Comprehensive Draft Genome Analyses of Three Rockfishes (Scorpaeniformes, Sebastiscus) via Genome Survey Sequencing.

Sebastiscus species, marine rockfishes, are of essential economic value. However, the genomic data of this genus is lacking and incomplete. Here, whole genome sequencing of all species of Sebastiscus was conducted to provide fundamental genomic information. The genome sizes were estimated to be 802.49 Mb (S. albofasciatus), 786.79 Mb (S. tertius), and 776.00 Mb (S. marmoratus) by using k-mer analyses. The draft genome sequences were initially assembled, and genome-wide microsatellite motifs were identified. The heterozygosity, repeat ratios, and numbers of microsatellite motifs all suggested possibly that S. tertius is more closely related to S. albofasciatus than S. marmoratus at the genetic level. Moreover, the complete mitochondrial genome sequences were assembled from the whole genome data and the phylogenetic analyses genetically supported the validation of Sebastiscus species. This study provides an important genome resource for further studies of Sebastiscus species.

Detection of heteroplasmy and nuclear mitochondrial pseudogenes in the Japanese spiny lobster Panulirus japonicus.

Partial mtDNA cytochrome oxidase subunit I (COI) fragments and near entire stretch of 12S rDNA (12S) and control region (Dloop) of the Japanese spiny lobster (Panulirus japonicus) (n = 3) were amplified by PCR and used for direct nucleotide sequencing and for clone library-based nucleotide sequence analysis. Nucleotide sequences of a total of 75 clones in COI, 77 in 12S and 92 in Dloop were determined. Haplotypes of the clones matched with those obtained by direct sequencing were determined to be genuine mtDNA sequence of the individual. Phylogenetic analysis revealed several distinct groups of haplotypes in all three regions. Genuine mtDNA sequences were observed to form a group with their closely related variables, and most of these variables may be due to amplification error but a few to be heteroplasmy. Haplotypes determined as nuclear mitochondrial pseudogenes (NUMTs) formed distinct groups. Nucleotide sequence divergence (K2P distance) between genuine haplotypes and NUMTs were substantial (7.169-23.880% for COI, 1.336-23.434% for 12S, and 7.897-71.862% for Dloop). These values were comparable to or smaller than those between species of the genus Panulirus, indicating that integration of mtDNA into the nuclear genome is a continuous and dynamic process throughout pre- and post-speciation events. Double peaks in electropherograms obtained by direct nucleotide sequencing were attributed to common nucleotides shared by multiple NUMTs. Information on the heteroplasmy and NUMTs would be very important for addressing their impact on direct nucleotide sequencing and for quality control of nucleotide sequences obtained.

Molecular Identification of Mid to Final Stage Slipper Lobster Phyllosoma Larvae of the Genus Chelarctus (Crustacea: Decapoda: Scyllaridae) Collected in the Pacific with Descriptions of Their Larval Morphology.

Morphological descriptions of phyllosoma larvae are essential for correct species identification and investigating the spatiotemporal distribution and recruitment process of spiny and slipper lobsters. Species identification of the phyllosoma larvae in the Scyllarinae subfamily is particularly difficult because of the morphological similarities among species and the scarcity of morphological information describing correct species identity. We extracted mid-to final-stage (V to VIII) phyllosoma larvae (n = 12) belonging to the subfamily Scyllarinae from several plankton samples collected in the Pacific and then performed molecular species identification using mitochondrial DNA COI and 16S rDNA sequence analyses. Three larvae collected around the Ryukyu Archipelago were identified as Chelarctus aureus (stage VI to VIII), and four collected around the Ryukyu Archipelago and Ogasawara Islands were identified as C. virgosus (V to VIII). One larva (V) collected in the central South Pacific was determined to be a subspecies of C. crosnieri. DNA barcodes could not be made for the remaining four larvae (V to VIII) collected around the Ryukyu Archipelago (designated by ?Chelarctus sp-1). Based on the morphological characteristics of the C. virgosus phyllosoma described in this study and the adult distributions reported to date, C. cultrifer phyllosomas previously reported in Japanese and Taiwanese waters are likely to be C. virgosus. This paper also presents a set of diagnostic morphological characteristics that can be used to discriminate among these four species of Chelarctus and from other genera in the subfamily Scyllarinae.

Comparative analysis of the complete mitochondrial genomes of three rockfishes (Scorpaeniformes, Sebastiscus) and insights into the phylogenetic relationships of Sebastidae.

Mitochondrial genome is a powerful molecule marker to provide information for phylogenetic relationships and revealing molecular evolution in ichthyological studies. Sebastiscus species, a marine rockfish, are of essential economic value. However, the taxonomic status and phylogenetic relationships of Sebastidae have been controversial so far. Here, the mitochondrial genomes (mitogenomes) of three species, S. tertius, S. albofasciatus, and S. marmoratus, were systemically investigated. The lengths of the mitogenomes' sequences of S. tertius, S. albofasciatus, and S. marmoratus were 16910, 17056, and 17580 bp, respectively. It contained 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNA (tRNA) genes, and one identical control region (D-loop) among the three species. The genetic distance and Ka/Ks ratio analyses indicated 13 PCGs were suffering purifying selection and the selection pressures were different from certain deep-sea fishes, which were most likely due to the difference in their living environment. The phylogenetic tree was constructed by Bayesian Inference (BI) and Maximum Likelihood (ML). Most interestingly, the results indicated that Sebastidae and Scorpaenidae were grouped into a separate branch, so the taxonomic status of Sebastidae should be classified into subfamily Sebastinae. Our results may lead to a taxonomic revision of Scorpaenoidei.

Inbreeding between Deep-Sea Snailfishes Careproctus pellucidus and Careproctus rastrinus in the Northwestern Pacific Ocean.

Genetic deviation between two deep-sea snailfishes, Careproctus pellucidus from the Pacific Ocean and Careproctus rastrinus from the Okhotsk Sea, of the Careproctus rastrinus species complex was analyzed, based on the nucleotide sequences of the mitochondrial cytochrome b (Cytb) gene. Our sampling revealed the occurrence of individuals with C. rastrinus-type Cytb genes off northeastern Japan, in the northwestern Pacific. Most of these individuals were collected from an area off Miyagi Prefecture, while few individuals were collected from areas to its north (off Iwate Prefecture) and south (off Fukushima and Ibaraki Prefectures). Phylogeographic analyses based on nucleotide sequences of the first intron region of the nuclear S7 ribosomal protein gene (S7) and 11 microsatellite loci indicated little genetic deviation between individuals with C. pellucidus-type and C. rastrinus-type Cytb genes in the area off Miyagi Prefecture. Significant genetic differences between the Pacific Ocean and the Okhotsk Sea populations may be attributed to unidirectional migration due to a greater current from the latter to the former. In addition, peritoneum and stomach colors of 10 and 17 specimens collected from the Pacific Ocean and the Okhotsk Sea, respectively, were evaluated quantitatively. The colors were significantly different for the two populations; however, they appeared to overlap. Although only one Pacific individual with a C. rastrinus-type Cytb gene was available for color evaluation, it was more similar to Pacific individuals than to Okhotsk Sea individuals. These results suggest inbreeding between C. pellucidus and C. rastrinus in the Pacific Ocean, off Miyagi Prefecture.

Close-up observations on the spawning behavior of a captive Japanese flying squid (Todarodes pacificus).

The spawning behavior of a Japanese flying squid (Todarodes pacificus) is described based on up-close observation of a captive female. The squid was first transferred from a 10-ton tank to a polystyrene plastic box containing 45 liters of seawater. About one hour later, the mantle-contraction rate increased rapidly, followed by a brief convulsion of the mantle and arms and a whitening of the body. The mantle contractions become shallow and rapid, and several seconds later, semitransparent jelly presumably from the nidamental glands emerged from the funnel and passed between the ventral pair of arms. Approximately 90 seconds after the egg mass first emerged, the female began ejecting oocytes through the funnel into the egg mass using rapid, powerful mantle contractions. Soon after the oocytes were ejected, translucent strands (presumably sperm) emanated from the buccal membrane. The female continued to eject oocytes for approximately two minutes, after which the mantle convulsed, and the mantle-contraction rate decreased slowly for about one minute until the contractions stopped. The squid died soon afterwards.

Molecular diet analysis of Anguilliformes leptocephalus larvae collected in the western North Pacific.

Natural diets of leptocephalus larvae have been enigmatic. In this study, we collected DNA samples from the gut contents and body surface of leptocephali belonging to the five Anguilliform families (Anguillidae, Chlopsidae, Congridae, Muraenidae, and Serrivomeridae) from the northwest Pacific and performed next-generation 18S rDNA sequencing. Wide variety of eukaryotes was detected in both samples, from which eight eukaryotic groups (jellyfish, conoid parasite, tunicate, copepod, krill, segmented worm, fungi, and dinoflagellate) were selected on the basis of abundance. All groups except conoid parasites were common in both the samples. Cnidarian 18S rDNA reads were the most abundant in both the samples; however, the number of samples having cnidarian reads and the read counts were significantly higher in the body surface scraping samples than in the gut content samples, regardless of careful rinsing of the body surface. These results indicate that the cnidarian DNAs are most likely found because of cross contamination from the body surface and/or environment. 18S rDNA read counts of copepod and tunicate in the gut contents were greater than or comparable with those in the body surface scraping samples, which may correspond to the previous observations of fecal pellets and larvacean houses in the leptocephali gut. Thus, the present study supports previous implications that leptocephali utilize detritus materials, so called marine snow.

Population genomics reveals possible genetic evidence for parallel evolution of Sebastiscus marmoratus in the northwestern Pacific Ocean.

Understanding patterns of population diversity and structuring among marine populations is of great importance for evolutionary biology, and can also directly inform fisheries management and conservation. In this study, genotyping-by-sequencing was used to assess population genetic diversity and connectivity of Sebastiscus marmoratus. Based on 130 individuals sampled from 10 locations in the northwestern Pacific Ocean, we identified and genotyped 17 653 single-nucleotide polymorphisms. The patterns of genetic diversity and population differentiation suggested that the Okinawa Trough might be the ancestral centre of S. marmoratus after the Last Glacial Maximum. A shallow genetic structure was observed among sampled populations based on the implemented structuring approaches. Surprisingly, we detected genetic homogeneity in two population pairs (i.e. Xiamen-Niigata and Zhuhai-Iki Island), in which populations have large geographical and latitudinal intervals. Population structure and allele frequency distribution based on outlier loci also mirrored the observed genetic homogeneity in the above-mentioned population pairs. Integrated with biological, environmental and genomic data, our results provide possible genetic evidence for parallel evolution. Our study also provides new perspectives on the population structure of S. marmoratus, which could have important implications for sound management and conservation of this fishery species.

Phylogeographic analysis of the genus Platycephalus along the coastline of the northwestern Pacific inferred by mitochondrial DNA.

BACKGROUND: Flathead fishes of the genus Platycephalus are economically important demersal fishes that widely inhabit the continental shelves of tropical and temperate sea waters. This genus has a long history of taxonomic revision, and recently four Platycephalus species (Platycephalus sp. 1, Platycephalus sp. 2, P. indicus, and P. cultellatus) in the northwestern Pacific Ocean (NWP) have been recognized and redescribed. However, many aspects of their systematics and evolutionary history are unclear. RESULTS: A total of 411 individuals were sampled from 22 different sites across their distributions in the NWP. Three mitochondrial loci were sequenced to clarify the phylogeny and phylogeographic history of the fishes. The results showed significant differentiation of four Platycephalus species in the NWP with well-supported clades in which Platycephalus sp. 1 and Platycephalus sp. 2 were the closest, clustered with P. cultellatus, while their genetic relationship with P. indicus was the furthest. There were significant genealogical branches corresponding to P. indicus but not to other Platycephalus lineages. We further examined the phylogeographic patterns of 16 Platycephalus sp. 1 populations along the coastlines of China and Japan. A total of 69 haplotypes were obtained, with 23 shared among populations. One dominant haplotypic group, with a modest lineage structure and low levels of haplotype diversity and nucleotide diversity, was observed among Platycephalus sp. 1 populations. The demographic history reconstruction suggested a Platycephalus sp. 1 population expansion event dating back to the late Pleistocene. CONCLUSIONS: Distributional rang variations may be the crucial factors shaping the genetic relationships of the genus Platycephalus. Reproductive schooling and potential egg/larval dispersal ability, coupled with the effects of ocean currents, are responsible for the present phylogeographic pattern of Platycephalus sp. 1.

Population genetic structure of Marbled Rockfish, Sebastiscusmarmoratus (Cuvier, 1829), in the northwestern Pacific Ocean.

Sebastiscusmarmoratus is an ovoviviparous fish widely distributed in the northwestern Pacific. To examine the gene flow and test larval dispersal strategy of S.marmoratus in Chinese and Japanese coastal waters, 421 specimens were collected from 22 localities across its natural distribution. A 458 base-pair fragment of the mitochondrial DNA (mtDNA) control region was sequenced to examine genetic diversity and population structure. One-hundred-six variable sites defined 166 haplotypes. The populations of S.marmoratus showed high haplotype diversity with a range from 0.8587 to 0.9996, indicating a high level of intrapopulation genetic diversity. Low non-significant genetic differentiation was estimated among populations except those of Hyogo, Behai, and Niiigata, which showed significant genetic differences from the other populations. The demographic history examined by neutrality tests, mismatch distribution analysis, and Bayesian skyline analysis suggested a sudden population expansion dating to the late Pleistocene. Recent population expansion in the last glacial period, wide dispersal of larvae by coastal currents, and the homogeneity of the environment may have important influences on the population genetic pattern. Knowledge of genetic diversity and genetic structure will be crucial to establish appropriate fishery management of S.marmoratus.

Levels and patterns of genetic variation in Japanese whiting (Sillago japonica) based on mitochondrial DNA control region.

Japanese whiting (Sillago japonica) is a commercially important demersal fish distributed along the coasts of East Asia and becomes recently an aquaculture species. Despite its commercial importance, the levels and patterns of population genetic variation remain poorly understood. In this study, 346 specimens were collected from 14 localities along the coastal waters of China, Korea and Japan and their genetic variation was analyzed with mtDNA control region (D-loop) sequences. A total of 131 polymorphic sites were detected which determined 294 haplotypes. A pattern with high levels of haplotype diversity (h = 0.999 ± 0.001) and nucleotide diversity (л = 0.030 ± 0.015) was detected in the examined range. Analyses of analysis of molecular variance (AMOVA) and Fst showed that no significant genetic differentiation existed among China, Korea and Japan populations, excepting for the populations between Ise Bay (IBP) sample and the other ones. Minimum spanning tree (MST), neutrality tests, mismatch distribution and Bayesian skyline analyses indicated that the species along coastline of China, Korea and Japan have experienced population expansions originated in its most recent history at about 106-423 kya during the late Pleistocene glaciations and deglaciations periods.

Assessing the Speciation of a Cold Water Species, Japanese Sand Lance Ammodytes personatus, in the Northwestern Pacific by AFLP Markers.

The use of molecular techniques in biodiversity research increasingly results in the recognition of multiple divergent mitochondrial DNA (mtDNA) lineages below the morphospecies level. However, the overlapping distribution of multiple divergent lineages raises the question of whether some of these lineages are in fact cryptic species. Assessing the status of these divergent lineages, delimiting evolutionarily significant units (ESUs), and identifying the dominant evolutionary and ecological drivers are critical components of successful wildlife conservation and management strategies. Amplified fragment length polymorphism (AFLP) markers were applied to characterize the phylogeography pattern of a cold water species, the Japanese sand lance Ammodytes personatus, in warm and cold ocean currents. A total of 211 individuals sampled from 12 populations through the species' range, including samples from Kuroshio Current, Oyashio Current, Tsushima Current, and Yellow Sea, were analyzed. The Bayesian assignment probability test and Neighbor joining (NJ) analysis divided these populations into two genetically and geographically distinct clades (northern and southern clades) characterized by different sea surface temperatures. The incongruence between nuclear clades and previous mitochondrial lineages suggested that A. personatus is indeed composed of at least two genetically divergent cryptic species. Pleistocene glaciation isolation after secondary contact, local thermal adaptation, and isolation by distance may explain the observed geographic pattern of two cryptic species and genetic structure within clades.

Restricted Gene Flow for Gadus macrocephalus from Yellow Sea Based on Microsatellite Markers: Geographic Block of Tsushima Current.

The Pacific cod Gadus macrocephalus is a demersal, economically important fish in the family Gadidae. Population genetic differentiation of Pacific cod was examined across its northwestern Pacific range by screening variation of eight microsatellite loci in the present study. All four populations exhibited high genetic diversity. Pairwise fixation index (Fst) suggested a moderate to high level of genetic differentiation among populations. Population of the Yellow Sea (YS) showed higher genetic difference compared to the other three populations based on the results of pairwise Fst, three-dimensional factorial correspondence analysis (3D-FCA) and STRUCTURE, which implied restricted gene flow among them. Wilcoxon signed rank tests suggested no significant heterozygosity excess and no recent genetic bottleneck events were detected. Microsatellite DNA is an effective molecular marker for detecting the phylogeographic pattern of Pacific cod, and these Pacific cod populations should be three management units.

Lack of population genetic differentiation of a marine ovoviviparous fish Sebastes schlegelii in Northwestern Pacific.

Sebastes schlegelii is one of the fishes that aggregate around drifting seaweed during early development. To examine the population genetic structure of S. schlegelii, a 452-bp fragment of the mtDNA control region was sequenced and used to interpret life history characteristics and larval dispersal strategy. Two-hundred and twenty-one individuals from 13 sites across the entire range of S. schlegelii in China, Japan and Korea were analyzed. A neighbor-joining tree and network showed that there were no significant genealogical structures corresponding to sampling locations. AMOVA, pair-wise FST and exact test revealed no significant genetic differentiation among locations. The migration rate among locations was high based on the result of LAMARC. We conclude that larval dispersal with drifting seaweed and the current environmental factors may play an important role in shaping the contemporary phylogeographic pattern and genetic homogeneity of S. schlegelii.

Population genetic structure of chub mackerel Scomber japonicus in the Northwestern Pacific inferred from microsatellite analysis.

Marine pelagic fishes are usually characterized by subtle but complex patterns of genetic differentiation, which are influenced by both historical process and contemporary gene flow. Genetic population differentiation of chub mackerel, Scomber japonicus, was examined across most of its range in the Northwestern Pacific by screening variation of eight microsatellite loci. Our genetic analysis detected a weak but significant genetic structure of chub mackerel, which was characterized by areas of gene flow and isolation by distance. Consistent with previous estimates of stock structure, we found genetic discontinuity between Japan and China samples. Local-scale pattern of genetic differentiation was observed between samples from the Bohai Sea and North Yellow Sea and those from the East China Sea, which we ascribed to differences in spawning time and migratory behavior. Furthermore, the observed homogeneity among collections of chub mackerel from the East and South China Seas could be the result of an interaction between biological characteristics and marine currents. The present study underlies the importance of understanding the biological significance of genetic differentiation to establish management strategies for exploited fish populations.

The complete mitochondrial genome of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae) from Japan.

The complete mitochondrial genome of marbled rockfish Sebastiscus marmoratus collected from Japan was determined by next-generation sequencing. The mitogenome is a circular molecule 17,301 bp in length, including the typical structure of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and a control region. The ETAS, central CSB and CSB were detected in the control region. The gene contents of the mitogenome are identical to those observed in most bony fishes.

Genetic differentiation of Trachurus japonicus from the Northwestern Pacific based on the mitochondrial DNA control region.

Trachurus japonicus is a pelagic fish widely distributed from China, Korea to Japan. To examine the gene flow of T. japonicus between Chinese and Japanese coastal waters, eight populations were collected for the present study. Eighty-seven variable sites defined 168 haplotypes. The populations of T. japonicus showed high haplotype diversity (h) with a range from 0.964 ± 0.027 to 1.000 ± 0.016 and low nucleotide diversity with a range from 0.011 ± 0.006 to 0.015 ± 0.008. The topology of the neighbour-joining tree showed no significant genealogical branches or clusters corresponding to sampling localities. The starburst structure of the minimum spanning tree suggested a very recent origin for most haplotypes. Both pairwise F st and analysis of molecular variance revealed no significant genetic differentiation throughout the range examined. The results of neutrality tests and mismatch distributions indicated that T. japonicus may experience a recent population demographic expansion.

Complete mitochondrial genome of Japanese Konosirus punctatus (Clupeiformes: Clupeidae).

The long polymerase chain reaction and primer walking methods were employed for the complete mitochondrial genome (mitogenome) sequence of Japanese Konosirus punctatus. The mitogenome is a circular molecule 16,698 bp in length, including the typical structure of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 non-coding regions (L-strand replication origin and control region), the gene contents of which are identical to those observed in most bony fishes. Within the control region, we identified the termination-associated sequence domain, the central conserved sequence block domains (CSB-F, CSB-E, CSB-D, CSB-C, and CSB-B), and the conserved sequence block domains (CSB-2 and CSB-3).

Genetic stock structure of walleye pollock (Theragra chalcogramma) inferred by PCR-RFLP analysis of the mitochondrial DNA and SNP analysis of nuclear DNA.

Walleye pollock, Theragra chalocogramma, is one of the most important species in the North Pacific and Bering Sea ecosystems. However genetic population structuring of walleye pollock is uncertain. In the present study, genetic variation of walleye pollock collected in several spawning areas ranging from the Japan Sea to the Gulf of Alaska was investigated by DNA analysis. Three regions of the spacer control region, the ND5 and ND6 region (ND complex), and the ND1 and 16S rRNA region (rDNA complex) were amplified using the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) was conducted on these PCR products and composite haplotypes were calculated. Furthermore, several nuclear DNA regions (actin, Calmodulin, S7 ribosomal protein, creatin kinase, and SypI gene) were investigated to study the stock structure of walleye pollock. It was considered that Calmodulin gene was one of good genetic marker, therefore we conducted SNP analysis for Calmodulin gene by SnaPshot kits. In RFLP analyses, there were no area-specific fragment patterns in the three regions, control region, ND complex and rDNA complex of mtDNA. However compositions of the fragment patterns for the three digested sets, control region/HinfI, rDNA complex/MspI and ND complex/MspI indicated that there are significant differences between around the Japan (Sado-Funka Bay-Wakkanai-Rausu) and the Bering Sea (Western Bering Sea-Nabarin-Atka I.-Bogoslof I). Furthermore, in the case of haplotype frequency, composition showed also significant genetic difference between two areas. Moreover, in Calmodulin analyses, haplotype compositions were changing from western area to eastern area gradually and the results of AMOVA analysis showed that there are interesting differences between western Pacific, western Bering Sea, and eastern Bering Sea. Judging from these results, it was considered that there are three populations of walleye pollock in the Northern Ocean. However, area-specific pattern was not found in some populations in the Northern Ocean. Therefore, we suggested that these populations were related by weak gene flow, and the walleye pollock was formed with meta-population around the Japan and the Bering Sea.