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 evidence supporting the existence of two distinct species in the genusGasterosteus around Japan

Synopsis The genetic and morphological features ofGasterosteus aculeatus were investigated for 29 populations around Japan. Allozyme analyses recognized two groups (Pacific Ocean group and Japan Sea group) that had distinct characteristic features, and showed high genetic differentiation between them (D = 0.482). The Pacific Ocean group had a wide range, from North America to Japan, along the Pacific coast. The distribution of the Japan Sea group was limited around the Sea of Japan and the Sea of Okhotsk. The distribution of these groups were found to be sympatric on the Pacific coast of Hokkaido Island, Japan. From this area, genetic analyses demonstrated that the sympatric populations of the two groups formed independent breeding stocks, and it is considered that the two groups were reproductively isolated from each other. Additionally, each group had distinctive morphological features of lateral plates and caudal keels in the sympatric area. These results suggested that these two groups of the threespine stickleback comprise different species and that the Japan Sea group is taxonomically distinguishable fromG. aculeatus.     

Distinct spawning migration patterns in sympatric Japan Sea and Pacific Ocean forms of threespine stickleback Gasterosteus aculeatus

Spawning migration patterns were compared between sympatric populations of the anadromous Japan Sea (JS) form and Pacific Ocean (PO) form of the threespine stickleback Gasterosteus aculeatus in the Lake Akkeshi–Bekanbeushi River system, eastern Hokkaido, Japan. In Akkeshi Bay and Lake Akkeshi, the JS form had longer collecting peak periods and more collection peaks than the PO form. Furthermore, although the PO form migrated upstream in freshwater habitats, the greater part of the JS form stayed in brackish waters before breeding. These findings suggest that the 2 forms have distinctly different spawning migration patterns and breeding grounds.     

Intraspecific differences in patterns of courtship behaviours between the Pacific Ocean and Japan Sea forms of the three-spined stickleback Gasterosteus aculeatus

The courtship behaviour of the Pacific Ocean and Japan Sea forms of the three-spined stickleback Gasterosteus aculeatus were studied in seven populations. The intraspecific difference in behavioural patterns, zigzag dance and weak dorsal pricking in the Pacific Ocean form and lateral display and vigorous dorsal pricking in the Japan Sea form, was clearly distinguishable regardless of the type of habitat the males lived in.     

Taxonomic review of the genus <Emphasis Type="Italic">Cottiusculus</Emphasis> (Cottoidei: Cottidae) with description of a new species from the Sea of Japan

A taxonomic review of the cottid genus Cottiusculus Jordan and Starks 1904 established three species, C. nihonkaiensis sp. nov., C. schmidti, and C. gonez. Cottiusculus nihonkaiensis sp. nov., which had been previously confused with C. schmidti, is described on the basis of 32 specimens (44.8–77.9 mm, SL) collected from the Sea of Japan. The new species is very similar to C. schmidti in having a curved barbless uppermost preopercular spine and the lateral line extending past the caudal fin base, but is distinguishable as follows: nasal spines simple or sometimes weakly bicuspid (vs. deeply bicuspid in C. schmidti); first dorsal fin not elongated in either males or females (vs. elongated in males); ventral lateral and lateral line cirri present (vs. absent). The former is known from the Sea of Japan coasts of Hokkaido, Honshu, Kyushu and the Korean Peninsula, and Volcano Bay, Hokkaido, and the latter from the Pacific coast of Tohoku District, Japan. Cottiusculus gonez, known from the Sea of Japan, Sea of Okhotsk and Pacific coast of Hokkaido, is characterized by having a simple nasal spine, the uppermost preopercular spine almost straight with two small cusps dorsally, posterior cusp barbed, and the lateral line almost reaching to the caudal fin base. A lectotype of C. gonez is designated here. Sequence differences in the cytochrome b gene among the above three species of Cottiusculus are also presented.     

Population structure of the sailfin sandfish, Arctoscopus japonicus (Trichodontidae), in the Sea of Japan

We analyzed patterns of genetic diversity in the sailfin sandfish (Arctoscopus japonicus), focusing on population subdivisions within the Sea of Japan. We observed 270 specimens from nine sampling sites in 1999–2000, i.e., seven sites in the Sea of Japan and two sites from the Pacific coast of Hokkaido. An additional site (30 specimens) was sampled from eastern Korea in the spawning season of 2004 for comparison. Forty haplotypes, compiled into three haplogroups (A–C), were detected based on the comparison of a 400-bp sequence of the anterior part of the mitochondrial control region. In accordance with previous hypotheses from morphological and molecular analyses, genetic discontinuity between the Sea of Japan and the Pacific coast of Hokkaido was conspicuous. Within the Sea of Japan, eight sampling sites were not genetically uniform, and most of the variations among sites were detected between eastern Korea [the “eastern Korea” (EK) population: distributed from the Korean Peninsula to Mishima, Yamaguchi Prefecture] and the other sites along the coast of Japan [the “western Japan” (WJ) population: from Oki Islands to western Hokkaido] (Φ _CT  = 0.096, P = 0.0183). The WJ population, having lower genetic variability, showed significant departure from neutrality, indicating influences through a recent population expansion. The period of the expansion can be estimated to have begun on the order of 10⁴ years ago. We consider that the present Japan Sea populations have been formed through the invasion of a small ancestral stock to the Sea of Japan and its population expansion during the last glacial period or later. On the other hand, we failed to detect distinct evidence of a population expansion in the EK population. Haplogroup C, detected in a high frequency in this population, was estimated to have mixed with haplogroup A after rapid differentiations of the latter. Therefore, the EK population, strongly influenced by such a mixture, might possess haplogroup C in a higher frequency and a different haplotype composition from the WJ population.     

Extensive Introgression of Mitochondrial DNA Found between two Genetically Divergent Forms of Threespine Stickleback, Gasterosteus Aculeatus, around Japan

The genetic structure and phylogenetic relationships of Gasterosteus aculeatus around Japan were investigated for 15 populations of the Japan Sea and Pacific Ocean forms previously detected by allozyme analyses. PCR-RFLP analysis of mtDNA ND5/6 gene indicated two major divergent clades: (1) Alaskan population and (2) Russian and Japanese populations. Japanese populations were clearly subdivided into G. aculeatus populations and hariyo (G. aculeatus leiurus). However, the relationships among Japanese G. aculeatus populations did not support the genetic divergence between Japan Sea and Pacific Ocean forms shown by allozyme analyses. This suggests that mtDNA genetype similarity between the two forms could have resulted from complete replacement of mtDNA from one form to the other. MtDNA introgression is possible in two direction: from Japan Sea form to Pacific Ocean form and the reverse. The haplotype composition of each population observed in the present study suggests that the second direction is more likely. The migrant number of females per generation (Nm) between populations of the two forms was remarkablely large (9.083–infinite), suggesting that gene flow is taking place between the populations at the present time or might have occurred until recent years.     

Phylogeographic analysis reveals two periods of divergence in large-scaled redfin <Emphasis Type="Italic">Tribolodon hakonensis</Emphasis> (Pisces,Cyprinidae)

Analysis of mtDNA variation in one of the amphidromic Far Eastern redfins, Tribolodon hakonensis, revealed the presence of three considerably genetically different mtDNA phylogroups in the individuals from the Russian part of the range. These data suggest the presence of the two periods of divergent evolution in the history of the species examined. Comparison of the haplotype distributions from different phylogroups over the species range revealed geographic localization of only one phylogroup in the population samples from southern continental coastal regions of the Sea of Japan. At the same time, two other phylogroups were found in almost equal ratios in northern continental samples and near the Sakhalin Island. These results suggest that the first stage of the divergent evolution, which occured between Pliocene and Pleistocene, resulted in the formation of genetically isolated form (probably, a species) in the region of the Sea of Japan. The second, later period of divergence, probably associated with the separation of the Sea of Okhotsk from the Pacific Ocean then ended with the integration of earlier genetically separated forms into one species with the common gene pool.     

Ecological divergence and habitat isolation between two migratory forms of Japanese threespine stickleback (Gasterosteus aculeatus)

When two closely related species migrate to divergent spawning sites, divergent use of spawning habitats can directly reduce heterospecific mating. Furthermore, adaptations to divergent spawning habitats can promote speciation as a by‐product of ecological divergence. Here, we investigated habitat isolation and ecological divergence between two anadromous forms of threespine stickleback (Gasterosteus aculeatus), the Japan Sea and Pacific Ocean forms. In several coastal regions of eastern Hokkaido, Japan, these forms migrate to the same watershed to spawn. Our field surveys in a single watershed revealed that segregation of distinct spawning sites between the two forms was maintained within the watershed across multiple years. These spawning sites diverged in salinity and predator composition. Morphological and physiological divergence between the forms also occurs in the direction predicted by ecological differences between the spawning sites. Our data indicate that migration into divergent spawning habitats can be an important mechanism contributing to speciation and phenotypic divergence in anadromous fishes.     

Postglacial vegetation history around Torihama,Fukui Prefecture,Japan

A pollen analytical study of a core sample from the Torihama site in the Sea of Japan area of the Kinki region was carried out. This core, dated by three types of tephra, corresponded to the period between the early and the middle postglacial periods. In the early postglacial period, the cool-temperate deciduous forest was accompanied byCryptomeria japonica. In the middle postglacial period,C. japonica forests formed on the lowlands. Lucidophyllous forests developed after the deposition of K-Ah ash (6300 years BP) in the Sea of Japan area of the Kinki region, which was later than the development of those in the inland and Pacific Ocean areas.     

Phylogeographic analysis reveals two periods of divergence in large-scaled redfin Tribolodon hakonensis (Pisces, Cyprinidae)

Analysis of mtDNA variation in one of the amphidromic Far Eastern redfins, Tribolodon hakonensis, revealed the presence of three considerably genetically different mtDNA phylogroups in the individuals from the Russian part of the range. These data suggest the presence of the two periods of divergent evolution in the history of the species examined. Comparison of the haplotype distributions from different phylogroups over the species range revealed geographic localization of only one phylogroup in the population samples from southern continental coastal regions of the Sea of Japan. At the same time, two other phylogroups were found in almost equal ratios in northern continental samples and near the Sakhalin Island. These results suggest that the first stage of the divergent evolution, which occured between Pliocene and Pleistocene, resulted in the formation of genetically isolated form (probably, a species) in the region of the Sea of Japan. The second, later period of divergence, probably associated with the separation of the Sea of Okhotsk from the Pacific Ocean then ended with the integration of earlier genetically separated forms into one species with the common gene pool.     

A part of the Japan Sea form of the threespine stickleback, Gasterosteus aculeatus, spawns in the seawater tidal pools of western Hokkaido Island, Japan

The Japan sea form of the threespine stickleback, Gasterosteus aculeatus, was found to spawn in the seawater tidal pool of the Cape Benkei, western Hokkaido Island, Japan, in 2001 and 2003. Nest-guarding males, adult females and juveniles of this form were also observed in four tidal pools of the Capes Benkei, Kabuto and Kamui, in 2001–2003. Otolith Sr:Ca ratios in sticklebacks collected from these tidal pools were relative high from the core to the edge, suggesting they lived in seawater environments throughout its life cycle. These findings suggest that a part of the Japan Sea formȁ9s individuals in western Hokkaido Island have the potential to breed in the coastal sea and the life history style has evolved in dependence on the seawater environment.     

Pseudo-nitzschia pungens (Bacillariophyceae): A cosmopolitan diatom species?

Genetic, reproductive and morphological variation were studied in 193 global strains of the marine diatom species Pseudo-nitzschia pungens (Grunow ex Cleve) Hasle to assess potential intraspecific variation and biogeographic distribution patterns. Genetic differentiation between allo- and sympatric strains was investigated using the ITS1–5.8S–ITS2 rDNA region. Three ITS clades were found. Clones of opposite mating type were sexually compatible within clades I or II, and viable F1 hybrid offspring were produced in crosses between them. The molecular differences between these clades were correlated with slight but consistent morphological differences. At present, nothing can be said about morphology and mating behavior for clade III clones because only ITS data were available. The three ITS clades showed different geographic distributions. Clade II was restricted to the NE Pacific, whereas clones belonging to clade III originated from geographically widely separated areas (Vietnam, China and Mexico). ITS clade I was recovered in all locations studied: the North Sea (Belgium, The Netherlands, France), the eastern and western N Atlantic (Spain, Canada), the NW and S Pacific (Japan, New Zealand) and the NE Pacific (Washington State). Clade I thus appears to be globally distributed in temperate coastal areas and provides the first strong evidence to date for the global distribution of a biologically, genetically and morphologically defined diatom species.     

Contrasting patterns of nuclear microsatellite genetic structure of Fraxinus mandshurica var. japonica between northern and southern populations in Japan

Aim The aim of this study is to detect extant patterns of population genetic structure of Fraxinus mandshurica var. japonica in Japan, and to provide insights into the post‐glacial history of this species during the Holocene. Location Hokkaido and Honshu islands, Japan (including the Oshima and Shimokita peninsulas). Methods We examined nine polymorphic nuclear microsatellite loci to assess genetic variation within and among 15 populations across almost the entire range of the species in Japan. Extant patterns of geographical structure were analysed using Bayesian clustering, Monmonier’s algorithm, analysis of molecular variance, Mantel tests and principal coordinates analysis. Recent bottlenecks within populations and regional genetic variation were also assessed. Results Northern populations (Hokkaido Island and the Shimokita Peninsula) formed a single homogeneous deme, maintaining the highest level of allelic diversity on the Oshima Peninsula. By contrast, southern populations (Honshu Island) demonstrated strong substructure on both coasts. Specifically, populations on the Pacific side of Honshu exhibited significant bottlenecks and erosion of allelic diversity but preserved distinct subclusters diverging from widespread subclusters on the Japan Sea side of this island. Main conclusions Genetic evidence and life history traits suggest that F. mandshurica occupied cryptic northern refugia on the Oshima Peninsula during the Last Glacial Maximum, which is reflected in the species’ extant northern distribution. Strong geographical structure in southern populations, in agreement with fossil pollen records, suggests geographical isolation by mountain ranges running north–south along Honshu. Given that this tree species is cold‐adapted and found in riparian habitats, populations on the Pacific side of Honshu probably contracted into higher‐elevation swamps during warm post‐glacial periods, leading to a reduction of effective population sizes and rare allelic richness.     

Intraspecific Genetic Diversity of Undaria pinnatifida in Japan, based on the Mitochondrial cox3 Gene and the ITS1 of nrDNA

The intraspecific genetic diversity of the kelp Undaria pinnatifida (Harvey) Suringar (Laminariales, Phaeophyceae) was investigated using DNA sequences of the mitochondrial cytochrome oxidase subunit 3 (cox3) gene and internal transcribed spacer 1 (ITS1) of nuclear ribosomal DNA in plants collected from 21 localities along the Japanese coast between 2001 and 2003. Morphological variation was also examined and compared with the genetic diversity. Cox3 analyses of 106 plants revealed 9 haplotypes (I–IX) that differed from each other by 1–7 bp (all synonymous substitutions). Haplotype I was distributed in Hokkaido and the northern Pacific coast of Honshu, while haplotype III was found along the Sea of Japan coast of Honshu. Other types were found along the central and southern coast of Honshu. ITS1 analyses of 42 plants revealed 0–1.7% nucleotide differences, but plants from the Sea of Japan coast and northern Japan had similar sequences. The lower genetic differentiation along the Sea of Japan and northern coasts might be due to the recent establishment (after the middle of the last glacial period) of the Sea of Japan flora. The cox3 haplotype of cultivated plants was found in natural populations occurring close to cultivation sites (Naruto, Tokushima Pref., and Hokutan, Hyogo Pref.). This suggests that cultivated plants possibly escaped and spread or crossed with plants of natural populations. Morphological analyses of variation in 10 characters were conducted using 66 plants. The results showed no significant local variation owing to the wide variation in each population and did not support any forma previously described. No correlations between the morphological characters and cox3 haplotypes were detected.     

Molecular identification of Anisakis simplex sensu stricto and Anisakis pegreffii (Nematoda: Anisakidae) from fish and cetacean in Japanese waters

Parasites morphologically consistent with Anisakis simplex sensu lato collected from the coast of Japan and Western North Pacific Ocean were examined by PCR-RFLP of the ITS region (ITS1, 5.8 subunit rRNA gene and ITS2) and mtDNA cox1. The RFLP patterns of rDNA generated by HinfI and HhaI showed that 100% of the larvae collected from Hokkaido and 94% of adults collected from Western North Pacific Ocean were identified as A. simplex sensu stricto. On the other hand, 97% of the larvae collected from Fukuoka prefecture were identified as A. pegreffii. A hybrid genotype was found in adults in Western North Pacific Ocean and larva in Fukuoka prefecture. These findings revealed that A. simplexs. str. is primarily distributed in the North Pacific Ocean and A. pegreffii is primarily distributed in the southern Sea of Japan. RFLP analysis of mtDNA cox1 showed different patterns between A. simplex s. str. and A. pegreffii after digestion with HinfI. This polymorphism obtained by RFLP analysis of mtDNA cox1 proved the usefulness as new genetic markers to distinguish two sibling species.     

Comparative Analysis of Japanese Three-Spined Stickleback Clades Reveals the Pacific Ocean Lineage Has Adapted to Freshwater Environments while the Japan Sea Has Not

Divergent selection and adaptive divergence can increase phenotypic diversification amongst populations and lineages. Yet adaptive divergence between different environments, habitats or niches does not occur in all lineages. For example, the colonization of freshwater environments by ancestral marine species has triggered adaptive radiation and phenotypic diversification in some taxa but not in others. Studying closely related lineages differing in their ability to diversify is an excellent means of understanding the factors promoting and constraining adaptive evolution. A well-known example of the evolution of increased phenotypic diversification following freshwater colonization is the three-spined stickleback. Two closely related stickleback lineages, the Pacific Ocean and the Japan Sea occur in Japan. However, Japanese freshwater stickleback populations are derived from the Pacific Ocean lineage only, suggesting the Japan Sea lineage is unable to colonize freshwater. Using stable isotope data and trophic morphology, we first show higher rates of phenotypic and ecological diversification between marine and freshwater populations within the Pacific Ocean lineage, confirming adaptive divergence has occurred between the two lineages and within the Pacific Ocean lineage but not in the Japan Sea lineage. We further identified consistent divergence in diet and foraging behaviour between marine forms from each lineage, confirming Pacific Ocean marine sticklebacks, from which all Japanese freshwater populations are derived, are better adapted to freshwater environments than Japan Sea sticklebacks. We suggest adaptive divergence between ancestral marine populations may have played a role in constraining phenotypic diversification and adaptive evolution in Japanese sticklebacks.     

EST and Mitochondrial DNA Sequences Support a Distinct Pacific Form of Salmon Louse, <Emphasis Type="Italic">Lepeophtheirus salmonis</Emphasis>

Nuclear deoxyribonucleic acid sequences from approximately 15,000 salmon louse expressed sequence tags (ESTs), the complete mitochondrial genome (16,148bp) of salmon louse, and 16S ribosomal ribonucleic acid (rRNA) and cytochrome oxidase subunit I (COI) genes from 68 salmon lice collected from Japan, Alaska, and western Canada support a Pacific lineage of Lepeophtheirus salmonis that is distinct from that occurring in the Atlantic Ocean. On average, nuclear genes are 3.2% different, the complete mitochondrial genome is 7.1% different, and 16S rRNA and COI genes are 4.2% and 6.1% different, respectively. Reduced genetic diversity within the Pacific form of L. salmonis is consistent with an introduction into the Pacific from the Atlantic Ocean. The level of divergence is consistent with the hypothesis that the Pacific form of L. salmonis coevolved with Pacific salmon (Onchorhynchus spp.) and the Atlantic form coevolved with Atlantic salmonids (Salmo spp.) independently for the last 2.5–11 million years. The level of genetic divergence coincides with the opportunity for migration of fish between the Atlantic and Pacific Ocean basins via the Arctic Ocean with the opening of the Bering Strait, approximately 5 million years ago. The genetic differences may help explain apparent differences in pathogenicity and environmental sensitivity documented for the Atlantic and Pacific forms of L. salmonis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inter-male associations in a wild Japanese macaque troop on Yakushima Island,Japan

Adult male association and its annual change were studied in a wild population of Japanese macaques (Macaca fuscata yakui) on Yakushima Island, Japan. Unlike many other Japanese macaque troops, adult troop males frequently maintained proximity and exchanged grooming with one another in both the mating and non-mating seasons, and the dominance relationship rarely appeared in such inter-male associations. The few cases of agonistic interactions occurred mostly when estrous females or food resources were immediately concerned. Although troop males were very intolerant to newly appeared solitary males (new males) during the mating season, close associations were formed between troop males and new males as soon as the mating season terminated. The consort of new males and lower-ranking troop males with estrous females was frequently disturbed, but these males could copulate no less frequently than higher-ranking males. A comparison among macaque species suggests the existence of two forms of inter-male association: (1) the frequent association based on the symmetrical exchange of social behaviors; and (2) the infrequent and asymmetrical association related to the dominance relationship. The form of inter-male association seems to be influenced by whether or not males can keep close associations with females throughout the year.