Complete F mitochondrial genomes of two freshwater mussels from the Lake Biwa system in Japan: Beringiana fukuharai and Sinanodonta tumens.

We determined the complete mitochondrial sequences of female-transmitted (F) mitogenomes of six unionid specimens from the Lake Biwa system, Japan. Their gene contents and orders agreed with those of the typical F mitogenome of freshwater mussels. Molecular phylogenetic analysis using fifteen previously identified partial COI and 12 (six previously identified and six newly determined) whole mitogenome sequences revealed that five of the six mitogenomes (LC592401, LC592402, LC592403, LC592408, and LC592410) were those of Beringiana fukuharai, while the remaining one (LC592406) was Sinanodonta tumens.

Complete female-transmitted mitochondrial genomes of two freshwater mussels from the Lake Biwa system in Japan: Nodularia douglasiae and N. nipponensis.

We determined the complete mitochondrial sequences of female-transmitted (F) mitogenomes of two unionid specimens from the Lake Biwa system, Japan. Their gene contents and orders agreed with those of the typical F mitogenome of freshwater mussels. Molecular phylogenetic analysis using 20 previously identified partial COI and seven (five previously identified and two newly determined) whole mitogenome sequences revealed that one of the two mitogenomes was that of Nodularia douglasiae, while the other was N. nipponensis.

Complete F mitochondrial genomes of the Biwa pearl mussel, Hyriopsis schlegelii: the first report from the species' native lake in Japan.

We determined two complete mitochondrial sequences of female-transmitted (F) mitogenomes of two Hyriopsis schlegelii specimens from this species' original habitat, Lake Biwa. The mitogenomes were both 15,951 bp in length, and the gene contents and orders agreed with those of the typical F mitogenome of Hyriopsis. Molecular phylogenetic analysis based on the previously identified 13 partial sequences confirmed that the two mitogenomes both belonged to H. schlegelii and not to a closely related Chinese species, Hyriopsis cumingii. The same analysis revealed that two mitogenomes (HQ641406 and FJ529186) previously published as H. schlegelii and H. cumingii might be misidentified.

Structure and variation of the mitochondrial genome of fishes.

BACKGROUND: The mitochondrial (mt) genome has been used as an effective tool for phylogenetic and population genetic analyses in vertebrates. However, the structure and variability of the vertebrate mt genome are not well understood. A potential strategy for improving our understanding is to conduct a comprehensive comparative study of large mt genome data. The aim of this study was to characterize the structure and variability of the fish mt genome through comparative analysis of large datasets. RESULTS: An analysis of the secondary structure of proteins for 250 fish species (248 ray-finned and 2 cartilaginous fishes) illustrated that cytochrome c oxidase subunits (COI, COII, and COIII) and a cytochrome bc1 complex subunit (Cyt b) had substantial amino acid conservation. Among the four proteins, COI was the most conserved, as more than half of all amino acid sites were invariable among the 250 species. Our models identified 43 and 58 stems within 12S rRNA and 16S rRNA, respectively, with larger numbers than proposed previously for vertebrates. The models also identified 149 and 319 invariable sites in 12S rRNA and 16S rRNA, respectively, in all fishes. In particular, the present result verified that a region corresponding to the peptidyl transferase center in prokaryotic 23S rRNA, which is homologous to mt 16S rRNA, is also conserved in fish mt 16S rRNA. Concerning the gene order, we found 35 variations (in 32 families) that deviated from the common gene order in vertebrates. These gene rearrangements were mostly observed in the area spanning the ND5 gene to the control region as well as two tRNA gene cluster regions (IQM and WANCY regions). Although many of such gene rearrangements were unique to a specific taxon, some were shared polyphyletically between distantly related species. CONCLUSIONS: Through a large-scale comparative analysis of 250 fish species mt genomes, we elucidated various structural aspects of the fish mt genome and the encoded genes. The present results will be important for understanding functions of the mt genome and developing programs for nucleotide sequence analysis. This study demonstrated the significance of extensive comparisons for understanding the structure of the mt genome.

Complete mitochondrial genome of the parrotfish Calotomus japonicus (Osteichthyes: Scaridae) with implications based on the phylogenetic position.

The complete mitochondrial genome sequence was determined for a specimen of Calotomus japonicus, a temperate parrotfish endemic to coastal East Asia. It was compared phylogenetically with previously published partial sequences from this species and other parrotfishes. The obtained tree indicated that the three cytb sequences of C. japonicus from a recent molecular study (LC068806-8) probably resulted from introgression through intergeneric hybridization, or possibly from sample confusion. Taking the presently obtained mitogenome as representative of C. japonicus, the species most closely related to this one among congeners is C. zonarchus, which is endemic to the Hawaiian islands.

Complete mitochondrial genomes of five introduced strains of common carp (Cyprinus carpio) in Japan with 29 diagnostic SNPs distinguishable by restriction enzyme analysis.

Complete mitochondrial genome sequences were determined for three common carp individuals captured at Lake Kasumigaura, Japan. They represent three of the five introduced strains of common carp in Japan, having the mtDNA D-loop haplotypes c1, d2 and e1. The three obtained mitogenome sequences were compared with two previously published mitogenomes that are identical in the D-loop region to haplotypes b1 and f1, representing the remaining two of the five introduced strains in Japan. Alignment (16 585 base pairs) of these five mitogenomes revealed 29 SNPs that are diagnostic for the five strains, detected through positive digestion by "rare-cutter" restriction enzymes.

Revision of the systematics of the cardinalfishes (Percomorpha: Apogonidae) based on molecular analyses and comparative reevaluation of morphological characters.

Molecular analyses were conducted based on 120 of the estimated 358 species of the family Apogonidae with 33 of 40 genera and subgenera, using three gobioids and one kurtid as collective outgroups. Species of Amioides, Apogon, Apogonichthyoides, Apogonichthys, Archamia, Astrapogon, Brephamia, Cercamia, Cheilodipterus, Fibramia n. gen., Foa, Fowleria, Glossamia, Gymnapogon, Jaydia, Lachneratus, Nectamia, Ostorhinchus, Paroncheilus, Phaeoptyx, Pristiapogon, Pristicon, Pseudamia, Pterapogon, Rhabdamia, Siphamia, Sphaeramia, Taeniamia, Verulux, Vincentia, Yarica, Zapogon and Zoramia were present in the molecular analyses; species of Bentuviaichthys, Holapogon, Lepidamia, Neamia, Paxton, Pseudamiops and Quinca were absent from the analyses. Maximum-likelihood (ML), Bayesian (BA), and Maximum parsimony (MP) analyses based on two mitochondrial (12S rRNA-tRNAVal-16S rRNA, ca. 1500 bp; COI, ca. 1500 bp) and two nuclear DNA (RAG1, ca. 1300 bp; ENC1, ca. 800 bp) fragments reproduced two basal clades within the monophyletic family: one including a single species, Amioides polyacanthus, and the other comprising species of Pseudamia. All the other apogonid species formed a large well-established monophyletic group, in which almost identical 12 major clades were reproduced, with phylogenetic positions of four species (Glossamia aprion, Ostorhinchus margaritophorus, Pterapogon kauderni, and Vincentia novaehollandiae) left unsettled. Apogon sensu lato and recent Ostorhinchus (excepting O. margaritophorus) were divided into six and three major clades, respectively. Each of the recognized clades in the family was then evaluated for morphological characters to identify synapomorphies. Based on the results of the molecular analyses and the reevaluation of morphological characters, four subfamilies were proposed within the family: Apogoninae (including most of the species in the family), Amioidinae new subfamily (including Amioides, and based on morphology, Holapogon), Paxtoninae new subfamily (including Paxton, based only on morphology) and Pseudamiinae (including Pseudamia). Within the largest subfamily Apogoninae, twelve new tribes were proposed based on the 12 molecular clades and associated morphology: Apogonichthyini, Apogonini (mainly including species of Apogon sensu stricto), Archamiini, Cheilodipterini, Gymnapogonini, Ostorhinchini (including striped species of recent Ostorhinchus), Pristiapogonini, Rhabdamiini, Sphaeramiini (mainly including barred species of traditional Ostorhinchus, such as Apogonichthyoides, Jaydia and Nectamia), Siphamiini, Veruluxini, and Zoramiini. Two additional tribes are proposed based only on morphology: Glossamiini and Lepidamiini. For each of the 14 tribes, morphological characters were described. One new genus, Fibramia, type species Apogon thermalis, recently in Ostorhinchus, was described supported by morphology and molecular trees. A key to all genera is provided and all valid and uncertain status species are allocated to tribes and genera.

Mitogenomic circumscription of a novel percomorph fish clade mainly comprising "Syngnathoidei" (Teleostei).

Percomorpha, comprising about 60% of modern teleost fishes, has been described as the "(unresolved) bush at the top" of the tree, with its intrarelationships still being ambiguous owing to huge diversity (>15,000 species). Recent molecular phylogenetic studies based on extensive taxon and character sampling, however, have revealed a number of unexpected clades of Percomorpha, and one of which is composed of Syngnathoidei (seahorses, pipefishes, and their relatives) plus several groups distributed across three different orders. To circumscribe the clade more definitely, we sampled several candidate taxa with reference to the previous studies and newly determined whole mitochondrial genome (mitogenome) sequences for 16 percomorph species across syngnathoids, dactylopterids, and their putatively closely-related fishes (Mullidae, Callionymoidei, Malacanthidae). Unambiguously aligned sequences (13,872 bp) from those 16 species plus 78 percomorphs and two outgroups (total 96 species) were subjected to partitioned Bayesian and maximum likelihood analyses. The resulting trees revealed a highly supported clade comprising seven families in Syngnathoidei (Gasterosteiformes), Dactylopteridae (Scorpaeniformes), Mullidae in Percoidei and two families in Callionymoidei (Perciformes). We herein proposed to call this clade "Syngnathiformes" following the latest nuclear DNA studies with some revisions on the included families.

The complete mitochondrial genome of the Japanese ornamental koi carp (Cyprinus carpio) and its implication for the history of koi.

Complete mitochondrial genome (mitogenome) sequences were determined for two individuals of Japanese ornamental koi carp. Interestingly, the obtained mitogenomes (16,581 bp) were both completely identical to the recently reported mitogenome of Oujiang color carp from China. Control region (CR) sequences in DNA database demonstrated that more than half (65%) of the koi carp individuals so far reported had partial or complete CR sequences identical to those from Oujiang color carp. These results might suggest that the Japanese koi carp has been originated from Chinese Oujiang color carp, contrary to the belief in Japan that the koi carps have been developed directly from carp stocks in Japan. In any case, the present results emphasize the importance of analyzing Oujiang color carp when studying the origin of koi carp.

Complete mitochondrial genome sequence of the thorny seahorse Hippocampus histrix (Gasterosteiformes: Syngnathidae).

We determined the complete mitochondrial genome (mitogenome) sequence of the thorny seahorse Hippocampus histrix. The total length of H. histrix mitogenome is 16,523 bp, which consists of 13 protein coding, 22 tRNA and 2 rRNA genes and 1 control region. It has the typical vertebrate mitochondrial gene arrangement. This mitogenome sequence provides the basis for taxonomic and conservation studies of this and several closely related species.

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.

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.

Hidden diversity in a reef-dwelling sea slug, Pteraeolidia ianthina (Nudibranchia, Aeolidina), in the Northwestern Pacific.

The population genetic structure of a reef-dwelling aeolid nudibranch, Pteraeolidia ianthina ( Angas, 1864 ) (Gastropoda, Mollusca), was investigated by analyzing the nuclear ribosomal RNA gene region, including 18S and 5.8S rRNA genes and the ITS1 region (545 bp). Among 235 individuals from 10 localities in the northwestern Pacific, two genetically distinct groups were detected: Groups A and B, which are separated by a minimum sequence difference of 5.3% (p-distance). The marked genetic differentiation of these two groups, even in localities where both co-occur, suggests that they are distinct species, although intergroup hybrids were observed in very low frequency. Within-population genetic variability was low in Group A, but high in Group B. Geographic analysis of between-population genetic distance and "allele" sharing data indicated three subgroups within Group A (temperate Japan, Ryukyus + Philippines, and Guam), whose distribution ranges appear to have some relationship to climate zones (temperate and subtropics + tropics) and ocean currents (Kuroshio and North Equatorial Currents). Within Group B detected only from three subtropic and tropic localities, however, genetic distances between populations were much smaller than those in Group A, and thus no significant geographic pattern was found. These differences in genetic variability and population structure between Groups A and B may reflect differences in biological features between them, such as the duration of the pelagic larval stage. Furthermore, some morphological differences were observed between Groups A and B. These observations provide further support for the suggestion that the two groups are separate species.

Complete mitochondrial genome sequence of the dragonet Callionymus curvicornis (Perciformes: Callionymoidei: Callionymidae).

We determined the complete mitochondrial genome (mitogenome) sequence of the dragonet Callionymus curvicornis. The total length of C. curvicornis mitogenome is 16,406 bp, which consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region. It has the typical vertebrate mitochondrial gene arrangement. This is the first report of a complete mitochondrial genome in the fish suborder Callionymoidei.

Evolutionary history of Otophysi (Teleostei), a major clade of the modern freshwater fishes: Pangaean origin and Mesozoic radiation.

BACKGROUND: Freshwater harbors approximately 12,000 fish species accounting for 43% of the diversity of all modern fish. A single ancestral lineage evolved into about two-thirds of this enormous biodiversity (≈ 7900 spp.) and is currently distributed throughout the world's continents except Antarctica. Despite such remarkable species diversity and ubiquity, the evolutionary history of this major freshwater fish clade, Otophysi, remains largely unexplored. To gain insight into the history of otophysan diversification, we constructed a timetree based on whole mitogenome sequences across 110 species representing 55 of the 64 families. RESULTS: Partitioned maximum likelihood analysis based on unambiguously aligned sequences (9923 bp) confidently recovered the monophyly of Otophysi and the two constituent subgroups (Cypriniformes and Characiphysi). The latter clade comprised three orders (Gymnotiformes, Characiformes, Siluriformes), and Gymnotiformes was sister to the latter two groups. One of the two suborders in Characiformes (Characoidei) was more closely related to Siluriformes than to its own suborder (Citharinoidei), rendering the characiforms paraphyletic. Although this novel relationship did not receive strong statistical support, it was supported by analyzing independent nuclear markers. A relaxed molecular clock Bayesian analysis of the divergence times and reconstruction of ancestral habitats on the timetree suggest a Pangaean origin and Mesozoic radiation of otophysans. CONCLUSIONS: The present timetree demonstrates that survival of the ancestral lineages through the two consecutive mass extinctions on Pangaea, and subsequent radiations during the Jurassic through early Cretaceous shaped the modern familial diversity of otophysans. This evolutionary scenario is consistent with recent arguments based on biogeographic inferences and molecular divergence time estimates. No fossil otophysan, however, has been recorded before the Albian, the early Cretaceous 100-112 Ma, creating an over 100 million year time span without fossil evidence. This formidable ghost range partially reflects a genuine difference between the estimated ages of stem group origin (molecular divergence time) and crown group morphological diversification (fossil divergence time); the ghost range, however, would be filled with discoveries of older fossils that can be used as more reasonable time constraints as well as with developments of more realistic models that capture the rates of molecular sequences accurately.

Multiple invasions into freshwater by pufferfishes (teleostei: tetraodontidae): a mitogenomic perspective.

Pufferfishes of the Family Tetraodontidae are the most speciose group in the Order Tetraodontiformes and mainly inhabit coastal waters along continents. Although no members of other tetraodontiform families have fully discarded their marine lives, approximately 30 tetraodontid species spend their entire lives in freshwaters in disjunct tropical regions of South America, Central Africa, and Southeast Asia. To investigate the interrelationships of tetraodontid pufferfishes and thereby elucidate the evolutionary origins of their freshwater habitats, we performed phylogenetic analysis based on whole mitochondrial genome sequences from 50 tetraodontid species and closely related species (including 31 newly determined sequences). The resulting phylogenies reveal that the family is composed of four major lineages and that freshwater species from the different continents are independently nested in two of the four lineages. A monophyletic origin of the use of freshwater habitats was statistically rejected, and ancestral habitat reconstruction on the resulting tree demonstrates that tetraodontids independently entered freshwater habitats in different continents at least three times. Relaxed molecular-clock Bayesian divergence time estimation suggests that the timing of these invasions differs between continents, occurring at 0-10 million years ago (MA) in South America, 17-38 MA in Central Africa, and 48-78 MA in Southeast Asia. These timings are congruent with geological events that could facilitate adaptation to freshwater habitats in each continent.

Divergence time of the two regional medaka populations in Japan as a new time scale for comparative genomics of vertebrates.

The southern and northern Japanese populations of the medaka fish provide useful tools to gain insights into the comparative genomics and speciation of vertebrates, because they can breed to produce healthy and fertile offspring despite their highly divergent genetic backgrounds compared with those of human-chimpanzee. Comparative genomics analysis has suggested that such large genetic differences between the two populations are caused by higher molecular evolutionary rates among the medakas than those of the hominids. The argument, however, was based on the assumption that the two Japanese populations diverged approximately at the same time (4.0-4.7 Myr ago) as the human-chimpanzee lineage (5.0-6.0 Myr ago). This can be misleading, because the divergence time of the two populations was calculated based on estimated, extremely higher molecular evolutionary rates of other fishes with an implicit assumption of a global molecular clock. Here we show that our estimate, based on a Bayesian relaxed molecular-clock analysis of whole mitogenome sequences from 72 ray-finned fishes (including 14 medakas), is about four times older than that of the previous study (18 Myr). This remarkably older estimate can be reconciled with the vicariant events of the Japanese archipelago, and the resulting rates of molecular evolution are almost identical between the medaka and hominid lineages. Our results further highlight the fact that reproductive isolation may not evolve despite a long period of geographical isolation.

Mitogenomic evaluation of the unique facial nerve pattern as a phylogenetic marker within the percifom fishes (Teleostei: Percomorpha).

Percomorpha has been described as the "(unresolved) bush at the top" of the teleostean phylogenies and its intrarelationships are intrinsically difficult to solve because of its huge diversity (>15,000 spp.) and ill-defined higher taxa. Patterns of facial nerves, such as those of the ramus lateralis accessorius (RLA), have been considered as one of the candidate characters to delimit a monophyletic group within the percomorphs. Six families of the suborder Percoidei (Arripidae, Dichistiidae, Kyphosidae, Terapontidae, Kuhliidae, and Oplegnathidae) and suborder Stromateoidei (including six families) share the unique pattern 10 of RLA and it has been suggested that those fishes form a monophyletic group across the two perciform suborders. To evaluate the usefulness of the RLA pattern 10 as a phylogenetic marker within the percomorphs, we newly determined whole mitochondrial genome (mitogenome) sequences for the 13 species having RLA pattern 10 and their putatively, closely-related species (5 spp.). Unambiguously aligned sequences (14,263 bp) from those 18 species plus 50 percomrphs and two outgroups (total 70 species) were subjected to partitioned maximum likelihood and Bayesian analyses. The resulting trees clearly indicated that there were at least two independent origins of the unique facial nerve pattern: one in a common ancestor of Kyphosidae, Terapontidae, Kuhliidae, and Oplegnathidae and another one in that of the percoid Arripidae and Stromateoidei. Thus further detailed anatomical studies are needed to clarify the homology of this character between the two lineages. It should be noted that the latter two taxa (Arripidae and Stromateoidei) formed an unexpected, highly-supported monophyletic group together with Scombridae and possibly Chiasmodontidae and Bramidae, all lacking RLA pattern 10 (the former two are members of other perciform suborders Scombroidei and Trachinoidei, respectively). This novel, trans-subordinal clade has never been suggested by any morphological studies, although they share a common ecological characteristic, dwelling in the pelagic realm and often associated with long-distance migrations.

Explosive speciation of Takifugu: another use of fugu as a model system for evolutionary biology.

Although the fugu Takifugu rubripes has attracted attention as a model organism for genomic studies because of its compact genome, it is not generally appreciated that there are approximately 25 closely related species with limited distributions in the waters of East Asia. We performed molecular phylogenetic analyses and constructed a time tree using whole mitochondrial genome sequences from 15 Takifugu species together with 10 outgroups to examine patterns of diversification. The resultant time tree showed that the modern Takifugu species underwent explosive speciation during the Pliocene 1.8-5.3 Ma, which is comparable with that of the Malawi cichlids and tropheine cichlids in Lake Tanganyika. Considering their limited distributions and remarkable variations in coloration, morphology, and behavior, the results of the present study strongly suggest that Takifugu species are strong candidates as a model system for evolutionary studies of speciation mechanisms in marine environments where few such organisms are available.