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.

Fish classifications, as those of most other taxonomic groups, are being transformed drastically as new molecular phylogenies provide support for natural groups that were unanticipated by previous studies. A brief review of the main criteria used by ichthyologists to define their classifications during the last 50 years, however, reveals slow progress towards using an explicit phylogenetic framework. Instead, the trend has been to rely, in varying degrees, on deep-rooted anatomical concepts and authority, often mixing taxa with explicit phylogenetic support with arbitrary groupings. Two leading sources in ichthyology frequently used for fish classifications (JS Nelson’s volumes of Fishes of the World and W. Eschmeyer’s Catalog of Fishes) fail to adopt a global phylogenetic framework despite much recent progress made towards the resolution of the fish Tree of Life. The first explicit phylogenetic classification of bony fishes was published in 2013, based on a comprehensive molecular phylogeny ( www.deepfin.org ). We here update the first version of that classification by incorporating the most recent phylogenetic results. The updated classification presented here is based on phylogenies inferred using molecular and genomic data for nearly 2000 fishes. A total of 72 orders (and 79 suborders) are recognized in this version, compared with 66 orders in version 1. The phylogeny resolves placement of 410 families, or ~80% of the total of 514 families of bony fishes currently recognized. The ordinal status of 30 percomorph families included in this study, however, remains uncertain (incertae sedis in the series Carangaria, Ovalentaria, or Eupercaria). Comments to support taxonomic decisions and comparisons with conflicting taxonomic groups proposed by others are presented. We also highlight cases were morphological support exist for the groups being classified. This version of the phylogenetic classification of bony fishes is substantially improved, providing resolution for more taxa than previous versions, based on more densely sampled phylogenetic trees. The classification presented in this study represents, unlike any other, the most up-to-date hypothesis of the Tree of Life of fishes.

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.

Environmental DNA (eDNA) metabarcoding has emerged as a potentially powerful tool to assess aquatic community structures. However, the method has hitherto lacked field tests that evaluate its effectiveness and practical properties as a biodiversity monitoring tool. Here, we evaluated the ability of eDNA metabarcoding to reveal fish community structures in species-rich coastal waters. High-performance fish-universal primers and systematic spatial water sampling at 47 stations covering ~11 km² revealed the fish community structure at a species resolution. The eDNA metabarcoding based on a 6-h collection of water samples detected 128 fish species, of which 62.5% (40 species) were also observed by underwater visual censuses conducted over a 14-year period. This method also detected other local fishes (≥23 species) that were not observed by the visual censuses. These eDNA metabarcoding features will enhance marine ecosystem-related research, and the method will potentially become a standard tool for surveying fish communities.

Although adequate resolution of higher-level relationships of organisms apparently requires longer DNA sequences than those currently being analyzed, limitations of time and resources present difficulties in obtaining such sequences from many taxa. For fishes, these difficulties have been overcome by the development of a PCR-based approach for sequencing the complete mitochondrial genome (mitogenome), which employs a long PCR technique and many fish-versatile PCR primers. In addition, recent studies have demonstrated that such mitogenomic data are useful and decisive in resolving persistent controversies over higher-level relationships of teleosts. As a first step toward resolution of higher teleostean relationships, which have been described as the “(unresolved) bush at the top of the tree,” we investigated relationships using mitogenomic data from 48 purposefully chosen teleosts, of which those from 38 were newly determined during the present study (a total of 632,315 bp), using the above method. Maximum-parsimony and maximum-likelihood analyses were conducted with the data set that 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) from the 48 species. The resultant two trees from the two methods were well resolved and largely congruent, with many internal branches supported by high statistical values. The tree topologies themselves, however, exhibited considerable variation from the previous morphology-based cladistic hypotheses, with most of the latter being confidently rejected by the mitogenomic data. Such incongruence resulted largely from the phylogenetic positions or limits of long-standing problematic taxa, which were quite unexpected from previous morphological and molecular analyses. We concluded that the present study provided a basis of and guidelines for future investigations of teleostean evolutionary mitogenomics and that purposeful higher-density taxonomic sampling, subsequent sequencing efforts, and phylogenetic analyses of their mitogenomes may be decisive in resolving persistent controversies over higher-level relationships of teleosts, the most diversified group of all vertebrates, comprising over 23,500 extant species.