Tetsuya Sado

MitoFish and MitoAnnotator: a mitochondrial genome database of fish with an accurate and automatic annotation pipeline.

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.

Mitogenomic evolution and interrelationships of the Cypriniformes (Actinopterygii: Ostariophysi): the first evidence toward resolution of higher-level relationships of the world's largest freshwater fish clade based on 59 whole mitogenome sequences.

Fishes of the order Cypriniformes are almost completely restricted to freshwater bodies and number > 3400 species placed in 5 families, each with poorly defined subfamilies and/or tribes. The present study represents the first attempt toward resolution of the higher-level relationships of the world’s largest freshwater-fish clade based on whole mitochondrial (mt) genome sequences from 53 cypriniforms (including 46 newly determined sequences) plus 6 outgroups. Unambiguously aligned, concatenated mt genome sequences (14,563 bp) were divided into 5 partitions (first, second, and third codon positions of the protein-coding genes, rRNA genes, and tRNA genes), and partitioned Bayesian analyses were conducted, with protein-coding genes being treated in 3 different manners (all positions included; third codon positions converted into purine [R] and pyrimidine [Y] [RY-coding]; third codon positions excluded). The resultant phylogenies strongly supported monophyly of the Cypriniformes as well as that of the families Cyprinidae, Catostomidae, and a clade comprising Balitoridae + Cobitidae, with the 2 latter loach families being reciprocally paraphyletic. Although all of the data sets yielded nearly identical tree topologies with regard to the shallower relationships, deeper relationships among the 4 major clades (the above 3 major clades plus Gyrinocheilidae, represented by a single species Gyrinocheilus aymonieri in this study), were incongruent depending on the data sets. Treatment of the rapidly saturated third codon–position transitions appeared to be a source of such incongruities, and we advocate that RY-coding, which takes only transversions into account, effectively removes this likely “noise” from the data set and avoids the apparent lack of signal by retaining all available positions in the data set.

Reconstructing the phylogenetic relationships of the earth's most diverse clade of freshwater fishes—order Cypriniformes (Actinopterygii: Ostariophysi): A case study using multiple nuclear loci and the mitochondrial genome

The order Cypriniformes is the most diverse clade of freshwater fishes and is natively distributed on all continents except South America, Australia, and Antarctica. Despite the diversity of the group and the fundamental importance of these species in both ecosystems and human culture, relatively little has been known about their relationships relative to their diversity. In recent years, with an international effort investigating the systematics of the group, more information as to their genealogical relationships has emerged and species discovery and their descriptions have increased. One of the more interesting aspects of this group has been a traditional lack of understanding of the relationships of the families, subfamilies, and other formally or informally identified groups. Historical studies have largely focused on smaller groups of species or genera. Because of the diversity of this group and previously published whole mitochondrial genome evidence for relationships of major clades in the order, this clade serves as an excellent group to investigate the congruence between relationships reconstructed for major clades with whole mitogenome data and those inferred from a series of nuclear gene sequences. As descent has resulted in only one tree of life, do the phylogenetic relationships of these major clades converge on similar topologies using the large number of available characters through this suite of nuclear genes and previously published mitochondrial genomes? In this study we examine the phylogenetic relationships of major clades of Cypriniformes using previously published mitogenomes and four putative single-copy nuclear genes of the same or closely related species. Combined nuclear gene sequences yielded 3810bp, approximately 26% of the bp found in a single mitogenome; however homoplasy in the nuclear genes was measurably less than that observed in mitochondrial sequences. Relationships of taxa and major clades derived from analyses of nuclear and mitochondrial sequences were nearly identical and both received high support values. While some differences of individual gene trees did exist for species, it is predicted that these differences will be minimized with increased taxon sampling in future analyses.

Deep-ocean origin of the freshwater eels

Of more than 800 species of eels of the order Anguilliformes, only freshwater eels (genus Anguilla with 16 species plus three subspecies) spend most of their lives in freshwater during their catadromous life cycle. Nevertheless, because their spawning areas are located offshore in the open ocean, they migrate back to their specific breeding places in the ocean, often located thousands of kilometres away. The evolutionary origin of such enigmatic behaviour, however, remains elusive because of the uncertain phylogenetic position of freshwater eels within the principally marine anguilliforms. Here, we show strong evidence for a deep oceanic origin of the freshwater eels, based on the phylogenetic analysis of whole mitochondrial genome sequences from 56 species representing all of the 19 anguilliform families. The freshwater eels occupy an apical position within the anguilliforms, forming a highly supported monophyletic group with various oceanic midwater eel species. Moreover, reconstruction of the growth habitats on the resulting tree unequivocally indicates an origination of the freshwater eels from the midwater of the deep ocean. This shows significant concordance with the recent collection of mature adults of the Japanese eel in the upper midwater of the Pacific, suggesting that they have retained their evolutionary origin as a behavioural trait in their spawning areas.

Systematics of the subfamily Danioninae (Teleostei: Cypriniformes: Cyprinidae)

The members of the cyprinid subfamily Danioninae form a diverse and scientifically important group of fishes, which includes the zebrafish, Danio rerio. The diversity of this assemblage has attracted much scientific interest but its monophyly and the relationships among its members are poorly understood. The phylogenetic relationships of the Danioninae are examined herein using sequence data from mitochondrial cytochrome b, mitochondrial cytochrome c oxidase I, nuclear opsin, and nuclear recombination activating gene 1. A combined data matrix of 4117 bp for 270 taxa was compiled and analyzed. The resulting topology supports some conclusions drawn by recent studies on the group and certain portions of the traditional classification, but our results also contradict key aspects of the traditional classification. The subfamily Danioninae is not monophyletic, with putative members scattered throughout Cyprinidae. Therefore, we restrict Danioninae to the monophyletic group that includes the following genera: Amblypharyngodon, Barilius, Cabdio, Chela, Chelaethiops, Danio, Danionella, Devario (including Inlecypris), Esomus, Horadandia, Laubuca, Leptocypris, Luciosoma, Malayochela, Microdevario, Microrasbora, Nematabramis, Neobola, Opsaridium, Opsarius, Paedocypris, Pectenocypris, Raiamas, Rasbora (including Boraras and Trigonostigma), Rasboroides, Salmostoma, Securicula, and Sundadanio. This Danioninae sensu stricto is divided into three major lineages, the tribes Chedrini, Danionini, and Rasborini, where Chedrini is sister to a Danionini-Rasborini clade. Each of these tribes is monophyletic, following the restriction of Danioninae. The tribe Chedrini includes a clade of exclusively African species and contains several genera of uncertain monophyly (Opsarius, Raiamas, Salmostoma). Within the tribe Rasborini, the species-rich genus Rasbora is rendered non-monophyletic by the placement of two monophyletic genera, Boraras and Trigonostigma, hence we synonymize those two genera with Rasbora. In the tribe Danionini, the miniature genus Danionella is recovered as the sister group of Danio, with D. nigrofasciatus sister to D. rerio.

MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species

We developed a set of universal PCR primers (MiFish-U/E) for metabarcoding environmental DNA (eDNA) from fishes. Primers were designed using aligned whole mitochondrial genome (mitogenome) sequences from 880 species, supplemented by partial mitogenome sequences from 160 elasmobranchs (sharks and rays). The primers target a hypervariable region of the 12S rRNA gene (163–185 bp), which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. To test versatility of the primers across a diverse range of fishes, we sampled eDNA from four tanks in the Okinawa Churaumi Aquarium with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing of the region using high-throughput next-generation sequencing technologies. Out of the 180 marine fish species contained in the four tanks with reference sequences in a custom database, we detected 168 species (93.3%) distributed across 59 families and 123 genera. These fishes are not only taxonomically diverse, ranging from sharks and rays to higher teleosts, but are also greatly varied in their ecology, including both pelagic and benthic species living in shallow coastal to deep waters. We also sampled natural seawaters around coral reefs near the aquarium and detected 93 fish species using this approach. Of the 93 species, 64 were not detected in the four aquarium tanks, rendering the total number of species detected to 232 (from 70 families and 152 genera). The metabarcoding approach presented here is non-invasive, more efficient, more cost-effective and more sensitive than the traditional survey methods. It has the potential to serve as an alternative (or complementary) tool for biodiversity monitoring that revolutionizes natural resource management and ecological studies of fish communities on larger spatial and temporal scales.

Evolutionary origin of the Scombridae (tunas and mackerels): members of a paleogene adaptive radiation with 14 other pelagic fish families.

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.

Phylogeny and polyploidy: Resolving the classification of cyprinine fishes (Teleostei: Cypriniformes)

Cyprininae is the largest subfamily (>1300 species) of the family Cyprinidae and contains more polyploid species (∼400) than any other group of fishes. We examined the phylogenetic relationships of the Cyprininae based on extensive taxon, geographical, and genomic sampling of the taxa, using both mitochondrial and nuclear genes to address the phylogenetic challenges posed by polyploidy. Four datasets were analyzed in this study: two mitochondrial gene datasets (465 and 791 taxa, 5604bp), a mitogenome dataset (85 taxa, 14,771bp), and a cloned nuclear RAG1 dataset (97 taxa, 1497bp). Based on resulting trees, the subfamily Cyprininae was subdivided into 11 tribes: Probarbini (new; Probarbus+Catlocarpio), Labeonini Bleeker, 1859 (Labeo & allies), Torini Karaman, 1971 (Tor, Labeobarbus & allies), Smiliogastrini Bleeker, 1863 (Puntius, Enteromius & allies), Poropuntiini (Poropuntius & allies), Cyprinini Rafinesque, 1815 (Cyprinus & allies), Acrossocheilini (new; Acrossocheilus & allies), Spinibarbini (new; Spinibarbus), Schizothoracini McClelland, 1842 (Schizothorax & allies), Schizopygopsini Mirza, 1991 (Schizopygopsis & allies), and Barbini Bleeker, 1859 (Barbus & allies). Phylogenetic relationships within each tribe were discussed. Two or three distinct RAG1 lineages were identified for each of the following tribes Torini, Cyprinini, Spinibarbini, and Barbini, indicating their hybrid origin. The hexaploid African Labeobarbus & allies and Western Asian Capoeta are likely derived from two independent hybridization events between their respective maternal tetraploid ancestors and Cyprinion.

Phylogeny of the gudgeons (Teleostei: Cyprinidae: Gobioninae)

The members of the cyprinid subfamily Gobioninae, commonly called gudgeons, form one of the most well-established assemblages in the family Cyprinidae. The subfamily is a species-rich group of fishes, these fishes display diverse life histories, appearances, and behavior. The phylogenetic relationships of Gobioninae are examined using sequence data from four loci: cytochrome b, cytochrome c oxidase I, opsin, and recombination activating gene 1. This investigation produced a data matrix of 4114 bp for 162 taxa that was analyzed using parsimony, maximum likelihood, and Bayesian inference methods. The phylogenies our analyses recovered corroborate recent studies on the group. The subfamily Gobioninae is monophyletic and composed of three major lineages. We find evidence for a Hemibarbus-Squalidus group, and the tribes Gobionini and Sarcocheilichthyini, with the Hemibarbus-Squalidus group sister to a clade of Gobionini-Sarcocheilichthyini. The Hemibarbus-Squalidus group includes those two genera; the tribe Sarcocheilichthyini includes Coreius, Coreoleuciscus, Gnathopogon, Gobiocypris, Ladislavia, Paracanthobrama, Pseudorasbora, Pseudopungtungia, Pungtungia, Rhinogobio, and Sarcocheilichthys; the tribe Gobionini includes Abbottina, Biwia, Gobio, Gobiobotia, Huigobio, Microphysogobio, Platysmacheilus, Pseudogobio, Romanogobio, Saurogobio, and Xenophysogobio. The monotypic Acanthogobio is placed into the synonymy of Gobio. We tentatively assign Belligobio to the Hemibarbus-Squalidus group and Mesogobio to Gobionini; Paraleucogobio and Parasqualidus remain incertae sedis. Based on the topologies presented, the evolution of swim bladder specializations, a distinctive feature among cyprinids, has occurred more than once within the subfamily.

Deep-sea mystery solved: astonishing larval transformations and extreme sexual dimorphism unite three fish families

The oceanic bathypelagic realm (1000–4000 m) is a nutrient-poor habitat. Most fishes living there have pelagic larvae using the rich waters of the upper 200 m. Morphological and behavioural specializations necessary to occupy such contrasting environments have resulted in remarkable developmental changes and life-history strategies. We resolve a long-standing biological and taxonomic conundrum by documenting the most extreme example of ontogenetic metamorphoses and sexual dimorphism in vertebrates. Based on morphology and mitogenomic sequence data, we show that fishes currently assigned to three families with greatly differing morphologies, Mirapinnidae (tapetails), Megalomycteridae (bignose fishes) and Cetomimidae (whalefishes), are larvae, males and females, respectively, of a single family Cetomimidae. Morphological transformations involve dramatic changes in the skeleton, most spectacularly in the head, and are correlated with distinctly different feeding mechanisms. Larvae have small, upturned mouths and gorge on copepods. Females have huge gapes with long, horizontal jaws and specialized gill arches allowing them to capture larger prey. Males cease feeding, lose their stomach and oesophagus, and apparently convert the energy from the bolus of copepods found in all transforming males to a massive liver that supports them throughout adult life.