns can provide valuable insight into the ecological factors associated with phenotypic diversification. Armor plate reduction in replicate freshwater populations of the threespine stickleback (Gasterosteus aculeatus) represents a well-known example of parallel evolution. Many freshwater populations in multiple regions of the Northern Hemisphere have reduced plate numbers, but not all freshwater populations exhibit plate reduction. In this study, we characterized plate number variation in Japanese freshwater populations and investigated the association between plate number and several abiotic environmental factors. We found that most freshwater populations have not reduced plate numbers in Japan. Plate reduction tends to occur in habitats with warmer winter temperatures at lower latitudes in Japan. In contrast, low dissolved calcium levels or water turbidity had no significant effects on plate reduction, although these were reported to be associated with plate reduction in Europe. Although our data are consistent with the hypothesis that winter temperatures are associated with plate reduction, further studies on the relationship between temperatures and fitness using sticklebacks with varying plate numbers are necessary to confirm this hypothesis and understand the factors causing variations in the extent of parallel evolution.

Gudgeon fish with and without genetically determined countershading coexist in heterogeneous littoral environments of an ancient lake.

Countershading, characterized by a darker dorsal surface and lighter ventral surface, is common among many animals. This dorsoventral pigment polarity is often thought to be adaptive coloration for camouflage. By contrast, noncountershaded (melanistic) morphs often occur within a species due to genetic color polymorphism in terrestrial animals. However, the polymorphism with either countershaded or melanistic morphs is poorly known in wild aquatic animals. This study explored the genetic nature of diverged color morphs of a lineage of gudgeon fish (genus Sarcocheilichthys) in the ancient Lake Biwa and propose this system as a novel model for testing hypotheses of functional aspects of countershading and its loss in aquatic environments. This system harbors two color morphs that have been treated taxonomically as separate species; Sarcocheilichthys variegatus microoculus which occurs throughout the littoral zone and Sarcocheilichthys biwaensis which occurs in and around rocky areas. First, we confirmed that the divergence of dorsoventral color patterns between the two morphs is under strict genetic control at the levels of chromatophore distribution and melanin-related gene expression under common garden rearing. The former morph displayed sharp countershading coloration, whereas the latter morph exhibited a strong tendency toward its loss. The crossing results indicated that this divergence was likely controlled by a single locus in a two-allele Mendelian inheritance pattern. Furthermore, our population genomic and genome-wide association study analyses detected no genome-wide divergence between the two morphs, except for one region near a locus that may be associated with the color divergence. Thus, these morphs are either in a state of intraspecific color polymorphism or two incipient species. Evolutionary forces underlying this polymorphism appear to be associated with heterogeneous littoral environments in this lake. Future ecological genomic research will provide insight into adaptive functions of this widespread coloration, including the eco-evolutionary drivers of its loss, in the aquatic world.

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.

Accumulation of Deleterious Mutations in Landlocked Threespine Stickleback Populations.

Colonization of new habitats often reduces population sizes and may result in the accumulation of deleterious mutations by genetic drift. Compared with the genomic basis for adaptation to new environments, genome-wide analysis of deleterious mutations in isolated populations remains limited. In the present study, we investigated the accumulation of deleterious mutations in five endangered freshwater populations of threespine stickleback (Gasterosteus aculeatus) in the central part of the mainland of Japan. Using whole-genome resequencing data, we first conducted phylogenomic analysis and confirmed at least two independent freshwater colonization events in the central mainland from ancestral marine ecotypes. Next, analyses of single nucleotide polymorphisms showed a substantial reduction of heterozygosity in freshwater populations compared with marine populations. Reduction in heterozygosity was more apparent at the center of each chromosome than the peripheries and on X chromosomes compared with autosomes. Third, bioinformatic analysis of deleterious mutations showed increased accumulation of putatively deleterious mutations in the landlocked freshwater populations compared with marine populations. For the majority of populations examined, the frequencies of putatively deleterious mutations were higher on X chromosomes than on autosomes. The interpopulation comparison indicated that the majority of putatively deleterious mutations may have accumulated independently. Thus, whole-genome resequencing of endangered populations can help to estimate the accumulation of deleterious mutations and inform us of which populations are the most severely endangered. Furthermore, analysis of variation among chromosomes can give insights into whether any particular chromosomes are likely to accumulate deleterious mutations.

Genomic and phenotypic consequences of two independent secondary contact zones between allopatric lineages of the anadromous ice goby Leucopsarion petersii.

Genetic and phenotypic analyses of independent secondary contact zones between certain pairs of divergent populations offer powerful opportunities to assess whether the consequences vary with different environmental backgrounds. Populations of the ice goby Leucopsarion petersii are distributed throughout the Japanese archipelago and comprise genetically and phenotypically divergent groups in the Japan Sea and the Pacific Ocean. In particular, populations in the Japan Sea have a larger body size and numbers of vertebrae than those in the Pacific Ocean. Herein, we performed integrated analyses of genotypes and phenotypes of two independent secondary contact zones and investigated their consequences. Population genetic analyses revealed asymmetric introgression of the mitochondrial genome of either lineage relative to little admixture of nuclear genomes in both secondary contact zones. On phenotype analyses, vertebral numbers were clearly explained by nuclear genomic ancestry in both secondary contact zones, whereas body size was not, suggesting that a little introgression of nuclear genes regulates body size. Actually, we observed biased introgression of a candidate gene, neuropeptide Y (NPY), which potentially controls body size in the ice goby. Moreover, the body size changes in the introgressed populations possibly affect the introgression patterns of mitochondrial genomes across these zones. Collectively, our results demonstrated that genomic and phenotypic consequences of secondary contact varied in marine variable environments.

A key metabolic gene for recurrent freshwater colonization and radiation in fishes.

Colonization of new ecological niches has triggered large adaptive radiations. Although some lineages have made use of such opportunities, not all do so. The factors causing this variation among lineages are largely unknown. Here, we show that deficiency in docosahexaenoic acid (DHA), an essential ω-3 fatty acid, can constrain freshwater colonization by marine fishes. Our genomic analyses revealed multiple independent duplications of the fatty acid desaturase gene Fads2 in stickleback lineages that subsequently colonized and radiated in freshwater habitats, but not in close relatives that failed to colonize. Transgenic manipulation of Fads2 in marine stickleback increased their ability to synthesize DHA and survive on DHA-deficient diets. Multiple freshwater ray-finned fishes also show a convergent increase in Fads2 copies, indicating its key role in freshwater colonization.

Genomic architecture of habitat-related divergence and signature of directional selection in the body shapes of Gnathopogon fishes.

Evolution of ecomorphologically relevant traits such as body shapes is important to colonize and persist in a novel environment. Habitat-related adaptive divergence of these traits is therefore common among animals. We studied the genomic architecture of habitat-related divergence in the body shape of Gnathopogon fishes, a novel example of lake-stream ecomorphological divergence, and tested for the action of directional selection on body shape differentiation. Compared to stream-dwelling Gnathopogon elongatus, the sister species Gnathopogon caerulescens, exclusively inhabiting a large ancient lake, had an elongated body, increased proportion of the caudal region and small head, which would be advantageous in the limnetic environment. Using an F2 interspecific cross between the two Gnathopogon species (195 individuals), quantitative trait locus (QTL) analysis with geometric morphometric quantification of body shape and restriction-site associated DNA sequencing-derived markers (1622 loci) identified 26 significant QTLs associated with the interspecific differences of body shape-related traits. These QTLs had small to moderate effects, supporting polygenic inheritance of the body shape-related traits. Each QTL was mostly located on different genomic regions, while colocalized QTLs were detected for some ecomorphologically relevant traits that are proxy of body and caudal peduncle depths, suggesting different degree of modularity among traits. The directions of the body shape QTLs were mostly consistent with the interspecific difference, and QTL sign test suggested a genetic signature of directional selection in the body shape divergence. Thus, we successfully elucidated the genomic architecture underlying the adaptive changes of the quantitative and complex morphological trait in a novel system.

A RAD-based linkage map and comparative genomics in the gudgeons (genus Gnathopogon, Cyprinidae).

BACKGROUND: The construction of linkage maps is a first step in exploring the genetic basis for adaptive phenotypic divergence in closely related species by quantitative trait locus (QTL) analysis. Linkage maps are also useful for comparative genomics in non-model organisms. Advances in genomics technologies make it more feasible than ever to study the genetics of adaptation in natural populations. Restriction-site associated DNA (RAD) sequencing in next-generation sequencers facilitates the development of many genetic markers and genotyping. We aimed to construct a linkage map of the gudgeons of the genus Gnathopogon (Cyprinidae) for comparative genomics with the zebrafish Danio rerio (a member of the same family as gudgeons) and for the future QTL analysis of the genetic architecture underlying adaptive phenotypic evolution of Gnathopogon. RESULTS: We constructed the first genetic linkage map of Gnathopogon using a 198 F2 interspecific cross between two closely related species in Japan: river-dwelling Gnathopogon elongatus and lake-dwelling Gnathopogon caerulescens. Based on 1,622 RAD-tag markers, a linkage map spanning 1,390.9 cM with 25 linkage groups and an average marker interval of 0.87 cM was constructed. We also identified a region involving female-specific transmission ratio distortion (TRD). Synteny and collinearity were extensively conserved between Gnathopogon and zebrafish. CONCLUSIONS: The dense SNP-based linkage map presented here provides a basis for future QTL analysis. It will also be useful for transferring genomic information from a "traditional" model fish species, zebrafish, to screen candidate genes underlying ecologically important traits of the gudgeons.

Molecular signatures of lineage-specific adaptive evolution in a unique sea basin: the example of an anadromous goby Leucopsarion petersii.

Climate changes on various time scales often shape genetic novelty and adaptive variation in many biotas. We explored molecular signatures of directional selection in populations of the ice goby Leucopsarion petersii inhabiting a unique sea basin, the Sea of Japan, where a wide variety of environments existed in the Pleistocene in relation to shifts in sea level by repeated glaciations. This species consisted of two historically allopatric lineages, the Japan Sea (JS) and Pacific Ocean (PO) lineages, and these have lived under contrasting marine environments that are expected to have imposed different selection regimes caused by past climatic and current oceanographic factors. We applied a limited genome-scan approach using seven candidate genes for phenotypic differences between two lineages in combination with 100 anonymous microsatellite loci. Neuropeptide Y (NPY) gene, which is an important regulator of food intake and potent orexigenic agent, and three anonymous microsatellites were identified as robust outliers, that is, candidate loci potentially under directional selection, by multiple divergence- and diversity-based outlier tests in comparisons focused on multiple populations of the JS vs. PO lineages. For these outlier loci, populations of the JS lineage had putative signals of selective sweeps. Additionally, real-time quantitative PCR analysis using fish reared in a common environment showed a higher expression level for NPY gene in the JS lineage. Thus, this study succeeded in identifying candidate genomic regions under selection across populations of the JS lineage and provided evidence for lineage-specific adaptive evolution in this unique sea basin.

Phylogeography and historical demography of the anadromous fish Leucopsarion petersii in relation to geological history and oceanography around the Japanese Archipelago.

Phylogeographical patterns of marine and diadromous organisms are often influenced by dynamic ocean histories. For example, the marine realm around the Japanese Archipelago is an interesting area for phylogeographical research because of the wide variation in the environments driven by repeated shifts in sea level in the Quaternary. We analysed mitochondrial cyt b gene and nuclear myh6 gene sequences for individuals collected from throughout the range of the anadromous fish Leucopsarion petersii to assess the lineage divergence, phylogeographical pattern and historical demography in relation to geological history and oceanographic features around the archipelago. Leucopsarion petersii has two major lineages (the Japan Sea and Pacific Ocean lineages), which diverged during the late-early to middle Pleistocene. Geographical distributions of the two lineages were closely related to the pathways of the two warm currents, the Tsushima Current and the Kuroshio Current, that flow past the archipelago. Evidence of introgressive hybridization between these lineages was found at two secondary contact zones. Demographic tests suggested that the Japan Sea and Pacific Ocean lineages carried the genetic signal of different historical demographic processes, and these signals are probably associated with differences in habitat stability during recent glacial periods. The Japan Sea lineage has a larger body-size and more vertebrae, probably in relation to severe habitat conditions through Pleistocene climatic oscillations. Thus, the two lineages have long independent evolutionary histories, and the phylogeographical structure and demography of this species have been influenced both by historical events and the present-day oceanography around the Japanese Archipelago.

PERMANENT GENETIC RESOURCES: Isolation and characterization of polymorphic trinucleotide and dinucleotide microsatellite loci for the ice goby, Leucopsarison petersii.

Eleven microsatellite markers were isolated and characterized for the ice goby, Leucopsarion petersii, from genomic libraries enriched for (ATG)(9) and (CA)(16) . Twenty individuals from a single population were used to screen polymorphism in these loci. The number of alleles per locus and observed heterozygosity ranged from four to 22 and from 0.35 to 1.00, respectively. All loci did not significantly deviated from Hardy-Weinberg equilibrium, and there was no evidence of linkage disequilibrium between all loci-pairs. These loci showed Mendelian inheritance in a full-sib family. The high level of polymorphism of these loci will be useful for studies of population genetics.