Linking Pedigree Information to the Gene Expression Phenotype to Understand Differential Family Survival Mechanisms in Highly Fecund Fish: A Case Study in the Larviculture of Pacific Bluefin Tuna.

Mass spawning in fish culture often brings about a marked variance in family size, which can cause a reduction in effective population sizes in seed production for stock enhancement. This study reports an example of combined pedigree information and gene expression phenotypes to understand differential family survival mechanisms in early stages of Pacific bluefin tuna, Thunnus orientalis, in a mass culture tank. Initially, parentage was determined using the partial mitochondrial DNA control region sequence and 11 microsatellite loci at 1, 10, 15, and 40 days post-hatch (DPH). A dramatic proportional change in the families was observed at around 15 DPH; therefore, transcriptome analysis was conducted for the 15 DPH larvae using a previously developed oligonucleotide microarray. This analysis successfully addressed the family-specific gene expression phenotypes with 5739 differentially expressed genes and highlighted the importance of expression levels of gastric-function-related genes at the developmental stage for subsequent survival. This strategy demonstrated herein can be broadly applicable to species of interest in aquaculture to comprehend the molecular mechanism of parental effects on offspring survival, which will contribute to the optimization of breeding technologies.

Draft Whole-Genome Sequence of Triparma laevis f. inornata (Parmales, Bolidophyceae), Isolated from the Oyashio Region, Western North Pacific Ocean.

We present the first draft whole-genome sequence for the Parmales (Bolidophyceae, Heterokonta), a picoplanktonic sister group of diatoms, using a Triparma laevis f. inornata strain that was isolated from the Oyashio region in the western North Pacific Ocean.

Whole-Genome Sequencing of 84 Japanese Eels Reveals Evidence against Panmixia and Support for Sympatric Speciation.

The Japanese eel (Anguilla japonica), European eel (Anguilla anguilla), and American eel (Anguilla rostrata) are migratory, catadromous, temperate zone fish sharing several common life cycle features. The population genetics of panmixia in these eel species has already been investigated. Our extensive population genetics analysis was based on 1400 Gb of whole-genome sequence (WGS) data from 84 eels. It demonstrated that a Japanese eel group from the Kuma River differed from other populations of the same species. Even after removing the potential adapted/selected single nucleotide polymorphism (SNP) data, and with very small differences (fixation index [Fst] = 0.01), we obtained results consistently indicating that panmixia does not occur in Japanese eels. The life cycle of the Japanese eel is well-established and the Kuma River is in the center of its habitat. Nevertheless, simple reproductive isolation is not the probable cause of non-panmixia in this species. We propose that the combination of spawning area subdivision, philopatry, and habitat preference/avoidance accounts for the non-panmixia in the Japanese eel population. We named this hypothesis the "reproductive isolation like subset mapping" (RISM) model. This finding may be indicative of the initial stages of sympatric speciation in these eels.

Rhodopsin gene copies in Japanese eel originated in a teleost-specific genome duplication.

BACKGROUND: Gene duplication is considered important to increasing the genetic diversity in animals. In fish, visual pigment genes are often independently duplicated, and the evolutionary significance of such duplications has long been of interest. Eels have two rhodopsin genes (rho), one of which (freshwater type, fw-rho) functions in freshwater and the other (deep-sea type, ds-rho) in marine environments. Hence, switching of rho expression in retinal cells is tightly linked with eels' unique life cycle, in which they migrate from rivers or lakes to the sea. These rho genes are apparently paralogous, but the timing of their duplication is unclear due to the deep-branching phylogeny. The aim of the present study is to elucidate the evolutionary origin of the two rho copies in eels using comparative genomics methods. RESULTS: In the present study, we sequenced the genome of Japanese eel Anguilla japonica and reconstructed two regions containing rho by de novo assembly. We found a single corresponding region in a non-teleostean primitive ray-finned fish (spotted gar) and two regions in a primitive teleost (Asian arowana). The order of ds-rho and the neighboring genes was highly conserved among the three species. With respect to fw-rho, which was lost in Asian arowana, the neighboring genes were also syntenic between Japanese eel and Asian arowana. In particular, the pattern of gene losses in ds-rho and fw-rho regions was the same as that in Asian arowana, and no discrepancy was found in any of the teleost genomes examined. Phylogenetic analysis supports mutual monophyly of these two teleostean synteny groups, which correspond to the ds-rho and fw-rho regions. CONCLUSIONS: Syntenic and phylogenetic analyses suggest that the duplication of rhodopsin gene in Japanese eel predated the divergence of eel (Elopomorpha) and arowana (Osteoglossomorpha). Thus, based on the principle of parsimony, it is most likely that the rhodopsin paralogs were generated through a whole genome duplication in the ancestor of teleosts, and have remained till the present in eels with distinct functional roles. Our result indicates, for the first time, that teleost-specific genome duplication may have contributed to a gene innovation involved in eel-specific migratory life cycle.

Mitochondrial genotyping of an endangered bitterling Acheilognathus typus (Cyprinidae).

Genotyping of endangered species is helpful for establishing and evaluating conservation strategies. Mitochondrial sequence data was analyzed from 541 individuals of a critically endangered fish, Acheilognathus typus from present-day range-wide localities to re-evaluate an in-progress restoration program around Lake Izunuma-Uchinuma, Miyagi, Japan. Acheilognathus typus showed low sequence diversity with only eight haplotypes and π and h values of 0.59129 and 0.00118 respectively. Genetic data suggests Acheilognathus typus is adapted to pulsed environments and prone to population flush and crash. Genotyping of populations in introduced localities revealed that their source is not from nearby localities.

Sequencing and analysis of the complete organellar genomes of Parmales, a closely related group to Bacillariophyta (diatoms).

We sequenced the complete plastid and mitochondrial genomes of the unicellular marine phytoplankton Triparma laevis, belonging to the order Parmales (Heterokonta). The cells of Parmales are surrounded by silicified cell walls, similar to Bacillariophyta (diatoms). T. laevis was recognized as a sister group of Bacillariophyta using a molecular phylogenetic analysis based on SSU rDNA and rbcL sequences. Bacillariophyta are the most successful group of phytoplankton in the modern ocean, but the origin and early evolution of them have not been clearly established. Detailed molecular analyses of T. laevis may increase our understanding of the evolutionary relationships among Parmales and Bacillariophyta. The gene contents of the plastid and mitochondrial genomes are similar between T. laevis and Bacillariophyta. The gene order of the plastid genome is also similar to Bacillariophyta, whereas the gene order of the mitochondrial genome is not conserved in Bacillariophyta, but the structure is more compact than Bacillariophyta. Phylogenetic analyses, using plastid-encoded concatenated amino acid datasets and mitochondria-encoded concatenated amino acid datasets suggest that T. laevis is a sister group of Bacillariophyta. These results suggest that the characteristics of the organellar genomes of T. laevis are similar and conserve ancestral characteristics more than Bacillariophyta.

Transcriptomic features associated with energy production in the muscles of Pacific bluefin tuna and Pacific cod.

Bluefin tuna are high-performance swimmers and top predators in the open ocean. Their swimming is grounded by unique features including an exceptional glycolytic potential in white muscle, which is supported by high enzymatic activities. Here we performed high-throughput RNA sequencing (RNA-Seq) in muscles of the Pacific bluefin tuna (Thunnus orientalis) and Pacific cod (Gadus macrocephalus) and conducted a comparative transcriptomic analysis of genes related to energy production. We found that the total expression of glycolytic genes was much higher in the white muscle of tuna than in the other muscles, and that the expression of only six genes for glycolytic enzymes accounted for 83.4% of the total. These expression patterns were in good agreement with the patterns of enzyme activity previously reported. The findings suggest that the mRNA expression of glycolytic genes may contribute directly to the enzymatic activities in the muscles of tuna.

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.

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.

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.

Molecular and morphological discrimination between an invasive ascidian, Ascidiella aspersa, and its congener A. scabra (Urochordata: Ascidiacea).

The solitary ascidian Ascidiella aspersa (Müller, 1776) has sometimes been regarded as conspecific with A. scabra (Müller, 1776), although previous detailed morphological comparisons have indicated that the two are distinguishable by internal structures. Resolution of this taxonomic issue is important because A. aspersa has been known as a notoriously invasive ascidian, doing much damage to aquaculture e.g. in Hokkaido, Japan. We collected many specimens from European waters (including the Swedish coast, near the type localities of these two species) and Hokkaido, Japan (as an alien population) and made molecular phylogenetic analyses using the mitochondrial cytochrome c oxidase subunit I (COI) gene, and found that in terms of COI sequences all the analyzed specimens were clustered into two distinct groups, one of which is morphologically referable to A. aspersa and the other to A. scabra. Thus, these two species should be regarded as distinct from each other.

Tetra-repeat microsatellite markers for the masu salmon (Oncorhynchus masou masou) and its application in cross-subspecies amplification.

We developed tetranucleotide-repeat microsatellite markers for the masu salmon (Oncorhynchus masou) complex. 454 pyrosequencing was used to discover repeat motifs, and seven polymorphic microsatellite-primer sets were identified. The number of alleles detected at each locus ranged from four to 24 and the expected heterozygosity varied from 0.57 to 0.92. Cross-subspecies amplification for O. m. masou, O. m. ishikawae and O. m. subsp. was successful. These microsatellites can be utilized in studies of genetic structure, genetic diversity, and intra- and inter-subspecific hybridization, making a contribution to conservation and management of the Oncorhynchus masou complex.

Evolutionary changes of multiple visual pigment genes in the complete genome of Pacific bluefin tuna.

Tunas are migratory fishes in offshore habitats and top predators with unique features. Despite their ecological importance and high market values, the open-ocean lifestyle of tuna, in which effective sensing systems such as color vision are required for capture of prey, has been poorly understood. To elucidate the genetic and evolutionary basis of optic adaptation of tuna, we determined the genome sequence of the Pacific bluefin tuna (Thunnus orientalis), using next-generation sequencing technology. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified five common fish visual pigment genes: red-sensitive (middle/long-wavelength sensitive; M/LWS), UV-sensitive (short-wavelength sensitive 1; SWS1), blue-sensitive (SWS2), rhodopsin (RH1), and green-sensitive (RH2) opsin genes. Sequence comparison revealed that tuna's RH1 gene has an amino acid substitution that causes a short-wave shift in the absorption spectrum (i.e., blue shift). Pacific bluefin tuna has at least five RH2 paralogs, the most among studied fishes; four of the proteins encoded may be tuned to blue light at the amino acid level. Moreover, phylogenetic analysis suggested that gene conversions have occurred in each of the SWS2 and RH2 loci in a short period. Thus, Pacific bluefin tuna has undergone evolutionary changes in three genes (RH1, RH2, and SWS2), which may have contributed to detecting blue-green contrast and measuring the distance to prey in the blue-pelagic ocean. These findings provide basic information on behavioral traits of predatory fish and, thereby, could help to improve the technology to culture such fish in captivity for resource management.

Population subdivision of Japanese flounder Paralichthys olivaceus in the Pacific Coast of Tohoku Japan detected by means of mitochondrial phylogenetic information.

This study deals with mitochondrial phylogenetic information of Japanese flounder in the Pacific coast of Tohoku Japan to estimate the genetic population subdivision that was undetectable by conventional population statistics. We determined complete sequences of mitochondrial NADH dehydrogenase subunit-2 (ND2) and subunit-5 (ND5) genes for 151 individuals from northern (Aomori and Iwate prefectures, 40-41°N) and southern (Miyagi and Fukushima prefectures, 37-38°N) waters. Samples from both waters showed high genetic diversity, including 126 haplotypes. These haplotypes were located at mixed and nested positions on an inferred phylogenetic tree, and traditional F-statistics indicated no significant population divergence (φ(ST) = -0.00335, p > 0.05), corroborating our previous study. Three variable sites, however, showed significant base composition heterogeneity between samples from the northern and southern waters (Fisher's exact-test, p < 0.01). Nucleotide substitutions at the three sites converged on an apical clade, which consisted of the five southern individuals, whereas its sister clade consisted only of the three northern individuals. This phylogenetic information corroborates previous ecological studies indicating the presence of separate stocks in the northern and southern waters.

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.

Limits and phylogenetic relationships of East Asian fishes in the subfamily Oxygastrinae (Teleostei: Cypriniformes: Cyprinidae).

The cyprinid subfamily Oxygastrinae is composed of a diverse group of fishes that has been taxonomically and phylogenetically problematic. Their great variation in appearance, life histories, and trophic diversity resulted in uncertainty regarding their relationships, which led to their historical classification across many disparate subfamilies. The phylogenetic relationships of Oxygastrinae are resolved based on sequence data from four loci: cytochrome b, cytochrome c oxidase I, opsin, and recombination activating gene 1. A combined data matrix consisting of 4114 bp for 144 taxa was compiled and analyzed using maximum likelihood and parsimony optimality criteria. The subfamily Oxygastrinae is recovered as a monophyletic group that includes Ancherythroculter, Aphyocypris, Candidia, Chanodichthys, Ctenopharyngodon, Culter, Distoechodon, Elopichthys, Hainania, Hemiculter, Hemiculterella, Hemigrammocypris, Hypophthalmichthys, Ischikauia, Macrochirichthys, Megalobrama, Metzia, Mylopharyngodon, Nicholsicypris, Nipponocypris, Ochetobius, Opsariichthys, Oxygaster, Parabramis, Parachela, Paralaubuca, Pararasbora, Parazacco, Plagiognathops, Pseudobrama, Pseudohemiculter, Pseudolaubuca, Sinibrama, Squaliobarbus, Toxabramis, Xenocyprioides, Xenocypris, Yaoshanicus, and Zacco. Of these genera, the following were found to be monophyletic: Aphyocypris, Distoechodon, Hypophthalmichthys, Nipponocypris, Opsariichthys, Parachela, Paralaubuca, Plagiognathops, Xenocyprioides, and Xenocypris. The following genera were not monophyletic: Metzia, Hemiculter, Toxabramis, Ancherythroculter, Chanodichthys, Culter, Megalobrama. The remainder are either monotypic or were represented by only a single species. Four genera not examined in this study are provisionally classified in Oxygastrinae: Anabarilius, Longiculter, Pogobrama, and Rasborichthys.

Molecular phylogeny of the cyprinid tribe Labeonini (Teleostei: Cypriniformes).

The cyprinid tribe Labeonini (sensuRainboth, 1991) is a large group of freshwater fishes containing around 40 genera and 400 species. They are characterized by an amazing diversity of modifications to their lips and associated structures. In this study, a total of 34 genera and 142 species of putative members of this tribe, which represent most of the generic diversity and more than one third of the species diversity of the group, were sampled and sequenced for four nuclear genes and five mitochondrial genes (totaling 9465bp). Phylogenetic relationships and subdivision of this tribe were investigated and the placement and status of most genera are discussed. Partitioned maximum likelihood analyses were performed based on the nuclear dataset, mitochondrial dataset, combined dataset, and the dataset for each nuclear gene. Inclusion of the genera Paracrossochilus, Barbichthys, Thynnichthys, and Linichthys in the Labeonini was either confirmed or proposed for the first time. None of the genera Labeo, Garra, Bangana, Cirrhinus, and Crossocheilus are monophyletic. Taxonomic revisions of some genera were made: the generic names Gymnostomus Heckel, 1843, Ageneiogarra Garman, 1912 and Gonorhynchus McClelland, 1839 were revalidated; Akrokolioplax Zhang and Kottelat, 2006 becomes a junior synonym of Gonorhynchus; the species Osteochilus nashii was found to be a member of the barbin genus Osteochilichthys. Five historical hypotheses on the classification of the Labeonini were tested and rejected. We proposed to subdivide the tribe, which is strongly supported as monophyletic, into four subtribes: Labeoina, Garraina, Osteochilina, and Semilabeoina. The taxa included in each subtribe were listed and those taxa that need taxonomic revision were discussed.

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.

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.

The role of propagule pressure in the invasion success of bluegill sunfish, Lepomis macrochirus, in Japan.

The bluegill sunfish, Lepomis macrochirus, is a widespread exotic species in Japan that is considered to have originated from 15 fish introduced from Guttenberg, Iowa, in 1960. Here, the genetic and phenotypic traits of Japanese populations were examined, together with 11 native populations of the USA using 10 microsatellite markers and six meristic traits. Phylogenetic analysis reconfirmed a single origin of Japanese populations, among which populations established in the 1960s were genetically close to Guttenberg population, keeping high genetic diversity comparable to the ancestral population. In contrast, genetic diversity of later-established populations significantly declined with genetic divergence from the ancestral population. Among the 1960s established populations, that from Lake Biwa showed a significant isolation-by-distance pattern with surrounding populations in which genetic bottlenecks increased with geographical distance from Lake Biwa. Although phenotypic divergence among populations was recognized in both neutral and adaptive traits, P(ST)-F(ST) comparisons showed that it is independent of neutral genetic divergence. Divergent selection was suggested in some populations from reservoirs with unstable habitats, while stabilizing selection was dominant. Accordingly, many Japanese populations of L. macrochirus appear to have derived from Lake Biwa population, expanding their distribution with population bottlenecks. Despite low propagule pressure, the invasion success of L. macrochirus is probably because of its drastic population growth in Lake Biwa shortly after its introduction, together with artificial transplantations. It not only enabled the avoidance of a loss in genetic diversity but also formed a major gene pool that supported local adaptation with high phenotypic plasticity.

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.