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We developed dbCNS (http://yamasati.nig.ac.jp/dbcns), a new database for conserved noncoding sequences (CNSs). CNSs exist in many eukaryotes and are assumed to be involved in protein expression control. Version 1 of dbCNS, introduced here, includes a powerful and precise CNS identification pipeline for multiple vertebrate genomes. Mutations in CNSs may induce morphological changes and cause genetic diseases. For this reason, many vertebrate CNSs have been identified, with special reference to primate genomes. We integrated ∼6.9 million CNSs from many vertebrate genomes into dbCNS, which allows users to extract CNSs near genes of interest using keyword searches. In addition to CNSs, dbCNS contains published genome sequences of 161 species. With purposeful taxonomic sampling of genomes, users can employ CNSs as queries to reconstruct CNS alignments and phylogenetic trees, to evaluate CNS modifications, acquisitions, and losses, and to roughly identify species with CNSs having accelerated substitution rates. dbCNS also produces links to dbSNP for searching pathogenic single-nucleotide polymorphisms in human CNSs. Thus, dbCNS connects morphological changes with genetic diseases. A test analysis using 38 gnathostome genomes was accomplished within 30 s. dbCNS results can evaluate CNSs identified by other stand-alone programs using genome-scale data.

The genus Acropora comprises the most diverse and abundant scleractinian corals (Anthozoa, Cnidaria) in coral reefs, the most diverse marine ecosystems on Earth. However, the genetic basis for the success and wide distribution of Acropora are unknown. Here, we sequenced complete genomes of 15 Acropora species and 3 other acroporid taxa belonging to the genera Montipora and Astreopora to examine genomic novelties that explain their evolutionary success. We successfully obtained reasonable draft genomes of all 18 species. Molecular dating indicates that the Acropora ancestor survived warm periods without sea ice from the mid or late Cretaceous to the Early Eocene and that diversification of Acropora may have been enhanced by subsequent cooling periods. In general, the scleractinian gene repertoire is highly conserved; however, coral- or cnidarian-specific possible stress response genes are tandemly duplicated in Acropora. Enzymes that cleave dimethlysulfonioproprionate into dimethyl sulfide, which promotes cloud formation and combats greenhouse gasses, are the most duplicated genes in the Acropora ancestor. These may have been acquired by horizontal gene transfer from algal symbionts belonging to the family Symbiodiniaceae, or from coccolithophores, suggesting that although functions of this enzyme in Acropora are unclear, Acropora may have survived warmer marine environments in the past by enhancing cloud formation. In addition, possible antimicrobial peptides and symbiosis-related genes are under positive selection in Acropora, perhaps enabling adaptation to diverse environments. Our results suggest unique Acropora adaptations to ancient, warm marine environments and provide insights into its capacity to adjust to rising seawater temperatures.

Crown-of-thorns starfish, Acanthaster planci (COTS), are common in coral reefs of Indo-Pacific Ocean. Since they are highly fecund predators of corals, periodic outbreaks of COTS cause substantial loss of healthy coral reefs. Using complete mitochondrial DNA sequences, we here examined how COTS outbreaks in the Ryukyu Archipelago, Japan are reflected by the profile of their population genetics. Population genetics of the blue starfish, Linckia laevigata, which lives in the Ryukyu Archipelago, but not break out and the northern Pacific sea star, Asterias amurensis, which lives in colder seawater around the main Islands of Japan, were also examined as controls. Our results showed that As. amurensis has at least two local populations that diverged approximately 4.7 million years ago (MYA), and no genetic exchanges have occurred between the populations since then. Linckia laevigata shows two major populations in the Ryukyu Archipelago that likely diverged ∼6.8 MYA. The two populations, each comprised of individuals collected from coast of the Okinawa Island and those from the Ishigaki Island, suggest the presence of two cryptic species in the Ryukyu Archipelago. On the other hand, population genetics of COTS showed a profile quite different from those of Asterias and Linckia. At least five lineages of COTS have arisen since their divergence ∼0.7 MYA, and each of the lineages is present at the Okinawa Island, Miyako Island, and Ishigaki Island. These results suggest that COTS have experienced repeated genetic bottlenecks that may be associated with or caused by repeated outbreaks.

Comparative genome-scale analyses of protein-coding gene sequences are employed to examine evidence for whole-genome duplication and horizontal gene transfer. For this purpose, an orthogroup should be delineated to infer evolutionary history regarding each gene, and results of all orthogroup analyses need to be integrated to infer a genome-scale history. An orthogroup is a set of genes descended from a single gene in the last common ancestor of all species under consideration. However, such analyses confront several problems: 1) Analytical pipelines to infer all gene histories with methods comparing species and gene trees are not fully developed, and 2) without detailed analyses within orthogroups, evolutionary events of paralogous genes in the same orthogroup cannot be distinguished for genome-wide integration of results derived from multiple orthogroup analyses. Here I present an analytical pipeline, ORTHOSCOPE* (star), to infer evolutionary histories of animal/plant genes from genome-scale data. ORTHOSCOPE* estimates a tree for a specified gene, detects speciation/gene duplication events that occurred at nodes belonging to only one lineage leading to a species of interest, and then integrates results derived from gene trees estimated for all query genes in genome-wide data. Thus, ORTHOSCOPE* can be used to detect species nodes just after whole-genome duplications as a first step of comparative genomic analyses. Moreover, by examining the presence or absence of genes belonging to species lineages with dense taxon sampling available from the ORTHOSCOPE web version, ORTHOSCOPE* can detect genes lost in specific lineages and horizontal gene transfers. This pipeline is available at https://github.com/jun-inoue/ORTHOSCOPE_STAR.

Fish-like larvae were foundational to the chordate body plan, given the basal placement of free-living lancelets. That body plan probably made it possible for chordate ancestors to swim by beating a tail formed of notochord and bilateral paraxial muscles. In order to investigate the molecular genetic basis of the origin and evolution of paraxial muscle, we deduced the evolutionary histories of 16 contractile protein genes from paraxial muscle, based on genomic data from all five deuterostome lineages, using a newly developed orthology identification pipeline and a species tree. As a result, we found that more than twice as many orthologs of paraxial muscle genes are present in chordates, as in nonchordate deuterostomes (ambulacrarians). Orthologs of paraxial-type actin and troponin C genes are absent in ambulacrarians and most paraxial muscle protein isoforms diversified via gene duplications that occurred in each chordate lineage. Analyses of genes with known expression sites indicated that some isoforms were reutilized in specific muscles of nonvertebrate chordates via gene duplications. As orthologs of most paraxial muscle genes were present in ambulacrarians, in addition to expression patterns of related genes and functions of the two protein isoforms, regulatory mechanisms of muscle genes should also be considered in future studies of the origin of paraxial muscle.

Our ability to correlate biological evolution with climate change, geological evolution, and other historical patterns is essential to understanding the processes that shape biodiversity. Combining data from the fossil record with molecular phylogenetics represents an exciting synthetic approach to this challenge. The first molecular divergence dating analysis (Zuckerkandl and Pauling 1962) was based on a measure of the amino acid differences in the hemoglobin molecule, with replacement rates established (calibrated) using paleontological age estimates from textbooks (e.g., Dodson 1960). Since that time, the amount of molecular sequence data has increased dramatically, affording ever-greater opportunities to apply molecular divergence approaches to fundamental problems in evolutionary biology.

Tunicates or urochordates&mdash, comprising ascidians, larvaceans, and salps&mdash, are the only metazoans that can synthesize cellulose, a biological function usually associated with bacteria and plants but not animals. Tunicate cellulose or tunicine is a major component of the outer acellular coverage (tunic) of the entire body of these organisms. Previous studies have suggested that the prokaryotic cellulose synthase gene (CesA) was horizontally transferred into the genome of a tunicate ancestor. However, no convenient tools have been devised to determine whether only tunicates harbor CesA. ORTHOSCOPE is a recently developed tool used to identify orthologous genes and to examine the phylogenic relationship of molecules within major metazoan taxa. The present analysis with this tool revealed the presence of CesA orthologs in all sequenced tunicate genomes but an absence in other metazoan genomes. This supports an evolutionary origin of animal cellulose and provides insights into the evolution of this animal taxon.

Horizontal gene transfer (HGT) is the movement of genetic material between different species. Although HGT is less frequent in eukaryotes than in bacteria, several instances of HGT have apparently shaped animal evolution. One well-known example is the tunicate cellulose synthase gene, CesA, in which a gene, probably transferred from bacteria, greatly impacted tunicate evolution. A Glycosyl Hydrolase Family 6 (GH6) hydrolase-like domain exists at the C-terminus of tunicate CesA, but not in cellulose synthases of other organisms. The recent discovery of another GH6 hydrolase-like gene (GH6-1) in tunicate genomes further raises the question of how tunicates acquired GH6. To examine the probable origin of these genes, we analyzed the phylogenetic relationship of GH6 proteins in tunicates and other organisms. Our analyses show that tunicate GH6s, the GH6-1 gene, and the GH6 part of the CesA gene, form two independent, monophyletic gene groups. We also compared their sequence signatures and exon splice sites. All tunicate species examined have shared splice sites in GH6-containing genes, implying ancient intron acquisitions. It is likely that the tunicate CesA and GH6-1 genes existed in the common ancestor of all extant tunicates.

Background The striped catfish, Pangasianodon hypophthalmus, is a freshwater and benthopelagic fish common in the Mekong River delta. Catfish constitute a valuable source of dietary protein. Therefore, they are cultured worldwide, and P. hypophthalmus is a food staple in the Mekong area. However, genetic information about the culture stock, is unavailable for breeding improvement, although genetics of the channel catfish, Ictalurus punctatus, has been reported. To acquire genome sequence data as a useful resource for marker-assisted breeding, we decoded a draft genome of P. hypophthalmus and performed comparative analyses. Results Using the Illumina platform, we obtained both nuclear and mitochondrial DNA sequences. Molecular phylogeny using the mitochondrial genome confirmed that P. hypophthalmus is a member of the family Pangasiidae and is nested within a clade including the families Cranoglanididae and Ictaluridae. The nuclear genome was estimated at approximately 700 Mb, assembled into 568 scaffolds with an N50 of 14.29 Mbp, and was estimated to contain ~ 28,600 protein-coding genes, comparable to those of channel catfish and zebrafish. Interestingly, zebrafish produce gadusol, but genes for biosynthesis of this sunscreen compound have been lost from catfish genomes. The differences in gene contents between these two catfishes were found in genes for vitamin D-binding protein and cytosolic phospholipase A2, which have lost only in channel catfish. The Hox cluster in catfish genomes comprised seven paralogous groups, similar to that of zebrafish, and comparative analysis clarified catfish lineage-specific losses of A5a, B10a, and A11a. Genes for insulin-like growth factor (IGF) signaling were conserved between the two catfish genomes. In addition to identification of MHC class I and sex determination-related gene loci, the hypothetical chromosomes by comparison with the channel catfish demonstrated the usefulness of the striped catfish genome as a marker resource. Conclusions We developed genomic resources for the striped catfish. Possible conservation of genes for development and marker candidates were confirmed by comparing the assembled genome to that of a model fish, Danio rerio, and to channel catfish. Since the catfish genomic constituent resembles that of zebrafish, it is likely that zebrafish data for gene functions is applicable to striped catfish as well. Electronic supplementary material The online version of this article (10.1186/s12864-018-5079-x) contains supplementary material, which is available to authorized users.

With our increasing ability for generating whole-genome sequences, comparative analysis of whole genomes has become a powerful tool for understanding the structure, function, and evolutionary history of human and other vertebrate genomes. By virtue of their position basal to bony vertebrates, cartilaginous fishes (class Chondrichthyes) are a valuable outgroup in comparative studies of vertebrates. Recently, a holocephalan cartilaginous fish, the elephant shark, Callorhinchus milii (Subclass Holocephali: Order Chimaeriformes), has been proposed as a model genome, and low-coverage sequence of its genome has been generated. Despite such an increasing interest, the evolutionary history of the modern holocephalans-a previously successful and diverse group but represented by only 39 extant species-and their relationship with elasmobranchs and other jawed vertebrates has been poorly documented largely owing to a lack of well-preserved fossil materials after the end-Permian about 250 Ma. In this study, we assembled the whole mitogenome sequences for eight representatives from all the three families of the modern holocephalans and investigated their phylogenetic relationships and evolutionary history. Unambiguously aligned sequences from these holocephalans together with 17 other vertebrates (9,409 nt positions excluding entire third codon positions) were subjected to partitioned maximum likelihood analysis. The resulting tree strongly supported a single origin of the modern holocephalans and their sister-group relationship with elasmobranchs. The mitogenomic tree recovered the most basal callorhinchids within the chimaeriforms, which is sister to a clade comprising the remaining two families (rhinochimaerids and chimaerids). The timetree derived from a relaxed molecular clock Bayesian method suggests that the holocephalans originated in the Silurian about 420 Ma, having survived from the end-Permian (250 Ma) mass extinction and undergoing familial diversifications during the late Jurassic to early Cretaceous (170-120 Ma). This postulated evolutionary scenario agrees well with that based on the paleontological observations.

Bayesian inference provides a powerful framework for integrating different sources of information (in particular, molecules and fossils) to derive estimates of species divergence times. Indeed, it is currently the only framework that can adequately account for uncertainties in fossil calibrations. We use 2 Bayesian Markov chain Monte Carlo programs, MULTIDIVTIME and MCMCTREE, to analyze 3 empirical datasets to estimate divergence times in amphibians, actinopterygians, and felids. We evaluate the impact of various factors, including the priors on rates and times, fossil calibrations, substitution model, the violation of the molecular clock and the rate-drift model, and the exact and approximate likelihood calculation. Assuming the molecular clock caused seriously biased time estimates when the clock is violated, but 2 different rate-drift models produced similar estimates. The prior on times, which incorporates fossil-calibration information, had the greatest impact on posterior time estimation. In particular, the strategies used by the 2 programs to incorporate minimum- and maximum-age bounds led to very different time priors and were responsible for large differences in posterior time estimates in a previous study. The results highlight the critical importance of fossil calibrations to molecular dating and the need for probabilistic modeling of fossil depositions, preservations, and sampling to provide statistical summaries of information in the fossil record concerning species divergence times.

The continental distributions of freshwater fishes in the family Notopteridae (Osteoglossomorpha) across Africa, India, and Southeast Asia constitute a long standing and enigmatic problem of freshwater biogeography. The migrational pathway of the Asian notopterids has been discussed in light of two competing schemes: the first posits recent transcontinental dispersal while the second relies on distributions being shaped by ancient vicariance associated with plate-tectonic events. In this study, we determined complete mitochondrial DNA sequences from 10 osteoglossomorph fishes to estimate phylogenetic relationships using partitioned Bayesian and maximum likelihood methods and divergence dates of the family Notopteridae with a partitioned Bayesian approach. We used six species representing the major lineages of the Notopteridae and seven species from the remaining osteoglossomorph families. Fourteen more-derived teleosts, nine basal actinopterygians, two coelacanths, and one shark were used as outgroups. Phylogenetic analyses indicated that the African and Asian notopterids formed a sister group to each other and that these notopterids were a sister to a clade comprising two African families (Mormyridae and Gymnarchidae). Estimated divergence time between the African and Asian notopterids dated back to the early Cretaceous when India-Madagascar separated from the African part of Gondwanaland. Thus, estimated time of divergence based on the molecular evidence is at odds with the recent dispersal model. It can be reconciled with the geological and paleontological evidence to support the vicariance model in which the Asian notopterids diverged from the African notopterids in Gondwanaland and migrated into Eurasia on the Indian subcontinent from the Cretaceous to the Tertiary. However, we could not exclude an alternative explanation that the African and Asian notopterids diverged in Pangea before its complete separation into Laurasia and Gondwanaland, to which these two lineages were later confined, respectively.

We determined the whole mitochondrial genome sequence for spotted green pufferfish, Tetraodon nigroviridis (Teleostei: Tetraodontiformes). The genome (16,488 bp) contained 37 genes (two ribosomal RNA genes, 22 transfer RNA genes, and 13 protein-coding genes) plus control region as found in other vertebrates, with the gene order identical to that of typical vertebrates. The sequence was used to estimate phylogenetic relationships and divergence times among major lineages of fishes, including representative model organisms in fishes. We employed partitioned Bayesian approaches for these two analyses using two datasets that comprised concatenated amino acid sequences from 12 protein-coding genes (excluding the ND6 gene) and concatenated nucleotide sequences from the 12 protein-coding genes (without 3rd codon positions), 22 transfer RNA genes, and two ribosomal RNA genes. The resultant trees from the two datasets were well resolved and largely congruent with those from previous studies, with spotted green pufferfish being placed in a reasonable phylogenetic position. The approximate divergence times between spotted green pufferfish and model organisms in fishes were 85 million years ago (MYA) vs. torafugu, 183 MYA vs. three-spined stickleback, 191 MYA vs. medaka, and 324 MYA vs. zebrafish, all of which were about twice as old as the divergence times estimated by their earliest occurrences in fossil records.

Although the order Gonorynchiformes includes only 31 species assigned to seven genera and four families, it exhibits a large variety of anatomical structures, making difficult the reconstruction of phylogenetic relationships among its representatives. Within the basal teleosts, the Gonorynchiformes belong to the Otocephala where they have been alternatively placed as the sister group of the Otophysi and of the Clupeiformes. In this context, we investigated the phylogeny of the Gonorynchiformes using whole mitogenome sequences from 40 species (six being newly determined for this study). Our taxonomic sampling included at least one species of each gonorynchiform genus and of each other major otocephalan lineage. Unambiguously aligned, concatenated mitogenomic sequences (excluding the ND6 gene and control region) were divided into five partitions (1st, 2nd, and 3rd codon positions, tRNA genes, and rRNA genes) and partitioned Bayesian analyses were conducted. The resultant phylogenetic trees were fully resolved, with most of the nodes well supported by the high posterior probabilities. As expected, the Otocephala were recovered as monophyletic. Within this group, the mitogenome data supported the monophyly of Alepocephaloidei, Gonorynchiformes, Otophysi, and Clupeiformes. The Gonorynchiformes and the Otophysi formed a sister group, rending the Ostariophysi monophyletic. This result conflicts with previous mitogenomic phylogenetic studies, in which a sister relationship was found between Clupeiformes and Gonorynchiformes. We discussed the possible causes of this incongruence. Within the Gonorynchiformes, the following original topology was found: (Gonorynchus (Chanos (Phractolaemus (Cromeria (Grasseichthys (Kneria, Parakneria)))))). We confirmed that the paedomorphic species Cromeria nilotica and Grasseichthys gabonensis belong to the family Kneriidae; however, the two species together did not form a monophyletic group. This result challenges the value of reductive or absent characters as synapomorphies in this group.

The monophyly of Elopomorpha (eels and their relatives) has long been one of the most problematic issues in systematic ichthyology. Since Greenwood et al. (1966) established the Elopomorpha based on the existence of the leaf-like larval form, termed a leptocephalus, no one has corroborated their monophyly using character matrices derived from both morphological and molecular data during the last 30 years. We investigated their monophyly and interrelationships at the ordinal level using complete mitochondrial genomic (mitogenomic) data from 33 purposefully chosen species (data for nine species being newly determined during the study) that fully represent the major teleostean and elopomorph lineages. Partitioned Bayesian analyses were conducted with the two data sets that comprised concatenated nucleotide sequences from 12 protein-coding genes (with and without third codon positions), 22 transfer RNA genes, and two ribosomal RNA genes. The resultant trees were well resolved and largely congruent, with most internal branches being supported by high statistical values. Mitogenomic data strongly supported the monophyly of Elopomorpha, indicating the validity of the leptocephalus as an elopomorph synapomorphy. The order Elopiformes occupied the most basal position in the elopomorph phylogeny, with the Albuliformes and a clade comprising the Anguilliformes and the Saccopharyngiformes forming a sister group. The most parsimonious reconstruction of the three previously recognized, distinct larval types of elopomorphs onto the molecular phylogeny revealed that one of the types (fork-tailed type) had originated as the common ancestor of the Elopomorpha, the other two (filament-tailed and round-tailed types) having diversified separately in two more derived major clades.

Ostariophysi is the second largest superorder within Teleostei. It contains five orders: Gonorynchiformes, Cypriniformes, Characiformes, Siluriformes, and Gymnotiformes. Resolving the higher-level relationships among ostariophysan and related fishes will aid in resolving basal teleostean divergence and provide basis to historical biogeographic analysis of major freshwater fish groups. In this study, we report the complete mitochondrial (mt) DNA sequences for eleven ostariophysan fishes and the results of phylogenetic analyses including these species plus four other ostariophysan and nine non-ostariophysan teleostean fishes. Maximum likelihood and maximum parsimony analyses reconfirmed clupeiforms as the closest relatives of ostariophysans. However, gonorynchiforms were closer to clupeiforms than to otophysans (ostariophysan groups excluding gonorynchiforms), thus raising a question over the current definition of Ostariophysi. The lack of clarity in otocephalan (ostariophysans + clupeiforms) basal relationships implies that such divergence took place over a short period of time. The monophyly of cypriniforms, characiphysans (characiforms, siluriforms, and gymnotiforms), and orders or superorders outside the ostariophysans examined here were conceivably reconstructed. The phylogenetic hypothesis suggests a Pangean origin of otophysans. Within characiphysans, gymnotiforms and siluriforms have independent evolutionary origins and evolutionary histories comparable to or older than that of characiforms. This helps to explain the present geographic distribution of characiphysans.

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.

The complete nucleotide sequence of the mitochondrial genome for the Japanese anchovy Engraulis japonicus (Teleostei: Clupeiformes) was determined. The entire genome was purified by gene amplification using the long polymerase chain reaction (PCR) technique, and products were subsequently used as templates for PCR with 56 fish-versatile primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome (16 675 base pairs [bp]) contained the same 37 mitochondrial genes (two ribosomal RNA, 22 transfer RNA and 13 protein-coding genes) as those found in other vertebrates, with the gene order being identical to that in typical vertebrates. A major non-coding region between the tRNAPro and tRNAPhe genes (1024 bp) was considered to be the control (D-loop) region, as it has several conservative blocks characteristic to this region.

SUMMARY: We determined the complete nucleotide sequence of the mitochondrial genome for the Japanese eel Anguilla japonica (Teleostei: Anguilliformes). The entire genome was purified by gene amplification using a long polymerase chain reaction (PCR) technique, and the products were subsequently used as templates for PCR with 60 fish-versatile and six species-specific primers that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome [16 685 base pairs (bp)] contained the same 37 mitochondrial genes (two ribosomal RNA, 22 transfer RNA and 13 protein-coding genes) as found in other vertebrates, with the gene order identical to that in typical vertebrates. A major non-coding region between the tRNAPro and tRNAPhe genes (967 bp) was considered as the control region (D-loop), as it has several conservative blocks characteristic to this region.

SUMMARY: We determined the complete nucleotide sequence of the mitochondrial genome for a Japanese sardine, Sardinops melanostictus (Teleostei: Clupeiformes). The entire genome was purified by gene amplification using long polymerase chain reactions (PCR), and the products were subsequently used as templates for PCR with 30 sets of fish-versatile primers (including three species-specific primers) that amplify contiguous, overlapping segments of the entire genome. Direct sequencing of the PCR products demonstrated that the genome [16 881 base pairs (bp)] contained the same 37 mitochondrial structural genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as found in other vertebrates, with the gene order identical to that in typical vertebrates. A major non-coding region between the tRNAPro and tRNAPhe genes (1200 bp) was considered as the control (D-loop) region, as it has several conservative blocks characteristic to this region.

Skates, rays and allies (Batoidea) comprise more than half of the species diversity and much of the morphological disparity among chondrichthyan fishes, the sister group to all other jawed vertebrates. While batoids are morphologically well characterized and have an excellent fossil record, there is currently no consensus on the interrelationships of family-level taxa. Here we construct a resolved, robust and time-calibrated batoid phylogeny using mitochondrial genomes, nuclear genes, and fossils, sampling densely across taxa. Data partitioning schemes, biases in the sequence data, and the relative informativeness of each fossil are explored. The molecular phylogeny is largely congruent with morphology crownward in the tree, but the branching orders of major batoid groups are mostly novel. Body plan convergence appears to be widespread in batoids. A depressed, rounded pectoral disk supported to the snout tip by fin radials, common to skates and stingrays, is indicated to have been derived independently by each group, while the long, spiny rostrum of sawfishes similarly appears to be convergent with that of sawsharks, which are not batoids. The major extant batoid lineages are inferred to have arisen relatively rapidly from the Late Triassic into the Jurassic, with long stems followed by subsequent radiations in each group around the Cretaceous/Tertiary boundary. The fossil record indicates that batoids were affected with disproportionate severity by the end-Cretaceous extinction event.

Series Atherinomorpha, with its plentiful number of species and highly diversified ecological and morphological characters, is the most successful fish group at the surface layer of the ocean and many freshwater habitats, comprising 1552 species classified into three orders, six suborders, 21 families, and 193 genera. The group includes one of the most important research model organisms, the medaka (Oryzias latipes), together with diverse fishes with morphological, physiological, and ecological specializations, such as highly developed pectoral fins to glide, self-fertilization, and live-bearing. In this study, we examined the whole mitochondrial genomes (mitogenomes) from 17 species representing all of the three orders and six suborders within Atherinomorpha, with data from 70 additional percomorph species as ingroups, and two non-percomorph outgroup species. We subjected the unambiguously aligned mitogenome sequences to partitioned maximum likelihood and Bayesian phylogenetic analyses. The resulting phylogenies recovered a monophyletic Atherinomorpha within the Percomorpha, and demonstrated its phylogenetic affinity to the percomorph fishes (including cichlids) spawning demersal eggs with filaments. This study, further, provided the first molecular evidence for the monophyly of the respective atherinomorph orders (Atheriniformes, Beloniformes, and Cyprinodontiformes) with high posterior probabilities and mostly high bootstrap values, providing an important basis for the future studies on the phylogeny and evolution of this diverse group.

We determined the whole mitochondrial genome sequence for Indonesian coelacanth Latimeria menadoensis. The genome content and organization were identical to that of typical vertebrates including Comoran coelacanth, Latimeria chalumnae. The overall nucleotide differences between the two species (excluding the control region) was 4.28%. The divergence time between the two species was estimated using whole mitochondrial genome data from the two coelacanths and 26 actinopterygians that represent major actinopterygian lineages plus an outgroup. Partitioned Bayesian analyses were conducted with the two data sets that comprised concatenated amino acid sequences from 12 protein-coding genes (excluding ND6 gene) and concatenated nucleotide sequences from 12 protein-coding genes (without 3rd codon positions), 22 transfer RNA genes, and two ribosomal RNA genes. The molecular clock analysis was also conducted with the concatenated amino acid sequences from the 12 protein-coding genes after removing faster or more slowly evolving sequences. Using the sarcopterygian-actinopterygian split as a calibration point (450 Mya), divergence time estimation between L. menadoensis and L. chalumnae fell in the range of 40-30 Mya, which is much older than those of the previous studies (

Molecular phylogenetic analyses were conducted using the whole mitochondrial genome sequences of all 18 species/subspecies of the freshwater eels genus Anguilla to infer their phylogenetic relationships and to evaluate hypotheses about the possible dispersal routes of this genus. The Bayesian and maximum likelihood analyses using a total of 15,187 sites of mitochondrial DNA sequences suggested that A. mossambica was the most basal species of anguillid eel, and that the other species (except for A. borneensis) formed three geographic clades: Atlantic (two species), Oceania (three species), and Indo-Pacific (11 species). The present study clearly indicated a sister relationship between the Atlantic and Oceanian species, which now have distantly separated geographic distributions. This suggests that the previous hypotheses to estimate the dispersal route of anguillid eels into the Atlantic Ocean based on the current geographic distribution of species are unsupported by the present more complete analysis. Alternatively, the unique geographic distribution of the present day species in the genus Anguilla appears to have resulted from multiple dispersal events. Although the age of the beginning of speciation among anguillid eels was tentatively estimated as 20 million years ago using a calibration for bony fishes of 7.3x10(-4) substitutions/site/million years, it is possible that this divergence time was underestimated because of the ecological characteristics of these fishes. The results of the present study suggest that the hypotheses for the dispersal route and divergence time of the genus Anguilla should be reconsidered.

Recent studies have demonstrated that deviations from the typical vertebrate mitochondrial gene order are more frequent than initially thought. Such deviations, however, are minor, with inversions and/or translocations of a few genes being involved and tandem duplication of the gene regions followed by deletions of genes having been invoked as mechanisms originating in such novel gene order. During the course of molecular phylogenetic studies on the Elopomorpha (eels and their allies), we found that mitochondrial genomes (mitogenomes) from the two deep-sea gulper eels, Eurypharynx pelecanoides (Eurypharyngidae) and Saccopharynx lavenbergi (Saccopharyngidae), exhibit an identical gene order which greatly differs from that of any other vertebrates. Phylogenetic analysis using the mitogenomic data from 59 species of fish not only confirmed a single origin of such a gene order with confidence but also indicated that it had been derived from the typical vertebrate gene order. Detailed comparisons of the gulper eel gene order with that of typical vertebrates suggested that occurrence of a single step, large-scale duplication of gene region extending >12 kb, followed by deletions of genes in a common ancestor of the two species, most parsimoniously accounts for this unusual gene arrangement.

The basal actinopterygians comprise four major lineages (polypteriforms, acipenseriforms, lepisosteids, and Amia ) and have been collectively called “ancient fish.” We investigated the phylogeny of this group of fishes in relation to teleosts using mitochondrial genomic (mitogenomic) data, and compared this to the various alternative phylogenetic hypotheses that have been proposed previously. In addition to the previously determined complete mitochondrial DNA (mtDNA) sequences from 14 teleosts and two outgroups, we used newly determined mitogenomic sequences of 12 purposefully chosen species representing all the ancient fish lineages plus related teleosts. This data set comprised concatenated nucleotide sequences from 12 protein-coding genes (excluding the ND6 gene and third codon positions) and 22 transfer RNA (tRNA) genes (stem regions only) and these data were subjected to maximum parsimony, maximum likelihood, and Bayesian analyses. The resultant trees from the three methods were well resolved and largely congruent, with most internal branches being supported by high statistical values. Mitogenomic data strongly supported not only the monophyly of the teleosts (osteoglossomorphs and above), but also a sister-group relationship between the teleosts and a clade comprising the acipenseriforms, lepisosteids, and Amia , with the polypteriforms occupying the most basal position in the actinopterygian phylogeny. Although the tree topology differed from any of the previously proposed hypotheses based on morphology, it exhibited congruence with a recently proposed novel hypothesis based on nuclear markers.

A recent study demonstrated that mitochondrial genomic (mitogenomic) data comprising nucleotide sequences from the concatenated protein-coding (no 3rd codon positions) plus transfer RNA (stem regions only) genes reproduced the expected phylogeny of teleosts with high statistical support. We reexamined the interrelationships of the five major, basal teleostean lineages (Osteoglossomorpha, Elopomorpha, Clupeomorpha, Ostariophysi, and Protacanthopterygii; given various rankings) using mitogenomic data for which five alternative phylogenetic hypotheses have been previously proposed on the basis of both morphological and molecular analyses. In addition to previously determined complete mitochondrial DNA (mtDNA) sequences from eight basal teleosts and two outgroups, we determined the complete mtDNA sequences (excluding a portion of the control region) for two, purposefully chosen species of Osteoglossomorpha (Osteoglossum bicirrhosum and Pantodon buchholzi), and the data were subjected to maximumparsimony and maximum-likelihood analyses. The resultant tree topologies from the two methods were congruent, although they differed from any of the previously proposed hypotheses. Furthermore, the mitogenomic data confidently rejected all of these hypotheses with high statistical significance.

The complete nucleotide sequence of the mitochondrial genome was determined for a conger eel, Conger myriaster (Elopomorpha: Anguilliformes), using a PCR-based approach that employs a long PCR technique and many fish-versatile primers. Although the genome [18,705 base pairs (bp)] contained the same set of 37 mitochondrial genes [two ribosomal RNA (rRNA), 22 transfer RNA (tRNA), and 13 protein-coding genes] as found in other vertebrates, the gene order differed from that recorded for any other vertebrates. In typical vertebrates, the ND6, tRNA(Glu), and tRNA(Pro) genes are located between the ND5 gene and the control region, whereas the former three genes, in C. myriaster, have been translocated to a position between the control region and the tRNA(Phe) gene that are contiguously located at the 5' end of the 12S rRNA gene in typical vertebrates. This gene order is similar to the recently reported gene order in four lineages of birds in that the latter lack the ND6, tRNA(Glu), and tRNA(Pro) genes between the ND5 gene and the control region; however, the relative position of the tRNA(Pro) to the ND6-tRNA(Glu) genes in C. myriaster was different from that in the four birds, which presumably resulted from different patterns of tandem duplication of gene regions followed by gene deletions in two distantly related groups of organisms. Sequencing of the ND5-cyt b region in 11 other anguilliform species, representing 11 families, plus one outgroup species, revealed that the same gene order as C. myriaster was shared by another 4 families, belonging to the suborder Congroidei. Although the novel gene orders of four lineages of birds were indicated to have multiple independent origins, phylogenetic analyses using nucleotide sequences from the mitochondrial 12S rRNA and cyt b genes suggested that the novel gene orders of the five anguilliform families had originated in a single ancestral species.