Chromosomal DNA sequences of the Pacific saury genome: versatile resources for fishery science and comparative biology.

Pacific saury (Cololabis saira) is a commercially important small pelagic fish species in Asia. In this study, we conducted the first-ever whole genome sequencing of this species, with single molecule, real-time (SMRT) sequencing technology. The obtained high-fidelity (HiFi) long-read sequence data, which amount to ~30-folds of its haploid genome size that was measured with quantitative PCR (1.17 Gb), were assembled into contigs. Scaffolding with Hi-C reads yielded a whole genome assembly containing 24 chromosome-scale sequences, with a scaffold N50 length of 47.7 Mb. Screening of repetitive elements including telomeric repeats was performed to characterize possible factors that need to be resolved towards 'telomere-to-telomere' sequencing. The larger genome size than in medaka, a close relative in Beloniformes, is at least partly explained by larger repetitive element quantity, which is reflected in more abundant tRNAs, in the Pacific saury genome. Protein-coding regions were predicted using transcriptome data, which resulted in 22,274 components. Retrieval of Pacific saury homologs of aquaporin (AQP) genes known from other teleost fishes validated high completeness and continuity of the genome assembly. These resources are available at https://treethinkers.nig.ac.jp/saira/ and will assist various molecular-level studies in fishery science and comparative biology.

Mitochondrial genome of the garfish Hyporhamphus quoyi (Beloniformes: Hemiramphidae) and phylogenetic relationships within Beloniformes based on whole mitogenomes.

Mitochondrial DNA (mtDNA) can provide genome-level information (e.g. mitochondrial genome structure, phylogenetic relationships and codon usage) for analyzing molecular phylogeny and evolution of teleostean species. The species in the order Beloniformes have commercial importance in recreational fisheries. In order to further clarify the phylogenetic relationship of these important species, we determined the complete mitochondrial genome (mitogenome) of garfish Hyporhamphus quoyi of Hemiramphidae within Beloniformes. The mitogenome was 16,524 bp long and was typical of other teleosts mitogenomes in size and content. Thirteen PCGs started with the typical ATG codon (with exception of the cytochrome coxidase subunit 1 (cox1) gene with GTG). All tRNA sequences could be folded into expected cloverleaf secondary structures except for tRNASer (AGN) which lost a dihydrouracil (DHU) stem. The control region was 866 bp in length, which contained some conserved sequence blocks (CSBs) common to Beloniformes. The phylogenetic relationship between 26 fish Beloniformes species was analyzed based on the complete nucleotide and amino acid sequences of 13 PCGs by two different inference methods (Maximum Likelihood and Bayesian Inference). Phylogenetic analyses revealed Hemiramphidae as the sister group to Exocoetidae and it is a paraphyletic grouping. Our results may provide useful information on mitogenome evolution of teleostean species.

Exploring hidden diversity in Southeast Asia's Dermogenys spp. (Beloniformes: Zenarchopteridae) through DNA barcoding.

Members of the freshwater halfbeak genus Dermogenys are hard to identify to the species level, despite several previous attempts to isolate fixed meristic, morphometric and colour pattern differences. This has led to ongoing confusion in scientific literature, records of species occurrence, and entries in museum collections. Here, a DNA barcoding study was conducted on the genus to gain further understanding of its taxonomic status across the Southeast Asian region. Fish were collected from 33 localities, spanning freshwater and brackish habitats in Malaysia, Western Indonesia, Thailand and Vietnam. In total, 290 samples of Dermogenys spp. were amplified for a 651 base pair fragment of the mitochondrial cytochrome oxidase c subunit I (COI) gene. Analysis was able to successfully differentiate the three species: D. collettei, D. siamensis, D. sumatrana; reveal the presence of a new putative species, Dermogenys sp., that was sampled in sympatry with D. collettei at three locations; as well as uncovering two genetic lineages of a fifth species, D. bispina, that display non-overlapping geographical distributions in drainages of northern Borneo; Kudat and Sandakan. This study expands the barcode library for Zenarchopteridae, demonstrates the efficacy of DNA barcoding techniques for differentiating Dermogenys species, and the potential thereof in species discovery.

Population Genetic Structure of the Tropical Two-Wing Flyingfish (Exocoetus volitans).

Delineating populations of pantropical marine fish is a difficult process, due to widespread geographic ranges and complex life history traits in most species. Exocoetus volitans, a species of two-winged flyingfish, is a good model for understanding large-scale patterns of epipelagic fish population structure because it has a circumtropical geographic range and completes its entire life cycle in the epipelagic zone. Buoyant pelagic eggs should dictate high local dispersal capacity in this species, although a brief larval phase, small body size, and short lifespan may limit the dispersal of individuals over large spatial scales. Based on these biological features, we hypothesized that E. volitans would exhibit statistically and biologically significant population structure defined by recognized oceanographic barriers. We tested this hypothesis by analyzing cytochrome b mtDNA sequence data (1106 bps) from specimens collected in the Pacific, Atlantic and Indian oceans (n = 266). AMOVA, Bayesian, and coalescent analytical approaches were used to assess and interpret population-level genetic variability. A parsimony-based haplotype network did not reveal population subdivision among ocean basins, but AMOVA revealed limited, statistically significant population structure between the Pacific and Atlantic Oceans (ΦST = 0.035, p<0.001). A spatially-unbiased Bayesian approach identified two circumtropical population clusters north and south of the Equator (ΦST = 0.026, p<0.001), a previously unknown dispersal barrier for an epipelagic fish. Bayesian demographic modeling suggested the effective population size of this species increased by at least an order of magnitude ~150,000 years ago, to more than 1 billion individuals currently. Thus, high levels of genetic similarity observed in E. volitans can be explained by high rates of gene flow, a dramatic and recent population expansion, as well as extensive and consistent dispersal throughout the geographic range of the species.

Genetic diversity and population structure of Hyporhamphus sajori (Beloniformes: Hemiramphidae) inferred from mtDNA control region and msDNA markers.

This paper presents preliminary data on the genetic diversity and population structure of Hyporhamphus sajori by analysing a 510 bp sequence in the mitochondrial DNA (mtDNA) control region and eight polymorphic microsatellite DNA loci. The H. sajori individuals from different locations were indistinguishable from one another based on mtDNA variation, as demonstrated with a neighbour-joining tree and minimum spanning network analysis. Low level of genetic diversity and the absence of population structure in H. sajori from the north-west Pacific Ocean, combined with negative indices for neutral evolution in these populations, suggest that H. sajori underwent a population expansion after a recent bottleneck. The Structure analysis, discriminant analysis of principal components (DAPC) and the pair-wise ΦST values after Bonferroni correction using eight microsatellite loci provided no clear inference on the genetic differentiation and thus no evidence of population structure of H. sajori. The genetic connectivity among locations might be due to fairly high gene flow via transport of eggs and larvae by the Kuroshio and Tsushima warm current. This study revealed low levels of genetic diversity and suggested high level of contemporary gene flow among populations of H. sajori in the East (Japan) Sea and the Pacific Ocean.

Complete mitochondrial genome of the Asian pencil halfbeak Hyporhamphus intermedius (Beloniformes, Hemirhamphidae).

The complete mitochondrial genome of Hyporhamphus intermedius was determined to be 16,720 bp in length with (A + T) content of 56.3%, and it consists of 13 protein-coding genes, 22 tRNAs, two ribosomal RNAs, and a control region. The gene composition and the structural arrangement of the H. intermedius complete mtDNA were identical to most of the other vertebrates. Interestingly, two tandem repeat units were identified across tRNA-Pro and control region (2*41 bp), while in most of the fishes the tandem repeat units are located in the control region. The molecular data we presented here could play a useful role to study the evolutionary relationships and population genetics of Hemirhamphidae fish.

The complete mitochondrial genome of the Pacific needlefish Strongylura anastomella (Belonidae, Beloniformes) from Korea.

Belonidae is a good model for investigating speciation and biogeography. To obtain basic information on the phylogeny of Belonidae, we determined the complete mitogenome of Strongylura anastomella using next-generation sequencing. The complete mitogenome is 16,534 bp in length and consists of 2 rRNAs, 22 tRNAs, 13 protein-coding genes and a control region. The nucleotide composition is 31.1% A, 29.8% T, 14.5% G and 24.6% C, with an AT bias (60.9%). The gene direction and position were similar to those of other Beloniformes. Belonidae and Scomberesocidae were separated in the phylogenetic tree based on complete mitogenomes. Further study is required to clarify the phylogenetic relationships among Belonidae and families within Beloniformes.

Paleo-drainage basin connectivity predicts evolutionary relationships across three Southeast Asian biodiversity hotspots.

Understanding factors driving diversity across biodiversity hotspots is critical for formulating conservation priorities in the face of ongoing and escalating environmental deterioration. While biodiversity hotspots encompass a small fraction of Earth's land surface, more than half the world's plants and two-thirds of terrestrial vertebrate species are endemic to these hotspots. Tropical Southeast (SE) Asia displays extraordinary species richness, encompassing four biodiversity hotspots, though disentangling multiple potential drivers of species richness is confounded by the region's dynamic geological and climatic history. Here, we use multilocus molecular genetic data from dense multispecies sampling of freshwater fishes across three biodiversity hotspots, to test the effect of Quaternary climate change and resulting drainage rearrangements on aquatic faunal diversification. While Cenozoic geological processes have clearly shaped evolutionary history in SE Asian halfbeak fishes, we show that paleo-drainage re-arrangements resulting from Quaternary climate change played a significant role in the spatiotemporal evolution of lowland aquatic taxa, and provide priorities for conservation efforts.

Successful extraction of DNA from archived alcohol-fixed white-eye fish specimens using an ancient DNA protocol.

A protocol used routinely for rapid ancient DNA extraction was applied to fish tissue archived over 80 years ago. The method proved successful, whereas other extraction protocols failed. Researchers working on DNA from older archived fish samples are encouraged to continue to concentrate their efforts on 'white-eye' specimens, which indicate an alcohol-based fixative and are thus likely to yield viable DNA.

Molecular phylogeny of the medaka fishes genus Oryzias (Beloniformes: Adrianichthyidae) based on nuclear and mitochondrial DNA sequences.

The phylogenetic relationships among medaka fishes of 2 genera, Oryzias and Xenopoecilus, were studied using the nuclear tyrosinase and mitochondrial 12S and 16S rRNA genes. Of the 23 species currently described for these genera, 13 species of Oryzias and 2 species of Xenopoecilus were examined. The tree topologies obtained from the nuclear and mitochondrial data were consistent, indicating that Xenopoecilus is a polyphyletic genus nested within Oryzias. This result suggested the necessity for a systematic study and taxonomic revision of Xenopoecilus. The combined data analysis of all data partitions resulted in a well-resolved tree, with most internal branches supported by high statistical values. Based on our combined data phylogeny, we divided the Oryzias species into three major species groups, namely the latipes, javanicus, and celebensis groups. These three groups corresponded to the three chromosomal groups (biarmed, monoarmed, and fused chromosome groups) previously proposed from karyological analyses. The phylogeographic pattern suggests historical vicariance between Sulawesi Island and the continental shelf.

Molecular phylogenetics and biogeography of transisthmian and amphi-Atlantic needlefishes (Belonidae: Strongylura and Tylosurus): perspectives on New World marine speciation.

Phylogenetic relationships among New World and eastern Atlantic species in the belonid genera Strongylura and Tylosurus were hypothesized using 3689bp of nucleotide sequence; including the entire mitochondrial (mtDNA) ATP synthase 6 and 8 genes; partial cytochrome b; 12S and 16S ribosomal genes; and introns and exons, 2 and 3 of the nuclear-encoded creatine kinase B gene. Concordant mtDNA and nuclear genealogies permitted well-supported inference of species relationships within Strongylura and Tylosurus, and of the chronology of diversification in the two genera. Our phylogenetic hypothesis permitted an assessment of Rosen's [Syst. Zool. 24 (1975) 431] model of species diversification across the eastern Atlantic to eastern Pacific marine biogeographic track. The spatial predictions of the Rosen model were generally supported, but not the temporal predictions. Furthermore, long branches leading to terminal Belonidae indicated that many species have persisted for millions of years or that nucleotide substitution rates were elevated for some clades. Though heterogeneity of nucleotide substitution rate was indicated across some belonid lineages, molecular clock estimates were used to hypothesize biogeographic scenarios for Strongylura across the eastern Pacific and Atlantic region. Furthermore, use of a molecular clock indicated; that early diversification among contemporary Strongylura may have been initiated by changes in Atlantic Ocean circulation precipitated by closure of the Tethys Sea; and provided approximate dates for the isolation of the freshwater species on the American continents.

A new species of halfbeak, Hyporhamphus naos (Beloniformes: Hemiramphidae), from the tropical eastern Pacific

The tropical eastern Pacific halfbeak previously considered conspecific with the western Atlantic Hyporhamphus unifasciatus (Ranzani 1842) is described as a new species, H. naos. It resembles H. meeki from the Atlantic and Gulf coasts of the United States in number of gill rakers on the first arch (usually 32-36, mean 33.6), more than in H. unifasciatus (usually 29-32, mean 30.6), but fewer than in other sympatric species of eastern Pacific Hyporhamphus. Results of a three-treatment ANCOVA (H. naos, H. meeki, and H. unifasciatus) show significant differences in slopes and means for all 14 morphometric characters examined, 9 of 14 characters comparing H. naos with H. unifasciatus, and 7 of 14 comparing H. naos with H. meeki. Protein electrophoretic patterns clearly distinguish all three species with a number of fixed allelic differences.

Status of the eastern Pacific agujon needlefish Tylosurus pacificus (Steindachner, 1876) (Beloniformes: Belonidae)

Tylosurus pacificus (Steindachner, 1876) is confirmed to have full species rank based on: 1) sympatry with T. acus melanotus at Isla Gorgona and in Panamá; 2) level of morphological differentiation in numbers of vertebrae, dorsal and anal fin rays; and 3) level of mtDNA differentiation. The eastern Pacific agujon needlefish is found from the Gulf of California, Mexico, to Peru, including the Galápagos Islands.

A new species of halfbeak, Hyporhamphus naos (Beloniformes: Hemiramphidae), from the tropical eastern Pacific.

The tropical eastern Pacific halfbeak previously considered conspecific with the western Atlantic Hyporhamphus unifasciatus (Ranzani 1842) is described as a new species, H. naos. It resembles H. meeki from the Atlantic and Gulf coasts of the United States in number of gill rakers on the first arch (usually 32-36, mean 33.6), more than in H. unifasciatus (usually 29-32, mean 30.6), but fewer than in other sympatric species of eastern Pacific Hyporhamphus. Results of a three-treatment ANCOVA (H. naos, H. meeki, and H. unifasciatus) show significant differences in slopes and means for all 14 morphometric characters examined, 9 of 14 characters comparing H. naos with H. unifasciatus, and 7 of 14 comparing H. naos with H. meeki. Protein electrophoretic patterns clearly distinguish all three species with a number of fixed allelic differences.

Status of the eastern Pacific agujon needlefish Tylosurus pacificus (Steindachner, 1876) (Beloniformes: Belonidae).

Tylosurus pacificus (Steindachner, 1876) is confirmed to have full species rank based on: 1) sympatry with T. acus melanotus at Isla Gorgona and in Panamá; 2) level of morphological differentiation in numbers of vertebrae, dorsal and anal fin rays; and 3) level of mtDNA differentiation. The eastern Pacific agujon needlefish is found from the Gulf of California, Mexico, to Peru, including the Galápagos Islands.