Naoto Hanzawa

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.

Phylogeny and biogeography of highly diverged freshwater fish species (Leuciscinae, Cyprinidae, Teleostei) inferred from mitochondrial genome analysis.

The distribution of freshwater taxa is a good biogeographic model to study pattern and process of vicariance and dispersal. The subfamily Leuciscinae (Cyprinidae, Teleostei) consists of many species distributed widely in Eurasia and North America. Leuciscinae have been divided into two phyletic groups, leuciscin and phoxinin. The phylogenetic relationships between major clades within the subfamily are poorly understood, largely because of the overwhelming diversity of the group. The origin of the Far Eastern phoxinin is an interesting question regarding the evolutionary history of Leuciscinae. Here we present phylogenetic analysis of 31 species of Leuciscinae and outgroups based on complete mitochondrial genome sequences to clarify the phylogenetic relationships and to infer the evolutionary history of the subfamily. Phylogenetic analysis suggests that the Far Eastern phoxinin species comprised the monophyletic clades Tribolodon, Pseudaspius, Oreoleuciscus and Far Eastern Phoxinus. The Far Eastern phoxinin clade was independent of other Leuciscinae lineages and was closer to North American phoxinins than European leuciscins. All of our analysis also suggested that leuciscins and phoxinins each constituted monophyletic groups. Divergence time estimation suggested that Leuciscinae species diverged from outgroups such as Tincinae to be 83.3 million years ago (Mya) in the Late Cretaceous and leuciscin and phoxinin shared a common ancestor 70.7 Mya. Radiation of Leuciscinae lineages occurred during the Late Cretaceous to Paleocene. This period also witnessed the radiation of tetrapods. Reconstruction of ancestral areas indicates Leuciscinae species originated within Europe. Leuciscin species evolved in Europe and the ancestor of phoxinin was distributed in North America. The Far Eastern phoxinins would have dispersed from North America to Far East across the Beringia land bridge. The present study suggests important roles for the continental rearrangements during the Late Cretaceous to form the present-day distribution of organisms. Furthermore, the Late Cretaceous biotic turnover influenced for the modern terrestrial biodiversity.

Phylogenetic Characterization of Three Morphs of Mussels (Bivalvia, Mytilidae) Inhabiting Isolated Marine Environments in Palau Islands

Marine lakes in the Palau Islands are known to harbor unique marine fauna that have remained isolated since the formation of the lakes after the Last Glacial Maximum. We analyzed mussels from marine lakes located on different islands and conducted morphological, phylogenetic and population genetic characterization to clarify their evolutionary history. The mussels were morphologically classified into three differentiated morphs: NS, ON, and MC. Their common characteristics were consistent with the Brachidontes—Hormomya complex of the Mytilidae family. Phylogenetic analysis based on the nuclear 18S ribosomal RNA gene supported the taxonomic position of the mussels among the Mytilidae. In the mitochondrial cytochrome c oxidase subunit I (COI) gene lineage, NS-and MC-morphs were highly diverged from each other; their estimated time of divergence dates back to the mid-Pleistocene. ON-morph was more closely related to MC-morph, although the shell morphologies of ON- and MC-morphs were easily distinguishable. Population genetic analysis revealed the coexistence of highly diverged haplotypes within a population of ON-morph, indicating introgression of mtDNA among the morphs. Our data suggest that morphological differentiation of marine lake mussels can occur in a relatively short period under different environmental conditions. Thus, the marine lakes provide a unique site for the study of diversification in mussels.

Erratum to: Comparative genomic analysis of mammalian NKG2D ligand family genes provides insights into their origin and evolution

NKG2D is a major activating receptor of natural killer cells. Its ligands are major histocompatibility complex (MHC) class I-like molecules whose expression is induced by cellular stresses such as infections and tumorigenesis. Humans have two families of NKG2D ligands (NKG2DL): MHC class I-related chains (MIC) encoded in the MHC and UL16-binding proteins (ULBP) encoded outside the MHC. By contrast, mice have only the latter family of ligands; instead, they have non-MHC-encoded MILL molecules that are closely related to MIC, but do not function as NKG2DL. To gain insights into the origin and evolution of MIC, ULBP, and MILL gene families, we conducted comparative genomic analysis of NKG2DL family genes in five mammalian species. In the opossum MHC, we identified a ULBP-like gene adjacent to a previously described MIC-like gene, suggesting that ULBP genes were originally encoded in the MHC. The opossum genome also contained a transcribed MILL-like gene in a region syntenic to the rodent regions encoding MILL molecules. These observations indicate that MIC-, ULBP-, and MILL-like genes emerged before the divergence of placental and marsupial mammals. Comparison of the human, cattle, rat, mouse, and opossum genomes indicates that after emigration from the MHC, ULBP genes underwent extensive duplications in each species. In mice, some of the ULBP genes appear to have been translocated telomerically on the same chromosome, forming a major cluster of existent NKG2DL genes.

Cytochrome oxidase 1 gene sequence analysis in six flatfish species (Teleostei, Pleuronectidae) of Far East Russia with inferences in phylogeny and taxonomy

Mitochondrial DNA at the cytochrome oxidase 1 (Co-1) gene region was sequenced for six flatfish species (in total, 11 sequences of at least 539 base pairs) from the Far East of Russia and compared with other sequences of Pleuronectiformes, comprising altogether 26 flatfish sequences and two outgroup sequences (Perciformes). An analysis of the protein-coding Co-1 gene revealed a statistically substantiated bias in (T+C):(A+G) content, supporting earlier findings. Average scores of the p-distances for different scales of the evolutionary history at the Co-1 gene revealed a clear pattern of increased nucleotide diversity at four different levels: (1) intraspecies, (2) intragenus, (3) intrafamily, and (4) intra-order. Scores of average p-distances of the four categories of comparison in flatfishes were (1) 0.17 ± 0.09%, (2) 10.60 ± 1.57%, (3) 12.40 ± 0.27%, and (4) 19.93 ± 0.05%, respectively (mean ± standard error). These data jointly with current knowledge support the concept that speciation in the order Pleuronectiformes mostly follows a geographic mode through accumulation of numerous small genetic changes over a long period of time. A phylogenetic tree for 26 sequences of flatfishes and two other fishes belonging to ray-finned fishes (Actinopterigii) was developed using the Co-1 gene and four different analytical approaches: neighbour-joining, Bayesian (BA), maximum parsimony (MP), and maximum likelihood. The analysis revealed a monophyletic origin for the representatives of Pleuronectidae, which is the principal flatfish family investigated (73–100% support level in our MP and BA analyses). According to the current and literary data, the monophyletic origin for the six compared flatfish families was well supported. Species identification on a per-individual basis (barcoding tagging) was high.

Sequence diversity at cytochrome oxidase 1 (Co-1) gene among sculpins (Scorpaeniformes, Cottidae) and some other scorpionfish of Russia Far East with phylogenetic and taxonomic insights

Mitochondrial DNA (mtDNA) atCo-1 gene region was sequenced for 7 scorpion fish species (in total, 16 sequences of at least 552 bp) from the Far East of Russia and compared with 15 other sequences of Scorpaeniformes comprising altogether 29 scorpion-like fish sequences and two outgroup sequences (Cypriniformes). The analysis of the protein-codingCo-1 gene revealed statistically substantiated bias in the (T+C) ∶ (A+G) content, proving basic findings. The average scores ofp-distances for different scales of the evolutionary history atCo-1 gene revealed a clear pattern of increased nucleotide diversity at four different levels: (1) intraspecies, (2) intragenus, (3) intrafamily, and (4) intraorder. The scores of averagep-distances for the compared fish groups were: (1) 1.00±0.20%, (2) 3.80±0.20%, (3) 12.40±1.20%, and (4) 18.00±0.38%, respectively (mean±SE). These data support the concept that speciation in the order Scorpaeniformes, in most cases, follows a geographic mode through accumulation of numerous small genetic changes over a long period of time. However, intraspecies diversity was surprisingly high among scorpionfish. Phylogenetic trees for 29 sequences of scorpionfish and 2 other fishes belonging to ray-finned fishes (Actinopterigii) were developed usingCo-1 gene and four different analytical approaches: Bayesian (BA), maximum likelihood (ML), neighbour-joining (NJ), and maximum parsimony (MP). The analysis revealed a monophyletic origin for the representatives of Cottidae, which is the principal scorpionfish family investigated (100, 96, 98% support level in our BA, MP, and NJ analyses). Similarly, the monophyletic origin of up to the three compared scorpion-like fish genera was supported by molecular phylogenetic data. Species identification on individual basis (barcoding tagging) was high. A few taxonomic complications arose during the analysis and they are discussed here in.

Characterization of comparative genome-derived simple sequence repeats for acanthopterygian fishes.

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.

Phylogenetic relationships of Russian far eastern flatfish (Pleuronectiformes, Pleuronectidae) based on two mitochondrial gene sequences, Co-1 and Cyt-b, with inferences in order phylogeny using complete mitogenome data.

Abstract The systematics and phylogeny of flatfish is investigated on the complete sequence of nucleotides at subunit 1 cytochrome c oxidase (Co-1) and cytochrome b (Cyt-b) genes. In total 17 species from our collection and some species from GenBank were analyzed. Four types of trees were built: Bayesian (BA), maximum likelihood (ML), maximum parsimony (MP), and neighbor joining (NJ). These trees showed similar topology. Two separate clusters on the trees support subfamily Hippoglossoidinae and Hippoglossinae subdivision and monophyletic status of these taxa. The subfamily Pleuronectinae also can be considered monophyletic, if the tribe Microstomini is excluded from it and genus Lepidopsetta is moved into the tribe Pleuronectini. Mitogenomes represented by 25 complete sequences from NCBI GenBank were analyzed. After alignment two sets of nucleotide sequences were formed and investigated independently. One set included 13 structural genes (14,886 bp), the second set comprised by the mtDNA without ND6 gene (10,457 bp). Both data sets give congruent phylogenetic signal that agreed with conventional views on the taxonomy of the order Pleuronectiformes; however, the first set gives better topology. In BA gene tree there are two well supported nodes which include the representatives of suborders Pleuronectoidei and Psettoidei. Within Pleuronectoidei two superfamilies, Pleuronectoidea and Soleidea are highly supported in BA but in all four kinds of gene trees (BA, ML, MP and NJ) the only superfamily Pleuronectoidea is well supported. At the top of hierarchy, all flatfishes belonging to the order Pleuronectiformes forming also a monophyletic clade in our data, with support level of 100% but in BA tree only. The monophyly of the family Pleuronectidae is well supported both by single gene data and by complete mtDNA sequences.

Molecular systematics and DNA barcoding of Altai osmans, oreoleuciscus (pisces, cyprinidae, and leuciscinae), and their nearest relatives, inferred from sequences of cytochrome b (Cyt-b), cytochrome oxidase c (Co-1), and complete mitochondrial genome

Abstract Mitochondrial DNA (mtDNA) at the protein-coding Cyt-b gene along with data retrieved from GenBank for Co-1 gene fragments and complete mitochondrial genome (mitogenome) of Altai osmans and the nearest relatives of Leuciscinae fish species were compared for the estimation of variability and phylogenetic tree building. Phylogenetic trees were built by four techniques: Bayesian (BA), maximum likelihood (ML), maximum parsimony (MP), and neighbor-joining (NJ). Resolution of Cyt-b trees for species of two genera (Oreoleuciscus and Phoxinus) was quite distinct at all the approaches. For Tribolodon, the single gene trees were not well resolved; however, the mitogenome tree was resolved. Species identification on per individual basis (DNA barcoding) was high for both Cyt-b and Co-1 genes. The trees built using the data for 13 protein mitochondrial genes revealed a complicated phylogenetic pattern within the subfamily Leuciscinae. Scores of the average p-distances at three taxonomic levels were considerably different: (1) 1.16 ± 0.96, (2) 8.21 ± 1.01, and (3) 16.41 ± 0.85 for Cyt-b and (1) 1.04 ± 0.78, (2) 8.30 ± 0.92, and (3) 10.74 ± 0.79 for 13 protein genes of mitogenome, where (1) is intraspecies, (2) is intragenus, and (3) is intrasubfamily levels. Data on mitogenome distances were summarized for the taxonomic hierarchy for the first time. A concordant increase in distance score with growth of the rank of taxa (having the minimum score at the intraspecies level), both for a single gene and the whole mitogenome, substantiates the concept that speciation in the subfamily Leuciscinae in most cases follows the geographic mode. The distinct clustering of Altai osmans, Oreoleuciscus potanini and O. humilis, in the Cyt-b and Co-1 gene trees with small overall genetic distances, obtained for both genes, allows us to consider these taxa as separate but genetically sister species.

Geographical distribution and genetic diversity of Gymnogobius sp. “Chokai-endemic species” (Perciformes: Gobiidae)

The geographical distribution and intraspecific genetic diversity of an endangered freshwater goby, Gymnogobius sp. “Chokai-endemic species”, was surveyed based on mitochondrial cytochrome b gene sequences in Yamagata Prefecture and adjacent areas on the island of Honshu, Japan. An extensive field survey showed that the species is distributed in 11 locations from Gosen, Niigata Prefecture to Yuza, Yamagata Prefecture. The species was not collected together with Gymnogobius sp. “widely distributed species” at each location, although the distribution of these two species overlaps. A maximum likelihood phylogenetic analysis showed that Gymnogobius sp. “Chokai-endemic species” is genetically divergent from other Gymnogobius castaneus species complex and the species consists of Niigata and Yamagata sub-clades. The result suggests that the Niigata and Yamagata regional groups can be distinguished from each other by their sequence divergence and the two groups should be treated as provisionally distinct “evolutionarily significant units”. The indices of intra-population genetic diversity were noticeably low, suggesting that each population has probably experienced bottleneck events. The geographical distance between the two populations in the Yamagata group, where the haplotypes are completely different from each other, was within 3 km. This suggests that habitat loss, fragmentation and isolation occurred in the Yamagata area due to some artificial cause. Conservation measures may be needed not only to protect individual habitats but also to promote genetic exchange among habitats in the Yamagata area.