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The ammonia monooxygenase subunit A (amoA) gene has been used to investigate the phylogenetic diversity, spatial distribution, and activity of ammonia-oxidizing archaeal (AOA) and bacterial (AOB) populations that contribute greatly to the nitrogen cycle in various ecosystems. Amplicon sequencing ofamoAgenes is a widely used method; however, it generally produces inaccurate results owing to the lack of a ‘universal’ primer set. Moreover, currently available primer sets suffer from amplification biases, which can lead to severe misinterpretation. Although shotgun metagenomic and metatranscriptomic analyses are alternative approaches without amplification bias, the low concentration of target genes in heterogeneous environmental DNA restricts comprehensive analysis to a realizable sequencing depth. Here, we developed a method foramoAgene enrichment sequencing using a probe capture hybridization technique. Using mock community samples, our approach effectively enrichedamoAgenes with low compositional changes, outperforming amplification and meta-omics sequencing analyses. Following the analysis of marine metatranscriptome samples, we predicted 79 AOA and AOB operational taxonomic units (OTUs), of which 33 (42%) and 60 (76%) were unidentified by simple metatranscriptomic and amplicon sequencing, respectively. Mapped read ratios were significantly higher for the capture samples (50.4 ± 27.6%) than for non-capture samples (0.05 ± 0.02%), demonstrating the high enrichment efficiency of the method. The analysis also revealed the spatial diversity of AOA ecotypes with high sensitivity and phylogenetic resolution, which are difficult to examine using conventional approaches.ImportanceThe nitrogen cycle is largely driven by prokaryotes via nitrogen metabolism, including ammonia oxidation. To understand the phylogenetic diversity, spatial distribution, and potential activity of these ammonia-oxidizing prokaryotes, the ammonia monooxygenase subunit A (amoA) gene has been used. However, current conventional approaches, such asamoAamplicon sequencing and meta-omics (metagenomics and metatranscriptomics), generally produce inaccurate results owing to PCR biases and low concentrations of target genes in the samples. Here, as an alternative, we developed a novel enrichment sequencing method that condenses theamoAgene in metagenomic/metatranscriptomic samples via probe capture hybridization for high-throughput sequencing. We demonstrated that this method is applicable to both artificial mock and oceanic samples and generates reliable results with high phylogenetic resolution and sensitivity.

The distribution and early growth of juvenile Pacific bluefin tuna (PBF) Thunnus orientalis were studied based on trawl surveys conducted around Sado Island, eastern Sea of Japan, from late August to early September in 2017 and 2018. Juvenile PBF were mainly collected in an area between the Tsushima Warm Current and land where the surface water (5-m depth) temperature ranged from 22.8 to 27.2 °C. The fork length (FL) of the fish ranged from 12 to 320 mm, and there were modes at 140–170 mm FL in the frequency distribution for both years. In 2018, there was also a mode at around 50 mm FL. Otolith microstructure analysis revealed that hatch date mainly peaked in early and mid July in both years, and in early and mid August in 2018, and corresponded with the modes of FL. In addition, differences in growth were observed between year classes, and between size classes in 2018. Total gill raker number increased with FL and was almost constant at around 100 mm FL. This study provides an update of the distribution of juvenile PBF in the Sea of Japan, which was found to be related to the Tsushima Warm Current, and indicates the importance of the eastern Sea of Japan as a nursery ground for PBF that hatch from July to August.

Many organisms exhibit phenotypic plasticity that changes their traits in response to their environment. Although whether or not this plasticity contributes to adaptive evolution is a fundamental question in evolutionary biology, various studies report that natural populations adapt to rapid environmental changes via plasticity, which leads to novel adaptive traits as “triggers.” Namely, phenotypic plasticity has considered allowing an accumulation of genetic mutations to fix the alternative phenotypes induced by nongenetic perturbations that include gene expression noise or epigenetic modification caused by environmental change. However, because the molecular mechanism of phenotypic plasticity is unknown, verification of the process from phenotypic plasticity to genetic fixation remains insufficient. Here we show that decrease in methylated CpG sites leads to loss of plasticity, which triggers genetic fixation of novel traits, in medaka fish (Oryzias latipes). We found that the gut length was correlated with the number of methylated CpG sites upstream of the Plxnb3 gene, which is involved in the developmental process of nerve axons. The medaka, in which the methylated DNA region is deleted by CRISPR/Cas9, showed a loss of plasticity in gut length and a lower survival rate caused by nonoptimal feeding environments. Moreover, standing variation in the promoter region of another gene, Ppp3r1, which is also related to nerve axon development, raised the gene expression and made a longer gut stably in wild medaka groups that lost the gut-length plasticity. Furthermore, our phylogenetic analysis revealed the timing of these evolutionary events, indicating that the loss of phenotypic plasticity by nucleotide substitutions initiates the process of genetic fixation of the novel trait. That is, while phenotypic plasticity plays a role as a buffer of evolution and contributes to environmental adaptation, as previously thought, our molecular data suggest that mutation on CpG site causing the loss of phenotypic plasticity, is the trigger for a generation of novel traits.

Seabirds are long-lived birds that invest in offspring at very high levels, for which male parental care is indispensable. These characteristics are thought to explain seabirds’ generally low level of extra-pair paternity (EPP). Although the Streaked Shearwater (Calonectris leucomelas) is a socially monogamous seabird, it is known to copulate outside its social pair bond, which implies the frequent occurrence of EPP. In the closely related Cory’s Shearwater Calonectris borealis, cuckoldry is related to body size of the social male. To determine whether body-size-related EPP occurs among Streaked Shearwaters, we established 39 new microsatellite markers for parentage analysis and compared body size between cuckolded and non-cuckolded males. With the new markers, we found that extra-pair males sired 17 (15.0%) of 113 offspring during the 2014–2016 study period, which included three 1.5-month chick-rearing periods. This percentage is among the highest recorded for seabirds. We also found the bill and wing length of cuckolded males to be significantly shorter than those of non-cuckolded males, and that females can reject attempted copulations. These observations imply that EPP in this species is size related and involves female acceptance.

We studied the phylogeny, population structure, and demographic history of Scolopsis taenioptera in the western Pacific Ocean. Using the 80 samples collected from four locations, we obtained the nucleotide sequences of mitochondrial cytochrome c oxidase subunit I and cytochrome b genes. We identified two distinct lineages showing a clear phylogeographic break that was possibly due to the Pleistocene sea-level change. One lineage was distributed in Iloilo (Philippines) and the other in Terengganu (Malaysia), Rayong (Thailand), and Ha Long Bay (Vietnam). The Terengganu and Rayong populations showed clear signs of demographic expansion; the Iloilo and Ha Long Bay populations were relatively stable or spatially expanded as geographically subdivided populations.

Predicting speciation is a fundamental goal of research in evolutionary ecology. The probability of speciation is often positively correlated with ecosystem size. Although the mechanisms driving this correlation are generally difficult to identify, a shared geographical and ecological context provides a suitable condition to study the mechanisms that promote speciation in large ecosystems by reducing the number of factors to be considered. Here, we determined the correlation between speciation and ecosystem size, and discuss the underlying mechanisms of this relationship, using a probable parallel ecotype formation for freshwater fish. Our population genetic analysis revealed that speciation of the landlocked goby, Rhinogobius sp. YB, of the Ryukyu Archipelago, Japan, from its migratory ancestor, R. brunneus, occurred in parallel across five islands. Logistic regression analysis showed that speciation probability could be predicted using island size. The results suggest that ecosystem size predicts the occurrence of adaptation and reproductive isolation, probably through its association with three possible factors: divergent selection strength, population persistence, and occurrence probability of habitat separation.

In dispersive species with continuous distributions, genetic differentiation between local populations is often absent or subtle and thus difficult to detect. To incorporate such subtle differentiation into management plans, it may be essential to analyse many samples from many localities using adequate numbers of high-resolution genetic markers. Here, we evaluated the usefulness of dense locality sampling in resolving genetic population structure in the ayu (Plecoglossus altivelis), a dispersive fish important in Japanese inland fisheries. Genetic variability in, and differentiation between, ayu populations around the Japan-Ryukyu Archipelago were investigated in 4746 individuals collected from 120 localities by genotyping 12 microsatellite markers. These individuals represented the two subspecies of ayu, namely the Ryukyuan subspecies (Plecoglossus altivelis ryukyuensis) and both amphidromous and landlocked forms of the nominotypical subspecies (P. a. altivelis) along the archipelago. We successfully detected an absence of genetic differentiation within the landlocked form and subtle but significant differentiation and clear geographic patterns of genetic variation among populations of the amphidromous form, which had been considered genetically homogeneous. This suggests that dense locality sampling effectively resolves subtle differences in genetic population structure, reducing stochastic deviation in the detection of genetic differentiation and geographic patterns in local populations of this dispersive species. Resampling analyses based on empirical data sets clearly demonstrate the effectiveness of increasing the number of locality samples for stable and reliable estimations of genetic fixation indices. The genetic population structure observed within the amphidromous form provides useful information for identifying management or conservation units in ayu.

Reliably rooted phylogenetic trees play irreplaceable roles in clarifying diversification in the patterns of species and populations. However, such trees are often unavailable in phylogeographic studies, particularly when the focus is on rapidly expanded populations that exhibit star-like trees. A fundamental bottleneck is known as the random rooting effect, where a distant outgroup tends to root an unrooted tree “randomly.” We investigated whether parallel mitochondrial genome (mitogenome) sequencing alleviates this effect in phylogeography using a case study on the Sea of Japan lineage of the intertidal goby Chaenogobius annularis. Eighty-three C. annularis individuals were collected and their mitogenomes were determined by high-throughput and low-cost parallel sequencing. Phylogenetic analysis of these mitogenome sequences was conducted to root the Sea of Japan lineage, which has a star-like phylogeny and had not been reliably rooted. The topologies of the bootstrap trees were investigated to determine whether the use of mitogenomes alleviated the random rooting effect. The mitogenome data successfully rooted the Sea of Japan lineage by alleviating the effect, which hindered phylogenetic analysis that used specific gene sequences. The reliable rooting of the lineage led to the discovery of a novel, northern lineage that expanded during an interglacial period with high bootstrap support. Furthermore, the finding of this lineage suggested the existence of additional glacial refugia and provided a new recent calibration point that revised the divergence time estimation between the Sea of Japan and Pacific Ocean lineages. This study illustrates the effectiveness of parallel mitogenome sequencing for solving the random rooting problem in phylogeographic studies.

Genetic and morphological differences among the three species of the genus Rastrelliger, viz. Rastrelliger kanagurta, Rastrelliger brachysoma and Rastrelliger faughni were clarified on the basis of 150 specimens collected from Panay Island, the Philippines, thereby providing fundamental information for accurate species identification. Specimens were separated into three distinct clades on the Bayesian phylogenetic tree constructed from mitochondrial DNA sequences, which largely corresponded to R. kanagurta, R. brachysoma and R. faughni according to the number of gill rakers and body depth. These results indicated that they were reproductively isolated, separate species. Further morphological differences were evident among the three clades, including separation by principal component analysis on 29 measurements. Apart from such interspecific divergences, intraspecific variations of several morphological characters relative to body size [fork length (FL)] were revealed for each species. Despite a slight increase in the number and the length of gill rakers with growth, R. faughni was invariably distinguished from the other two species by fewer and shorter gill rakers throughout the entire body size range. In stark contrast, some larger (FL > 200 mm) R. kanagurta individuals attained a proportionately deeper body that had been thought to be characteristic of R. brachysoma. Likewise, some smaller individuals of R. brachysoma (FL

To better understand habitats of larval Pacific bluefin tuna (PBT; Thunnus orientalis) in the nursery grounds, the vertical distribution of larval PBT was collected using a 1-m2 Tucker trawl and a 3.14-m2 ring net at depths of 0, 10, 20, and 30 m during the spawning seasons of 2016 and 2017. Most of the larvae in the Japan Sea (756 of 938 individuals) were collected from the surface layer using a ring net, while similar numbers were collected from 0 to 20 m depth in the Nansei area. The temperature at ≥20 m depths in the Japan Sea was cooler than the optima temperature, and the thermocline was steep at 10 m depth, and the cold subsurface water was suggested to make the PBT larvae aggregate near the surface. A weak ontogenetic and diel migration was observed; the body size at 10 m depth during the day and 0 m at night were significantly larger than those at 0 m during the day in the Japan Sea. These results suggested that some small larvae sink to 10 m and/or large larvae move to the surface at night. Our results showed that the larval PBT habitat is strongly controlled by water temperature, and that global warming would have a positive effect on PBT larvae in the Japan Sea, which was evidenced by the higher density of PBT larvae after 2010 compared to before 1990.

Ricefishes of the genus Oryzias, including Japanese medaka (O. latipes), are known as excellent model organisms for studies in various fields of science. Some species of the genus inhabit brackish water, and such species are recognized to be useful to investigate physiological phenomena in seawater. However, only a limited number of species have been recorded from brackish waters. In addition, there is no information about the genetic relationship among populations inhabiting sites with different salinities. Here we report the discovery of Oryzias fish in two locations near Haiphong, northern Vietnam, a brackish mangrove planting area and a freshwater pond. A phylogenetic analysis using mitochondrial 12S and 16S ribosomal RNA (rRNA) gene sequences indicated that the fish from the two localities are the same species, Hainan medaka, O. curvinotus. Population genetic analysis using the mitochondrial 12S and 16S rRNA gene sequences revealed a close genetic relationship between the two populations. These results suggest that O. curvinotus is adaptable to both hyperosmotic and hypoosmotic environments. Due to its osmotic adaptability and ease of rearing in the laboratory, this species is expected to become a model for marine environmental and toxicological studies, as well as for studies of osmotic adaptation mechanisms.

Marine fish generally show higher genetic diversity and shallower population structure than freshwater fish, but data for rare species are lacking. A latid fish, akame (Lates japonicus), is a rare, large predatory fish endemic to Japan, occurring mainly in the estuaries and coastal waters of Kochi and Miyazaki prefectures. Among species of Lates, akame is the only one adapted to temperate waters, while the others are distributed in tropical waters. Despite conservation concerns for this species, almost nothing is known about its population genetic characteristics. We used amplified fragment length polymorphism (AFLP) and mitochondrial DNA (mtDNA) markers to describe the genetic variation and its partitioning among juvenile samples collected from multiple localities and over different years. Twenty-four primer combinations detected a total of 522 AFLP loci, only 40 (7.7 %) of which were polymorphic. Sequence analysis of the entire mtDNA control region identified only five haplotypes defined by four variable sites. The level of genetic diversity revealed by both genetic markers was unusually low compared to other marine fish and comparable to levels observed for some endangered freshwater fish exhibiting some of the lowest levels of genetic diversity among vertebrate populations. Analysis of molecular variance for the AFLP data indicated a weak but significant population structure (F CT = 0.07, P

The three mackerel species, Rastrelliger kanagurta, Rastrelliger brachysoma, and Rastrelliger faughni, co-occur in the central part of the Indo-West Pacific Ocean, and together represent important fisheries resources. However, they have sometimes been collectively referred to as “Rastrelliger spp.” in the fisheries statistics, owing to their morphological similarity. Therefore, species-specific data on landings, effort, and population status are lacking, undermining the appropriate resource management. The present study provides a rapid and cost-effective method based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis to distinguish the three species. Digestion of the PCR products of 1247 base pairs of mitochondrial DNA encompassing the complete sequence of cytochrome b gene using two restriction enzymes yielded diagnostic restriction patterns for the three species, thereby enabling accurate identification.

In host–parasite coevolution, parasite innovations including the acquisition of new habitats and novel traits can trigger evolutionary breakthroughs and enhance parasite diversification via accumulation of new hosts. All species of the family Cymothoidae are obligate fish parasites, attaching to exterior body surfaces of fish, the buccal or opercular cavities, or burrowing into abdominal muscle tissue. In the present study, we constructed a molecular phylogeny of 27 cymothoid species that parasitise 38 fish species, combined with 2 prior cymothoid datasets, based on the sequences of mitochondrial 16S rRNA and COI genes. We explored the evolution of the host attachment mode, and the habitat shift from saline water to freshwater. Our evolutionary trees include two freshwater clades, an abdominal burrower clade, and cymothoid clades that are closer to the base of Cymothoidae than those initially analysed. We found that the basal clade of Cymothoidae was Elthusa sacciger, which is parasitic in the opercular cavity of synaphobranchid eels. This result suggests that cymothoids may have originated in deep seas, subsequently expanded to shallow seas, and then to brackish and/or freshwater, by shifting host species. Invasion of freshwater habitats has occurred at least twice; freshwater abdominal muscle burrowers living on armoured catfish constitute a clade allied to E. sacciger. The ancestral host attachment site, based on our dataset, was the opercular cavity, followed (sequentially) by buccal colonisation and attachment to the external body.

Cooperative breeding has been studied intensively in many species of birds and mammals but remain less well studied in fish. We report a remarkable new example of a cooperatively breeding cichlid from Lake Tanganyika, Neolamprologus obscurus. Using field observations and microsatellite DNA analyses, we studied group structure, helping behavior, relatedness, and dispersal of this species. We present four major observations. First, large territorial breeding males mated with one to eight breeding females, each of which was territorial and unrelated to another. Second, one to ten smaller fish (“subordinates”) of both sexes were allowed to stay inside the breeding females’ territories. Subordinates were often highly related to both the respective breeding male and female and performed territory defense and shelter maintenance, which is regarded as helping behaviors. Third, one to three subordinate males, similar in size to breeding females, were allowed to stay inside a breeding male’s territory but were not tolerated in the breeding females’ territories. Pairwise relatedness suggests these individuals are usually sons of the respective breeding male. Fourth, pairwise relatedness estimates suggest that juveniles delay dispersal and assist their mothers in raising offspring. As female subordinates grow up, they leave the father’s territory and disperse into other groups. In contrast, male subordinates leave their mother’s territory but remain within the territory of their father. The described social system makes N. obscurus a promising new model species to study the evolution of cooperative breeding.

他の生物に卵を預ける海産のカジカ科魚類8種について産卵宿主種（ホヤやカイメン）の特定に成功し、さらにはカジカ科魚類が産卵管を宿主種の種類や大きさに応じて進化させていることを世界で初めて発見しました。, Interspecific interactions between parasites and hosts can influence the evolution of behavioural and morphological adaptations of both parasites and their hosts. There is, however, little empirical evidence available regarding the evolution of reproductive traits driven by these interactions. In this paper, we investigated host selection and ovipositor length in nine sympatric marine sculpins that oviposit into tunicates or sponges....

Mitofish is a database of fish mitochondrial genomes (mitogenomes) that includes powerful and precise de novo annotations for mitogenome sequences. Fish occupy an important position in the evolution of vertebrates and the ecology of the hydrosphere, and mitogenomic sequence data have served as a rich source of information for resolving fish phylogenies and identifying new fish species. The importance of a mitogenomic database continues to grow at a rapid pace as massive amounts of mitogenomic data are generated with the advent of new sequencing technologies. A severe bottleneck seems likely to occur with regard to mitogenome annotation because of the overwhelming pace of data accumulation and the intrinsic difficulties in annotating sequences with degenerating transfer RNA structures, divergent start/stop codons of the coding elements, and the overlapping of adjacent elements. To ease this data backlog, we developed an annotation pipeline named MitoAnnotator. MitoAnnotator automatically annotates a fish mitogenome with a high degree of accuracy in approximately 5 min; thus, it is readily applicable to data sets of dozens of sequences. MitoFish also contains re-annotations of previously sequenced fish mitogenomes, enabling researchers to refer to them when they find annotations that are likely to be erroneous or while conducting comparative mitogenomic analyses. For users who need more information on the taxonomy, habitats, phenotypes, or life cycles of fish, MitoFish provides links to related databases. MitoFish and MitoAnnotator are freely available at http://mitofish.aori.u-tokyo.ac.jp/ (last accessed August 28, 2013); all of the data can be batch downloaded, and the annotation pipeline can be used via a web interface.

Stream fish with low mobility are often restricted to a narrow range of a stream course, and their morphological characters tend to be affected substantially by the environmental conditions of their habitat. We examined the relationships between environmental conditions and morphological characters of Japanese fluvial sculpin (Cottus pollux), a small, benthic fish with low mobility, at seven tributaries of the same river system in eastern Japan. Discriminant function analysis (DFA) indicated significant morphological differences in sculpins amongst the seven tributaries, and over 80% of individuals were classified correctly in each tributary. The strong positive correlation/loadings on the first discriminant function (DF1: 49.9% of total variation) were anterior caudal peduncle depth (0.436) and caudal peduncle truss (0.387), both of which could be regarded as an attribute of caudal peduncle depth. The second discriminant function (DF2: 16.5% of total variation) had a high negative loading for distance between the posterior first dorsal fin and anterior anal fin (−0.454), which could be regarded as an attribute of body depth. Correlation analysis indicated that sculpin tended to have a lower caudal peduncle depth under faster bottom current velocities at the tributary scale. Individuals deviating substantially from the centroid of DF2 in each tributary had a poorer body condition. Our results suggest the possibility of changes in the morphological characters of Japanese fluvial sculpin in relation to environmental conditions even at the tributary scale within the same river system.

To clarify food resource use and niche breadths of the Japanese fluvial sculpin Cottus pollux (large-egg type), listed by over half the prefectures as an endangered species, we examined the diet and food selectivity of 168 specimens at 7 locations in 3 tributaries that drain into the upper regions of the Chikuma River. A stomach pump was used to assess the stomach contents. In total, 2,833 aquatic organisms from more than 22 families were found in the stomachs. An index of relative importance (IRI) indicated that chironomid, baetid, and ephemerellid larvae were the major prey for the sculpin. Despite considerable variation in the composition of aquatic organisms among locations, sculpin exhibited strong food selectivity for baetid, glossosomatid, chironomid, and tipulid larvae at multiple locations. Each individual sculpin preyed upon 17.2 prey items on average, and the number of prey items found in the stomach positively correlated with the number of prey families, regardless of sculpin body size. Of 22 prey families found form all stomachs we examined, each individual sculpin preyed upon only 3.54 families on average. These findings suggest that sculpin may have relatively narrow niche breadths regardless of a considerable number of prey individuals in their stomachs. GLM results indicated that a model that incorporated water depth and sculpin body size was the most parsimonious model to explain the prey diversity index in the stomachs of the sculpins. The diversity index of aquatic organisms positively correlated with the mean water depth at each location. These data suggest that rivers with deeper water depths might allow colonization of a variety of aquatic organisms, which might be expected to enhance availability of prey potentially available for the sculpin.

We present a set of nuclear DNA SNPs useful for discriminating native Lake Biwa and introduced “Eurasian” strains of common carp, and further, for detecting their hybridization. Such SNPs were searched around short tandem repeat (STR) regions to be used later as HapSTR markers. A total of 62 microsatellite locus were screened with PCR and direct sequencing. The resultant nucleotide sequences from typical Lake Biwa (n = 30) and “Eurasian” (n = 24) specimens indicated that a total of 21 SNPs distributed across 7 loci would be useful for the above-mentioned purpose. All of the SNPs can be genotyped by direct sequencing, simultaneously with STR polymorphisms.

The distribution patterns of many fishes between the three continents (Africa, Australia, and South America) in the Southern Hemisphere have been uncovered to be influenced by mostly vicariance or historical dispersal. Although some demersal fishes with intercontinental distribution are suggested to be more influenced by current/recent dispersal, few genetic studies have been made for demersal fishes so far. To provide more information for such fishes, genetic divergence was analyzed for two pairs of gadiform species and subspecies distributed around Australasia and South America: the blue grenadier, Macruronus novaezelandiae (from New Zealand) and the Patagonian grenadier, M. magellanicus (from South America) as well as two subspecies of the southern blue whiting, Micromesistius australis pallidus (from New Zealand) and M. a. australis (from South America). The sequence analyses of two mitochondrial DNA regions showed no divergence between Australasian and South American populations of the grenadiers and the southern blue whiting. The microsatellite DNA analysis also indicated significant but very minimal genetic differentiation between the two geographic populations of each pair. These results imply rather recent separation of the two geographic populations. Current/recent dispersal may be an important common factor for determining the distribution of demersal fishes in the Southern Hemisphere. Nonetheless, low but significant genetic differentiation observed requires treating the two populations of the economically important grenadiers and southern blue whiting, respectively, as different stocks for proper resource management.

Organisms that migrate between rivers and the sea inevitably pass through estuarine habitats. Despite the potential importance of salinity and temperature fluctuations for metabolic adaptation, little is known about the impact of environmental changes in estuaries on the survival of residents. Ayu (Plecoglossus altivelis) is a migratory fish that inhabits estuarine brackish water in its early life stages. The recent decline in the abundance of populations ascending into rivers is of concern for local biodiversity. The present study aims to elucidate the ecological processes that determine the early success of Ayu larvae under variable environmental conditions. The effects of salinity and water temperature on the endogenous growth of newly hatched larvae from the same brood were examined experimentally based on morphological and metabolic characteristics. High salinity and high water temperature together appeared to require more energy for larval osmoregulation, resulting in the acceleration of yolk depletion and reduced growth of the notochord. Increasing the osmoregulative cost during the yolk-sac stage resulted in the faster induction of a state of starvation. Seawater is considered to have an adverse effect on the survival of newly hatched larvae, as it lowers the efficiency of foraging and predator avoidance due to an energetic tradeoff. More attention should be paid to the significance of estuarine environments involving brackish waters to ensure the early survival of amphidromous fish such as Ayu.

The swamp eel Monopterus albus is widely distributed in tropical and subtropical freshwaters ranging from Southeast Asia to East Asia, and is unique in its ability to breathe air through the buccal mucosa. To examine the genetic structure of this widespread species, molecular phylogenetic analyses of mitochondrial 16S rRNA sequence (514 bp) were conducted for 84 specimens from 13 localities in Southeast and East Asia. The analyses showed clearly that this species can be genetically delineated into three clades based on geographical populations [China-Japan (Honshu + Kyushu), Ryukyu Islands, and Southeast Asia clades], with each clade exhibiting its own reproductive behavior. Therefore, “M. albus” is believed to be composed of at least three species. The Southeast Asia clade with the highest genetic diversity may include more species. The Ryukyu clade was estimated to have diverged more than 5.7 million years ago, suggesting that the Ryukyuan “M. albus” is native. In contrast, in the China-Japan clade, all haplotypes from Japan were closely related to those from China, suggesting artificial introduction(s).

A microsatellite-enriched genomic library was obtained from the nine-spined stickleback, the freshwater type of Pungitius pungitius, and 13 dinucleotide markers were successfully isolated and characterized. Cross-species amplification of these markers in three Pungitius species and two Gasterosteus species (family Gasterosteidae) was performed under standard conditions. The mean observed and expected heterozygosity was 0.500–0.688 and 0.508–0.750, respectively. Low allelic diversity was noted among the microsatellite loci in P. tymensis. These results confirm the genetic vulnerability of Pungitius and the need for its conservation. The newly established microsatellite markers will facilitate population-genetic and ecological studies for the conservation of sympatric sticklebacks.

Artificial introduction and habitat fragmentation affect the indigenous gene pools of fluvial animals. To investigate the effect of human activities on the genetic population structure of vulnerable brook lamprey Lethenteron sp. S in a single river system, samples from 12 tributaries of the Jinzu River, Japan, were analyzed using mitochondrial and microsatellite DNA markers. Exogenous Lake Biwa (Japan) haplotypes and alleles were detected in lampreys from several Jinzu River tributaries. Since Lake Biwa is the source of the commercial ayu fish Plecoglossus altivelis that is introduced in the Jinzu River, the exogenous Lake Biwa lamprey genotypes in the Jinzu River probably originated from the Lake Biwa lampreys that were unintentionally introduced along with the ayu fish. Bayesian admixture and mitochondrial DNA analyses revealed various genetic disturbance phases of the exogenous genotypes in the Jinzu River, such as the six indigenous populations, four admixed populations with low frequencies (average admixture proportion = 0.02–0.04; exogenous haplotype proportion

We developed a novel single nucleotide polymorphism (SNP) marker on the control region of the mitochondrial genome of the ayu Plecoglossus altivelis; the marker can distinguish nucleotides A or G at a specific nucleotide site at which amphidromous and landlocked stocks of ayu are differentiated by different predominant nucleotides. The mitochondrial SNP marker was used to genetically trace landlocked ayu fish released into the Sho River (Toyama Prefecture) which was also inhabited by a native amphidromous population. Larvae, juveniles, and adults were collected throughout a year from the fluvial and marine habitats within and around the river, and subjected to DNA analysis. Mixing rate of landlocked stock was estimated for each life-stage on the basis of the frequency of the specific nucleotides at the SNP site. Though the estimation suggested considerable loss of landlocked ayu after release, mixing of drifting larvae reproduced by females of landlocked ayu was traced in the early spawning season. However, no evidence of survival in the offspring of the landlocked form was found from fish in the marine stage. The landlocked ayu was confirmed to be ineffective in the stock enhancement by releasing.

A total of 49 novel polymorphic microsatellite markers in the ayu fish, Plecoglossus altivelis, were successfully isolated and characterized. The number of alleles per locus, observed heterozygosity, and expected heterozygosity ranged from two to 25 (11.3 on average), from 0.125 to 1.000 (0.648 on average), and from 0.123 to 0.957 (0.751 on average), respectively. These microsatellite markers will be useful for both studying population structure and identifying quantitative trait loci that control evolutionarily and economically important traits of ayu.

Marine environments often allow interbreeding of individuals over the species range, and analyses using neutral molecular markers may lose extant genetic boundaries laid between geographic majorities. Ayu Plecoglossus altivelis has a typical amphidromous life history, migrating between rivers and the sea. In order to clarify reproductive elements of the species, migrants from 64 rivers and streams sampled over a wide latitudinal range were examined for their vertebral number (VN) and dorsal pterygiophore number (DPN) as morphological markers to estimate the temperature history. The irregular variability in VN without a geographic cline suggests that the ascending schools of fish are composed of conspecifics sharing incubation temperature or hatching site. The intersample difference in DPN indicates that larvae and juveniles of ayu spend their marine life inside a water body with a distinct temperature. Site tenacity during the marine stage may be hhelpful to enhance the opportunity to return to the river where they hatched. Each assemblage of fish ascending to rivers and streams is deemed to mostly represent a reproductive element, and therefore, each acts as an evolutionarily significant unit within a metapopulational structure.

Sequence analyses of the non-coding, control region (CR) and coding region of the ND4-tRNA(Ser) genes in the mitochondrial DNA (mtDNA) were conducted for populations of the ayu Plecoglossus altivelis altivelis and the Ryukyu-ayu P. a. ryukyuensis. The level of genetic differentiation between the two subspecies evaluated from the CR data was substantially low, when comparing with that estimated from ND4-tRNA(Ser) gene region data, as well as those from nuclear genome data sets. By contrast, the differentiation between subspecies in the ND4-tRNA(Ser) gene region was substantial, being consistent with the results from the previous nuclear genome analyses. Results of UPGMA and minimum spanning network analyses also implied the unexpected ceiling of genetic differentiation in the CR. These results suggest that the CR does not reflect accurately the level of overall genetic differentiation between the populations of the ayu, but other coding regions of the mtDNA do reflect it so that the mtDNA on the whole may function as a rich source of useful markers for genetic assessment of populations of this species.

The search for dinucleotide repeat microsatellites within scaffolds 1–25 of genome database JGI Fugu v3.0 for the pufferfish Takifugu rubripes revealed that 80% of microsatellite loci consisted of five to 13-fold repeats with locus-specific differences in density. Eleven out of 15 microsatellite loci isolated from the database with which genotyping using wild pufferfish was successfully performed showed polymorphism; that is, the means of the number of alleles and expected and observed heterozygosities at these 11 loci were 21.8, 0.915 and 0.829, respectively. It was confirmed that eight out of the 11 polymorphic loci were inherited through the Mendelian law and one pair of microsatellite loci derived from the same scaffold was linked. These results demonstrated that these loci are useful for constructing a linkage map in the pufferfish as DNA markers.

A recent preliminary study using complete mitochondrial DNA sequences from 48 species of teleosts has suggested that higher teleostean phylogenies should be reinvestigated on the basis of more intensive taxonomic sampling. As a second step towards the resolution of higher teleostean phylogenies, which have been described as the “(unresolved) bush at the top of the tree,” we reanalyzed their relationships using mitogenomic data from 100 purposefully chosen species that fully represented all of the higher teleostean orders, except for the Batrachoidiformes. Unweighted and weighted maximum parsimony analyses were conducted with the data set that comprised concatenated nucleotide sequences from 12 protein-coding genes (excluding 3rd codon positions) and 21 transfer RNA (tRNA) genes (stem regions only) from each species. The resultant trees were well resolved and largely congruent, with most internal branches being supported by high statistical values. All major, comprehensive groups above ordinal level as currently defined in higher teleosts (with the exception of the Neoteleostei and several monotypic groups), such as the Eurypterygii, Ctenosquamata, Acanthomorpha, Paracanthopterygii, Acanthopterygii, and Percomorpha, appeared to be nonmonophyletic in the present tree. Such incongruities largely resulted from differences in the placement and/or limits of the orders Ateleopodiformes, Lampridiformes, Polymixiiformes, Ophidiiformes, Lophiiformes, Beryciformes, Stephanoberyciformes, and Zeiformes, long-standing problematic taxa in systematic ichthyology. Of these, the resulting phylogenetic positions of the Ophidiiformes and Lophiiformes were totally unexpected, because, although they have consistently been considered relatively primitive groups within higher teleosts (Paracanthopterygii), they were confidently placed within a crown group of teleosts, herein called the Percomorpha. It should be noted that many unexpected, but highly supported relationships were found within the Percomorpha, being highly promising for the next investigative step towards resolution of this remarkably diversified group of teleosts.

Recent advances in next-generation sequencing (NGS) technology have accelerated the development of microsatellite markers for wildlife conservation genetics. Although the discovery of microsatellite-containing sequences based on NGS is more efficient with sequencing of a microsatellite-enriched library than with whole-genome shotgun sequencing, the process of constructing a microsatellite-enriched library is somewhat complicated. Therefore, many researchers prefer to use external services for the microsatellite-enrichment, which requires more time. To facilitate the rapid and effective development of novel microsatellite markers, we attempted to simplify the process of constructing a microsatellite-enriched library for multiplexed parallel sequencing. To capture microsatellite-containing sequences, we applied an easy-to-use commercially available kit for the hybridization and wash steps. After preparing shotgun libraries of 30 fish species for NGS, we captured microsatellite-containing DNA fragments directly from the shotgun libraries by using the commercially available kit. Next, three runs of multiplexed parallel sequencing were conducted on the 454 GS Junior platform. The resulting sequences for each species included high proportions of microsatellite-containing sequences (from 46 to 79%). Thus, sufficient numbers of primer sets, ranging from 1029 to 6606, were effectively designed for each species. Microsatellite capture and sequencing were completed in about a week, so the time required was substantially reduced. To validate the effectiveness of our strategy, we screened 44 potential primer sets designed for ayu (Plecoglossus altivelis). The results of polymorphisms revealed that allelic variability at 23 markers will be useful for studying population structure. These results prove the effectiveness of our improved approach for microsatellite marker development.

A microsatellite-enriched genomic library was obtained for the Japanese gudgeon Sarcocheilichthys variegatus microoculus, and 15 dinucleotide markers were successfully isolated and characterized. These markers were also available for other Japanese congeners, Sarcocheilichthys variegatus variegatus and Sarcocheilichthys biwaensis. In three populations of Sarcocheilichthys from Lake Biwa and the Ashida River in western Japan, seven to 27 alleles were observed for each locus. Linkage equilibrium was observed among most loci, and only one locus showed significant deviation from Hardy-Weinberg equilibrium in a population. These microsatellite markers will be useful for studies of the genetic structure of the Japanese gudgeons.

Twelve short tandem repeat markers were successfully isolated from a cichlid, Haplochromis chilotes, in Lake Victoria, and characterized in Haplochromis pyrrhocephalus. The microsatellite regions of these markers were found to have between two and 48 alleles with heterozygosity ranging from 0.07 to 0.97. No loci showed significant departures from the Hardy-Weinberg or linkage equilibrium after the Bonferroni correction (P > 0.05). Cross-species amplification in other cichlids of Lake Victoria, Haplochromis laparogramma, Lithochromis rubripinnis, L. rufus and Haplochromis sp. 'rockkribensis', was successful.

A microsatellite-enriched genomic library was obtained for the threatened brook lamprey, Lethenteron sp. N, and from it 16 dinucleotide markers were successfully isolated and characterized. These markers were found to have between 1 and 6 alleles with heterozygosity ranging from zero to 0.79. Cross-species amplification was successful, with eight loci amplifying products in the counterpart cryptic species, Lethenteron sp. S, as well as 15 loci in Lethenteron japonicum. One out of 10 primer pairs previously reported also amplified products in all the three species.

This article documents the addition of 142 microsatellite marker loci to the Molecular Ecology Resources database. Loci were developed for the following species: Agriophyllum squarrosum, Amazilia cyanocephala, Batillaria attramentaria, Fungal strain CTeY1 (Ascomycota), Gadopsis marmoratus, Juniperus phoenicea subsp. turbinata, Liriomyza sativae, Lupinus polyphyllus, Metschnikowia reukaufii, Puccinia striiformis and Xylocopa grisescens. These loci were cross-tested on the following species: Amazilia beryllina, Amazilia candida, Amazilia rutila, Amazilia tzacatl, Amazilia violiceps, Amazilia yucatanensis, Campylopterus curvipennis, Cynanthus sordidus, Hylocharis leucotis, Juniperus brevifolia, Juniperus cedrus, Juniperus osteosperma, Juniperus oxycedrus, Juniperus thurifera, Liriomyza bryoniae, Liriomyza chinensis, Liriomyza huidobrensis and Liriomyza trifolii. © 2013 John Wiley & Sons Ltd.

Background: A skewed assemblage of two epi-, meso- and bathypelagic fish families makes up the order Myctophiformes – the blackchins Neoscopelidae and the lanternfishes Myctophidae. The six rare neoscopelids show few morphological specializations whereas the divergent myctophids have evolved into about 250 species, of which many show massive abundances and wide distributions. In fact, Myctophidae is by far the most abundant fish family in the world, with plausible estimates of more than half of the oceans combined fish biomass. Myctophids possess a unique communication system of species-specific photophore patterns and traditional intrafamilial classification has been established to reflect arrangements of photophores. Myctophids present the most diverse array of larval body forms found in fishes although this attribute has both corroborated and confounded phylogenetic hypotheses based on adult morphology. No molecular phylogeny is available for Myctophiformes, despite their importance within all ocean trophic cycles, open-ocean speciation and as an important part of neoteleost divergence. This study attempts to resolve major myctophiform phylogenies from both mitogenomic sequences and corroborating evidence in the form of unique mitochondrial gene order rearrangements. Results: Mitogenomic evidence from DNA sequences and unique gene orders are highly congruent concerning phylogenetic resolution on several myctophiform classification levels, corroborating evidence from osteology, larval ontogeny and photophore patterns, although the lack of larval morphological characters within the subfamily Lampanyctinae stands out. Neoscopelidae is resolved as the sister family to myctophids with Solivomer arenidens positioned as a sister taxon to the remaining neoscopelids. The enigmatic Notolychnus valdiviae is placed as a sister taxon to all other myctophids and exhibits an unusual second copy of the tRNA-Met gene – a gene order rearrangement reminiscent of that found in the tribe Diaphini although our analyses show it to be independently derived. Most tribes are resolved in accordance with adult morphology although Gonichthyini is found within a subclade of the tribe Myctophini consisting of ctenoid scaled species. Mitogenomic sequence data from this study recognize 10 reciprocally monophyletic lineages within Myctophidae, with five of these clades delimited from additional rearranged gene orders or intergenic non-coding sequences. Conclusions: Mitogenomic results from DNA sequences and unique gene orders corroborate morphology in phylogeny reconstruction and provide a likely scenario for the phylogenetic history of Myctophiformes. The extent of gene order rearrangements found within the mitochondrial genomes of myctophids is unique for phylogenetic purposes. publishedVersion

The full-length of insulin-like growth factor (IGF) complementary (c)DNAs encoded by igf-I and igf-II from torafugu pufferfish Takifugu rubripes were cloned in the present study. The deduced amino acid sequences of the two genes showed c. 80% identity each with those of Igf-I and Igf-II from other teleosts, respectively. Two growth hormone (GH) receptors, ghr1 and ghr2, were also cloned in silico using the T. rubripes Fugu genome database. The transcripts of T. rubripes igf-I were detected in slow muscle, heart, skin, gill, liver and intestine but not in fast muscle, spleen and testis of adult fish, whereas those of igf-II were found in all tissues examined. Subsequently, the accumulated messenger (m)RNA levels of igf-I and igf-II were investigated in an F(2) population derived from a male of an apparent fast-growing T. rubripes strain and a wild female T. rubripes together with those of other growth-related genes encoding Gh, Ghr1 and Ghr2, and with those of prolactin (Prl) and leptin (Lep) previously reported. The accumulated mRNA levels of igf-I, gh and ghr1 were significantly correlated to growth rate at larval stages in the population, but not for those of igf-II, prl, ghr2 and lep. Although it is unclear whether or not this phenotype is directly related to the heredity of the fast-growing strain, the findings suggest that the expression of igf-I, gh and ghr1 is involved in the regulation of growth rate at larval stages in T. rubripes.

Twelve dinucleotide markers were successfully isolated and characterized from a microsatellite-enriched genomic library obtained for the gudgeon Squalidus chankaensis biwae. These markers were also available for the congeners S. c. tsuchigae and S. japonicus from Japan, which had five to 46 alleles and an expected heterozygosity ranging from zero to 0.946. Linkage equilibrium was observed at all loci, and most loci did not show significant deviation from Hardy-Weinberg equilibrium. The isolated microsatellite markers will be useful for genetic diversity studies of Squalidus populations.

We established an efficient and reliable method to identify the species and ploidy of Carassius fishes in Lake Biwa (C. cuvieri, C. buergeri, and the latter’s polyploid) using a combination of multiplex mitochondrial DNA haplotype-specific PCR and ploidy determination based on nuclear DNA microsatellite PCR. The high accuracy of ploidy determination was confirmed by flow cytometry. This method can also identify triploid clonal lines and provide information about their genetic diversity and structure. The method is applicable to ecological and evolutionary studies of Carassius fishes in Lake Biwa throughout their entire life history.

The migratory fish ayu, Plecoglossus altivelis, has two forms: an amphidromous form found throughout the Japanese islands and a landlocked form found in Lake Biwa. Because Lake Biwa is one of the sources of the Yodo River, which flows into Osaka Bay, it is likely that there has been contact between the two forms within the Yodo River system. To examine the effects of such contact, 573 ayu were collected from the Yodo River system and several other rivers and compared through an extensive investigation of microsatellite genotyping. Multivariate and individual admixture analyses of microsatellite data showed that samples of upstream migrants collected from the Yodo River in May 2003 included many individuals assigned to the amphidromous form, although fish collections in June and July 2003 were primarily composed of individuals of the landlocked form. Samples of drifting larvae, which we considered to be derived from eggs spawned in the Yodo River system, were mostly assigned to the amphidromous form. These results imply that an indigenous population of the amphidromous form exists and reproduces in the Yodo River system. It is likely that upstream migrants of the landlocked Lake Biwa form are supplied annually as well-developed larvae drifting out from Lake Biwa, but contribute very little to the next generation.

Uncertainties surrounding the evolutionary origin of the epipelagic fish family Scombridae (tunas and mackerels) are symptomatic of the difficulties in resolving suprafamilial relationships within Percomorpha, a hyperdiverse teleost radiation that contains approximately 17,000 species placed in 13 ill-defined orders and 269 families. Here we find that scombrids share a common ancestry with 14 families based on (i) bioinformatic analyses using partial mitochondrial and nuclear gene sequences from all percomorphs deposited in GenBank (10,733 sequences) and (ii) subsequent mitogenomic analysis based on 57 species from those targeted 15 families and 67 outgroup taxa. Morphological heterogeneity among these 15 families is so extraordinary that they have been placed in six different perciform suborders. However, members of the 15 families are either coastal or oceanic pelagic in their ecology with diverse modes of life, suggesting that they represent a previously undetected adaptive radiation in the pelagic realm. Time-calibrated phylogenies imply that scombrids originated from a deep-ocean ancestor and began to radiate after the end-Cretaceous when large predatory epipelagic fishes were selective victims of the Cretaceous-Paleogene mass extinction. We name this clade of open-ocean fishes containing Scombridae “Pelagia” in reference to the common habitat preference that links the 15 families.