Whole mitogenome analysis and phylogeny of freshwater fish red-finned catopra (Pristolepis rubripinnis) endemic to Kerala, India.

The freshwater leaf fish Pristolepis rubripinnis belongs to the family Pristolepididae, restricted to Pamba and Chalakudy rivers of Kerala, India. In the present study, we sequenced the complete mitogenome of P. rubripinnis and analysed its phylogeny in the order Anabantiformes. The 16622-bp long genome comprised of 13 protein-coding genes, two rRNA genes, 22 transfer RNAs (tRNAs) genes and had a noncoding control region. All the protein-coding genes, tRNA and rRNA were located on the heavy strand, except nad6 and eight tRNAs (glutamine, alanine, asparagine, cysteine, tyrosine, serine, glutamic acid and proline) transcribed from L strand. The genome exhibited an overlapping between atp8 and atp6 (2 bp), nad4 and nad4l (2 bp), tRNAIle and tRNAGln. (1 bp), tRNAThr and tRNAPro (1 bp). Around 157 bp, an intergenic spacer was identified. The overall GC-skews and AT-skews of the H-strand mitogenome were -0.35 and 0.079, respectively, revealing that the H-strand consisted of equal amounts of A and T and that the overall nucleotide composition was C skewed. All tRNA genes exhibited cloverleaf secondary structures, while the secondary structure of tRNASer lacked a discernible dihydrouridine stem. The phylogenetic analysis of available mitogenomes of Anabantiformes revealed a sister group relationship between Pristolepididae and Channidae. The whole mitogenome of Pristolepis rubripinnis will form a molecular resource for further taxonomic and conservation studies on this endemic freshwater fish.

Mitogenome analysis of dwarf pufferfish (Carinotetraodon travancoricus) endemic to southwest India and its implications in the phylogeny of Tetraodontidae.

The Tetraodontidae (pufferfishes), is primarily a family of marine and estuarine fishes with a limited number of freshwater species. Freshwater invasions can be observed in South America, Southeast Asia and central Africa. In the present study, we have analysed the complete mitogenome of freshwater pufferfish, Carinotetraodon travancoricus (dwarf pufferfish or Malabar pufferfish) endemic to southwest India. The genome is 16487 bp in length and consist of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one control region like all the other vertebrate mitogenomes. The protein-coding genes ranged from 165 bp (ATP synthase subunit 8) to 1812 bp (NADH dehydrogenase subunit 5) and comprised of 11310 bp in total, constituting 68.5% of the complete mitogenome. Some overlaps have been observed in protein-coding genes by a total of 7 bp. The AT skew (0.032166) and GC skew (-0.29746) of the mitogenome indicated that heavy strand consists equal amount of A and T, but the overall base composition was mainly C skewed. The noncoding D-loop region comprised 869 bp. The conserved motifs ATGTA and its complement TACAT associated with thermostable hairpin structure formation were identified in the control region. The phylogenetic analysis depicted a sister group relationship of C. travancoricus with euryhaline species Dichotomyctere nigroviridis and D. ocellatus with 100% bootstrap value rather than with the other freshwater members of Carinotetraodon species from Southeast Asia. The data from this study will be useful for proper identification, genetic differentiation, management and conservation of the dwarf Indian pufferfish.

Mitogenomic phylogeny of the Percichthyidae and Centrarchiformes (Percomorphaceae): comparison with recent nuclear gene-based studies and simultaneous analysis.

Delineation of the fish family Percichthyidae (Percomorphaceae) has a long and convoluted history, with recent morphological-based studies restricting species members to South American and Australian freshwater and catadromous temperate perches. Four recent nuclear gene-based phylogenetic studies, however, found that the Percichthyidae was not monophyletic and was nested within a newly discovered inter-familial clade of Percomorphaceae, the Centrarchiformes, which comprises the Centrarchidae and 12 other families. Here, we reexamined the systematics of the Percichthyidae and Centrarchiformes based on new mitogenomic information. Our mitogenomic results are globally congruent with the recent nuclear gene-based studies although the overall amount of phylogenetic signal of the mitogenome is lower. They do not support the monophyly of the Percichthyidae, because the catadromous genus Percalates is not exclusively related to the freshwater percichthyids. The Percichthyidae (minus Percalates) and Percalates belong to a larger clade, equivalent to the Centrarchiformes, but their respective sister groups are unresolved. Because all recent analyses recover a monophyletic Centrarchiformes but with substantially different intra-relationships, we performed a simultaneous analysis for a character set combining the mitogenome and 19 nuclear genes previously published, for 22 centrarchiform taxa. This analysis furthermore indicates that the Centrarchiformes are divided into three lineages and the superfamily Cirrhitoidea is monophyletic as well as the temperate and freshwater centrarchiform perch-like fishes. It also clarifies some of the relationships within the freshwater Percichthyidae.

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.

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.

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.

Unique patterns of pelvic fin evolution: a case study of balistoid fishes (Pisces: Tetraodontiformes) based on whole mitochondrial genome sequences.

Balistoid fishes have a unique and reduced pelvic fin structure, which does not exhibit paired structures. The pelvic complex exhibits reductive trends, but its rudimentary structure was retained among balistoids, and its unidirectional and parsimonious reduction in more derived lineages has been hypothesized based on morphology. We investigated the evolution of pelvic complex reduction in balistoids using whole mitochondrial genome (mitogenome) data from 33 species (27 newly determined during the study) that represent the entire morphological diversity of balistoids. Partitioned maximum likelihood and Bayesian analyses were conducted with two datasets that comprised concatenated nucleotide sequences from 13 protein-coding genes (all positions included; third codon positions converted into purine [R] and pyrimidine [Y] [RY-coding]) plus 22 transfer RNA and two ribosomal RNA genes. The resultant trees were well resolved and largely congruent, with most internal branches having high support values. The mitogenomic datasets strongly supported monophylies of both balistids and monacanthids, but rejected previous hypotheses on the intra-relationships in each family. The present tree topology revealed that highly reduced pelvic complexes had multiple origins, and optimization of the traits on the resultant tree strongly suggested the non-unidirectional and independent reduction of pelvic complexes in balistoids. The evolution of balistoid pelvic structure is very different among fishes that exhibit its reductive trends, and this uniqueness in pelvic evolution may be a link to their reproductive behaviors.

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.

Interrelationships of Atherinomorpha (medakas, flyingfishes, killifishes, silversides, and their relatives): The first evidence based on whole mitogenome sequences.

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.

A new perspective on phylogeny and evolution of tetraodontiform fishes (Pisces: Acanthopterygii) based on whole mitochondrial genome sequences: basal ecological diversification?

BACKGROUND: The order Tetraodontiformes consists of approximately 429 species of fishes in nine families. Members of the order exhibit striking morphological diversity and radiated into various habitats such as freshwater, brackish and coastal waters, open seas, and deep waters along continental shelves and slopes. Despite extensive studies based on both morphology and molecules, there has been no clear resolution except for monophyly of each family and sister-group relationships of Diodontidae + Tetraodontidae and Balistidae + Monacanthidae. To address phylogenetic questions of tetraodontiform fishes, we used whole mitochondrial genome (mitogenome) sequences from 27 selected species (data for 11 species were newly determined during this study) that fully represent all families and subfamilies of Tetraodontiformes (except for Hollardinae of the Triacanthodidae). Partitioned maximum likelihood (ML) and Bayesian analyses were performed on two data sets comprising concatenated nucleotide sequences from 13 protein-coding genes (all positions included; third codon positions converted into purine [R] and pyrimidine [Y]), 22 transfer RNA and two ribosomal RNA genes (total positions = 15,084). RESULTS: The resultant tree topologies from the two data sets were congruent, with many internal branches showing high support values. The mitogenomic data strongly supported monophyly of all families and subfamilies (except the Tetraodontinae) and sister-group relationships of Balistidae + Monacanthidae and Tetraodontidae + Diodontidae, confirming the results of previous studies. However, we also found two unexpected basal splits into Tetraodontoidei (Triacanthidae + Balistidae + Monacanthidae + Tetraodontidae + Diodontidae + Molidae) and Triacanthodoidei (Ostraciidae + Triodontidae + Triacanthodidae). CONCLUSION: This basal split into the two clades has never been reported and challenges previously proposed hypotheses based on both morphology and nuclear gene sequences. It is likely that the basal split had involved ecological diversification, because most members of Tetraodontoidei exclusively occur in shallow waters (freshwater, brackish and coastal waters, and open seas), while those of Triacanthodoidei occur mainly in relatively deep waters along continental shelves and slopes except for more derived ostraciids. This suggests that the basal split between the two clades led to subsequent radiation into the two different habitats.

Phylogenetic position of a whale-fall lancelet (Cephalochordata) inferred from whole mitochondrial genome sequences.

BACKGROUND: The lancelet Asymmetron inferum (subphylum Cephalochordata) was recently discovered on the ocean floor off the southwest coast of Japan at a depth of 229 m, in an anaerobic and sulfide-rich environment caused by decomposing bodies of the sperm whale Physeter macrocephalus. This deep sulfide-rich habitat of A. inferum is unique among the lancelets. The distinguishing adaptation of this species to such an extraordinary habitat can be considered in a phylogenetic framework. As the first step of reconstruction of the evolutionary processes in this species, we investigated its phylogenetic position based on 11 whole mitochondrial genome sequences including the newly determined ones of the whale-fall lancelet A. inferum and two coral-reef congeners. RESULTS: Our phylogenetic analyses showed that extant lancelets are clustered into two major clades, the Asymmetron clade and the Epigonichthys + Branchiostoma clade. A. inferum was in the former and placed in the sister group to A. lucayanum complex. The divergence time between A. inferum and A. lucayanum complex was estimated to be 115 Mya using the penalized likelihood (PL) method or 97 Mya using the nonparametric rate smoothing (NPRS) method (the middle Cretaceous). These are far older than the first appearance of large whales (the middle Eocene, 40 Mya). We also discovered that A. inferum mitogenome (mitochondrial genome) has been subjected to large-scale gene rearrangements, one feature of rearrangements being unique among the lancelets and two features shared with A. lucayanum complex. CONCLUSION: Our study supports the monophyly of genus Asymmetron assumed on the basis of the morphological characters. Furthermore, the features of the A. inferum mitogenome expand our knowledge of variation within cephalochordate mitogenomes, adding a new case of transposition and inversion of the trnQ gene. Our divergence time estimation suggests that A. inferum remained a member of the Mesozoic and the early Cenozoic large vertebrate-fall communities before shifting to become a whale-fall specialist.

Phylogenetic position of tetraodontiform fishes within the higher teleosts: Bayesian inferences based on 44 whole mitochondrial genome sequences.

Tetraodontiformes includes approximately 350 species assigned to nine families, sharing several reduced morphological features of higher teleosts. The order has been accepted as a monophyletic group by many authors, although several alternative hypotheses exist regarding its phylogenetic position within the higher teleosts. To date, acanthuroids, zeiforms, and lophiiforms have been proposed as sister-groups of the tetraodontiforms. The monophyly and sister-group status was investigated using whole mitochondrial genome (mitogenome) sequences from 44 purposefully-chosen species (26 sequences newly-determined during the study) that fully represent the major tetraodontiform lineages plus all the groups that have been hypothesized as being close relatives. Partitioned Bayesian analyses were conducted with the three datasets that comprised concatenated nucleotide sequences from 13 protein-coding genes (with and without, or with RY-coding, 3rd codon positions), plus 22 transfer RNA and two ribosomal RNA genes. The resultant trees were well resolved and largely congruent, with most internal branches being supported by high posterior probabilities. Mitogenomic data strongly supported the monophyly of tetraodontiform fishes, placing them as a sister-group of either Lophiiformes plus Caproidei or Caproidei only. The sister-group relationship between Acanthuroidei and Tetraodontiformes was statistically rejected using Bayes factors. These results were confirmed by a reanalysis of the previously published nuclear RAG1 gene sequences using the Bayesian method. Within the Tetraodontiformes, however, monophylies of the three superfamilies were not recovered and further taxonomic sampling and subsequent efforts should clarify these relationships.

The mitochondrial genome of spotted green pufferfish Tetraodon nigroviridis (Teleostei: Tetraodontiformes) and divergence time estimation among model organisms in fishes.

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.