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Barramundi (Lates calcarifer) is an important farmed marine food fish species. Its compact genome (~700 Mb) is among the smallest genomes of food fish species. We established a first-generation genetic linkage map of Barramundi with a mapping panel containing three parents (two males and one female) and 93 progeny. A total of 240 microsatellite markers were mapped into 24 linkage groups. Among these markers, 10 were located in ESTs and known genes. The total lengths of the female and male maps were 873.8 and 414.5 cM with an average marker spacing of 6.20 and 4.70 cM, respectively. Comparing the flanking sequences of the 240 Barramundi microsatellites with the assembled whole-genome sequences of Tetraodon nigrovidiris revealed 55 homologous sequences located in 19 of the 21 chromosomes of T. nigrovidiris. The map will not only enable the mapping of quantitative trait loci, but also provide new resources for understanding the evolution of fish genomes.

The genetic structure and history of the Indian scad mackerel, Decapterus russelli are studied to analyze the genetic breaks for marine fishes and invertebrates between the Indian and Pacific oceans. Results indicate that a Pleistocene drop in sea level caused a geographical isolation, secondary contact is the reason for the occurrence of two major mitochondrial lineages and low genetic diversities indicate a population bottleneck and subsequent speedy population expansion.

We present a phylogeographic analysis of an abundant New Zealand endemic sea-star, Patiriella regularis, to help pinpoint the location of an important biogeographic disjunction in central New Zealand. The analysis incorporates 284 mtDNA control region sequences (approximately 800 bp) of P. regularis from 22 coastal locations around New Zealand. We detected 132 haplotypes, with a mean divergence of 0.96%. AMOVA analysis of New Zealand samples is consistent with a north-south biogeographic disjunction across central New Zealand (among-group genetic variance = 6.10%; P = 0.0005). Cook Strait, the shallow marine strait separating the main islands, is not correlated with the disjunction: samples from northern South Island are genetically indistinguishable from North Island samples (variance = 1.69%; P = 0.073). These results are consistent with the hypothesis that upwelling zones south of Cook Strait constitute a significant barrier to larval dispersal.

The wahoo, Acanthocybium solandri (Cuvier, 1832), is a pelagic, highly migratory, scombroid fish, distributed worldwide throughout tropical and warm temperate seas. To evaluate population genetic and phylogeographic structure against a null hypothesis of panmixia, the entire mitochondrial DNA control-region (~890 base pairs) was sequenced for 231 wahoo. Samples were collected from 1997 to 2001 from seven sites: North Carolina (NC; n = 23), east central Florida (CF; n = 30), Bimini, Bahamas (BB; n = 40), southern tip of Florida (SF; n = 21), Cayman Islands (CI; n = 23), northern Gulf of Mexico (NG; n = 54), and Hawaii (HI; n = 40). Inter-annual samples were obtained from four of these locations (NC, BB, SF, NG). Seventeen haplotypes were shared by individuals within and among samples; 187 singleton haplotypes were observed. Within-sample haplotype diversities ranged from 0.995 to 1.000 (overall h = 0.999) and within-sample nucleotide diversities ranged from 0.049 to 0.055 (overall [pi] = 0.053). A neighborjoining tree based on inter-haplotypic distances revealed two monophyletic lineages differing by 13.6% nucleotide divergence. Nested within each major lineage were several, well-supported subclades. There was no evidence of temporal heterogeneity in haplotype distributions. Partitioning mtDNA variation, 99.75% of the variance was within samples and 0.25% (P = 0.307) between samples; the fixation index ([[PHI].sub.ST] = 0.0025) was not significant. Likewise, pairwise [[PHI].sub.ST] values were low or negative, and none were significant on a table-wide basis. Exact tests for sample differentiation in haplotypes were also nonsignificant. All population analyses were consistent with the null hypothesis of panmixia. However, analytical power was limited by sample size. Mismatch distributions were inconsistent with expected distributions based on sudden-expansion and static-growth models. Wahoo exhibit concurrently high haplotype and nucleotide diversities, presumably a consequence of secondary contact between historical subpopulations rather than a long, stable evolutionary history. Given the level of geographic and individual sampling, wahoo thus far represent the sole example of a scombroid or xiphioid fish exhibiting coarse-grain genetic homogeneity across a broad, inter-oceanic range despite a deeply coalescing genealogical structure. Accordingly, cooperative fishery management on a broad, inter-ocean scale may be warranted., Introduction The wahoo, Acanthocybium solandri, is a pelagic marine fish distributed worldwide in tropical and warm temperate oceanic waters (Collette and Nauen 1983). It is a member of the Perciform [...]

The group of subspecies of Patella ulyssiponensis, described by Christiaens, was widely known as Patella aspera until recently. The group extends throughout the Mediterranean, on all Macaronesian islands, along the North African coast, and in Europe, as far north as southern Norway. Throughout its range it displays great variation in shell sculpture and colour. The aim of this work was to re-examine the various subspecies proposed by Christiaens and, genetically, to test the hypothesis that European continental populations belong to a different species than the one composed by Macaronesian (north-west African) populations. In the present work, this group was studied by allozyme electrophoresis of 21 loci and by six morphological variables. The monomorphic locus of malate dehydrogenase (Mdh-1) was found to be diagnostic for distinguishing European continental populations from those of north-west African archipelagos, confirming the specific status of both groups. The allele observed at this locus in the Macaronesian populations was novel, while European continental populations showed a plesiomorphic one, shared with all other north-west Atlantic patellids with the exception of Patina pellucida, suggesting a more recent origin of the Macaronesian species from the continental forms. Both species showed a genetic identity of 0.730 [+ or -] 0.061, which allowed rough estimations of 6.5-3 Mya since speciation. The subspecies Patella ulyssiponensis deserta described by Christiaens was not confirmed by our genetic data. Other loci (glyceraldehyde-3-phosphate dehydrogenase and the second locus of lactate dehydrogenase) were partially diagnostic, in which both species showed different most common alleles. Morphologically, both species are easily recognised by shell characters and the results agree with previous findings, that continental populations are more homogeneous in shell morphology and radula characters than populations from the Macaronesia. This work supports retention of the earliest valid name, Patella ulyssiponensis Gmelin, with Lisbon, Portugal as type locality, for the European continental species, and Patella aspera Roding for the Macaronesian populations. Population subdivision within species was measured by theta, the estimator of [F.sub.st], showing in both P. aspera and P. ulyssiponensis a high degree of genetic structuring ([theta] = 0.226 and 0.182, respectively) mostly explained by the large distances separating the populations within species.

Phylogenetic and statistical methods were used to detect changes in diversification rate through time in representative marine fish clades. The results enabled to identify episodes of rapid cladogenesis in 14 of 34 studied datasets using different methods of chronogram reconstruction and to answer fundamental questions about tempo and mode of marine fish radiations.

The genetic structure of seven sailfish Istiophorus platypterus populations sampled from three locations inside and four locations outside the Arabian Gulf was determined by restriction fragment length polymorphism analysis of mitochondrial DNA of 147 individuals using eight restriction endonucleases. A total of 39 composite haplotypes derived from 27 presumptive restriction sites demonstrated significant differences in frequency between population groups inside and outside the Gulf (analysis of molecular variance 34.80%, P

Genetics diversity and differentiation was assessed in 15 samples of the sand goby Pomatoschistus minutus from four major habitats of the North Sea. The results showed indications of a complex and dynamic spatiotemporal structure that was determined by historical events and local oceanic currents.

Recently we discovered a previously uncharacterized gene with the characteristics of a membrane progestin receptor (mPR) in a fish model, spotted seatrout. Here, we report the identification, cloning, and characteristics of other members of this hitherto unknown family of putative mPRs from several vertebrate species, including human, mouse, pig, Xenopus, zebrafish, and Fugu, with highly conserved nucleotide and deduced amino acid sequences and similar structures to the spotted seatrout mPR. The 13 vertebrate genes identified seem to belong to an unknown gene family. Phylogenetic analysis indicates these cDNAs comprise three distinct groups (named [alpha], [beta], and [gamma]) within this gene family. Structural analyses of the translated cDNAs suggest they encode membrane proteins with seven transmembrane domains. The transcript sizes of the human [alpha], [beta], and [gamma] putative mPR mRNAs varied from 2.8 to 5.8 kb and showed distinct distributions in reproductive, neural, kidney and intestinal tissues, respectively. Recombinant human [alpha], [gamma], and mouse [beta] proteins produced in an Escherichia coli expression system demonstrated high affinity ([K.sub.d] = 20-30 nM) saturable binding for progesterone. Further analysis of binding to the [gamma]-subtype revealed binding was specific for progestins and was displaceable, with rapid rates of association and dissociation ([t.sub.1/2] = 2-8 min). These results suggest this is a new family of steroid receptors unrelated to nuclear steroid receptors, but instead having characteristics of G protein-coupled receptors.

Using eight microsatellite loci and a variety of analytical methods, we estimated genetic effective size ([N.sub.e]) of an abundant and long-lived marine fish species, the red drum (Sciaenops ocellatus), in the northern Gulf of Mexico (Gulf). The ratio [N.sub.e]/N, where short-term variance [N.sub.e] was estimated via the temporal method from shifts in allele-frequency data over four cohorts and where N reflected a current estimate of adult census size in the northern Gulf, was ~0.001. In an idealized population, this ratio should approximate unity. The extraordinarily low value of [N.sub.e]/N appears to arise from high variance in individual reproductive success and perhaps more importantly from variance in productivity of critical spawning and nursery habitats located in spatially discrete bays and estuaries throughout the northern Gulf. An estimate of [N.sub.e] based on a coalescent approach, which measures long-term, inbreeding effective size, was four orders of magnitude lower than the estimate of current census size, suggesting that factors presently driving [N.sub.e]/N to low values among red drum in the northern Gulf may have operated similarly in the past. Models that predict [N.sub.e]/N exclusively from demographic and life-history features will seriously overestimate [N.sub.e] if variance in reproductive success and variance in productivity among spatially discrete demes is underestimated. Our results indicate that these variances, especially variance in productivity among demes, must be large for red drum. Moreover, our study indicates that vertebrate populations with enormous adult census numbers may still be at risk relative to decline and extinction from genetic factors.

Determining the origin of genetic structure is of wide interest because of its use in stock discrimination in marine organisms. Schematically, genetic differentiation can result from historical patterns maintained over geological time or from present-day isolation attributable to biological characteristics of the species. We used a comparative approach to population genetic analysis based on allozyme polymorphism to determine the impact of reproductive strategy (i.e. biological origin) and habitat (i.e. historical origin) on the genetic structure of individuals sampled from five isolated islands in French Polynesia. Eight species of coral reef fishes from two families (Chaetodontidae and Pomacentridae) were selected to test the impact of sea-level change (historical origin) and reproductive strategy (biological origin) on genetic structure. Seven of the eight study species showed significant divergence in allelic frequencies computed over all sites. For these seven species, multilocus [F.sub.st] values ranged from 0.0114 to 0.0287. None of the eight species showed a significant relationship between genetic divergence and geographical distance between sites. Significant divergence (difference in allozyme frequencies) between some pairs of sites occurred but was unrelated to distances between them. These results suggest that the genetic structure of coral reef fish in French Polynesia is likely to be driven according to an island model in which migrations between populations are rare and random in space and time. Overall, none of the species showed congruent genetic structures between sites sampled. Genetic structure of the eight species did not appear significantly related either to reproductive strategy or habitat preference. Genetic diversity (heterozygosity) was significantly correlated with these two factors, with species laying benthic eggs and/or inhabiting lagoons showing significantly higher multilocus heterozygosity than species laying pelagic eggs and/or living on the outer reef slope. Overall, the absence of differences according to habitat and/or reproductive strategy did not provide any conclusive pattern regarding the origin of the genetic structure, but the limited divergence in allelic frequencies suggests recent differentiations.

Research has been conducted on the tropical marine fish Acanthochromis polyacanthus which has lost the broad-scale dispersal capacity among the coral reefs. The phylogeography of Acanthochromis polyacanthus with mitochondrial cytochrome b has been investigated and the results indicate that the sequence divergence is high between the major clades.

Research has been conducted on the evolutionary partitions in marine bonefish, Albula. The mitochondrial DNA cytochrome b have been investigated in examining the number and distribution of these partitions and the results are presented.

Barriers to gene flow have been studied in the marine fish, Embiotoca jacksoni, or black surfperch, lacking a pelagic larval stage. Major and minor phylogeographic breaks were found in some places, but none was found in the Point Conception region. The study, which compared data from Acanthochromis polyacanthus, indicated that the lack of a pelagic larval stage combined with barrier to adult dispersal may have had similar effects on the two species, for which gene flow levels were almost the same.

Spatial and temporal variation in restriction sites of mitochondrial DNA among seven species of marine fishes from the northern Gulf of Mexico and the southeast US Atlantic was investigated. The aim of the study was to find out whether population structure exists in species with an apparent large dispersal potential. Results showed that dispersal and gene flow in three sciaenids follow an isolation-by-distance model. Furthermore, population structuring was nonexistent in the three reef-associated species.

The population genetic structure of widely distributed pelagic marine fishes was investigated. The study focused on four marine fish species with circumtropical populations as well as four species of cosmopolitan temperate fishes with disjunct distributions. Results showed that continuously distributed marine fish species had population structures that are shallow but significant within an ocean basin. In contrast, greater intraspecific genetic divergence was found within discontinuously distributed marine fishes.

Shallow population genetic architectures in deep evolutionary lineages of marine fish species were investigated by reviewing genetic evidence from allozyme and mitochondrial DNA datasets for anchovies and sardines. In addition, factors that influence mitochondrial DNA diversity were examined to gain a better understanding of population regulation in marine fishes. Results showed that regional populations of anchovies and sardines are attenuated by periodic extinctions and recolonizations, apparently resulting in low levels of nucleotide diversity and shallow population histories.

The genetic variation between the orange roughy Hoplostethus atlanticus and the hoki Macruronus novaezelandiae species of New Zealand marine fishes can be studied through amplification and direct sequencing of the mitochondrial (mt) cytochrome b gene. Average sequence divergence and haplotype diversity is low in hoki and high in orange roughy, due to the orange roughy's low fecundity, slow maturation and long lifespan compared to the hoki. These traits enhance the survival of mtDNA variation by increasing the overlap of generations, reducing the variation in female reproductive success, and maintaining a high long-term effective population size of breeding females.

Dascyllus aruanus, collected from different areas of French Polynesia, exhibit homogeneity at lower spatial scales and low levels of differentiation among different archipelagoes. The evidence available rules out larval capacity for dispersal as a causal factor. Variations in sea level and low levels of polymorphism are possibly crucial factors affecting their distribution.

Dendrograms, developed by genetic-distance estimates through single-linkage, complete-linkage and neighbor-joining procedures, aids the study of genetic distances among various species of fish of the Siganus genus in the western Pacific Ocean. A study of the levels of genetic distinctions in various species reveals that certain species exhibit unique features of genetic development. Species are organized into 'ball clusters' according to their topologies. The results of the study are similar to predictions made previously in the study of Acanthuroidei.

Genetic variation in natural populations of the clonal killifish Rivulus marmoratus was investigated. Genetic variation is presumed tobe negligible in R. marmoratus, as it is the only vertebrate known to be a selfing hermaphrodite. DNA fingerprinting with microsatellite and minisatelliteprobes revealed very high levels of clonal diversity in seven populations of R.marmoratus in Florida. Since recombination is unknown in this species, the observed genetic diversity could mainly be due to mutations. The implications for the common notion that recombination has a predominant role in evolution was discussed.

A comparison of genetic diversity in captive and wild populations of two nearly extinct species of Goodeid fish Ameca splendens and Zoogoneticus tequila reveals that one is inbred in the wild. Both species showed clear evidence of a decline in effective population size, but the population size of A. splendens were smaller than that of the wild population but the wild population of Z. tequila did not show a significantly greater estimate.

Five species of near-shore fishes living on the Baja coastline of the Gulf of California share a large genetic break between northern and southern peninsular populations. It is seen that genetic divisions within Gulf of California fishes coincide with recognized biogeographic regions based on fish community composition and several environmental factors and adaptation to environment and present oceanographic circulation limit gene exchange and help maintain evidence of past vicariance.

A study conducted reveals that among marine fish, genetic monogamy, confirmed by molecular analysis of parentage, is reported. A documentary of all reported cases of monogamy in marine fishes is presented and the evidence that supports each of the proposal hypotheses are considered to synthesize the current understanding of monogamy into a single predictive framework.

Research has been conducted on reef fish species of the Gulf of California. The authors have tested the dispersal and vicariance hypotheses via the use of allozymes, DNA sequences, restriction fragment length polymorphisms, and via the use of phylogeorgaphic methods.

Unlike temperate fishes, Antarctic fishes of the notothenioid suborder, whose body temperatures (-2 to +1 [degrees] C) conform to the Southern Ocean, must express their genomes in an extremely cold thermal regime. To determine whether these fishes have evolved compensatory adjustments that maintain efficient gene transcription at low temperatures, we have initiated studies of the cis-acting regulatory elements that control globin gene expression in the Antarctic rockcod Notothenia coriiceps and in its close relative, the temperate New Zealand black cod N. angustata (habitat temperature = +6 to +15 [degrees] C). The genes encoding the major [Alpha]1 and [Beta] globins of these fishes are tightly linked in head-to-head (5' to 5') orientation. The intergenic regions separating the globin genes in the two fishes, ~4.3 kb in N. coriiceps and ~3.2 kb in N. angustata, are highly similar in sequence, the major difference being the absence of a 1.1-kb, repeat-containing segment in the latter. To assess the promoter and enhancer activities of the intergenic regions, each was cloned into the luciferase-reporter vector pGL3-Basic, and the constructs were transfected into MEL cells. Upon DMSO induction of MEL cell differentiation, each of the [Alpha]/[Beta]-intergenic regions functioned in both orientations as erythroid-responsive transcriptional regulators. However, expression of luciferase mediated by the N. coriiceps intergene was 6-fold greater in the [Alpha] orientation than that for the N. angustata intergene and 2-fold greater for the [Beta]. The greater transcription-stimulating activity of the N. coriiceps intergene can be attributed to two enhancers composed of combinations of CAC/Sp1 and GATA motifs and located in direct repeat elements. N. angustata, which lacked repetitive structure in its intergene, contained a single copy of the enhancer. We propose that cold adaptation of globin gene expression in N. coriiceps evolved in part through duplication and refinement of critical cis-acting regulatory elements as the Southern Ocean cooled during the past 25 million years.

The branchial epithelium in marine fish is the main site of acid-base transfers between the extracellular space and the environment. [Na.sup.+]/[H.sup.+] exchange proteins (NHE) are thought to contribute to net acid excretion in these animals. We have used molecular and biochemical methods to detect these antiporters in the branchial epithelium of two species of fish. 3'/5'-RACE reactions allowed us to clone and sequence for the first time a full length cDNA transcript for an NHE-2-like isoform from the gills of the marine long-horned sculpin (Myoxocephalus octodecimspinosus). The open reading frame is approximately 2890 bp in length and has a ~57% amino acid homology to the rat NHE-2 isoform with much lower homology at the 3' end. We have previously shown NHE-1 like immunoreactivity in the gills of the sculpin and the seawater adapted mummichog (Fundulus heteroclitus). Using Western analysis on gill membrane preparations from fresh and seawater adapted mummichogs, we have now observed proteins approximately 80 kDa and 77 kDa in size that are immunoreactive to antibodies made against mammalian NHE-1 (aa 514-818) and NHE-3 (aa 528-648), respectively. Following exposure of this species to 1% hypercapnia for one hour, an increase in expression was detected for both NHE-like protein isoforms in seawater adapted animals, while no difference in expression of NHE-1-like proteins could be detected in freshwater adapted fish. We hypothesize that during acidosis, apical gill NHE activity enhances net [H.sup.+] transfers to the water in these marine species. Supported by NSF-IBN-9808141 to JBC and AIMS.

The fishes of the Antarctic express their genomes in an extremely cold thermal environment (-1.86 to +1 [degrees] C). To determine the compensatory adjustments, if any, that adapt transcription to low temperatures, we are studying the structure, genomic organization, and expression of globin genes from an Antarctic rockcod, Notothenia coriiceps (Nc), and from a temperate relative, the New Zealand black cod IV. angustata (Na). The adult alpha- and beta-globin genes of these fishes are tightly linked in head-to-head (5' to 5') orientation. When assayed in MEL cells, the DNA sequences that separate the globin genes (4.3 kb in Nc and 3.2 kb in Na) function as bidirectional, erythroid-responsive promoters. However, the globin promoters of Nc are 2-6 fold more active than are the Na promoters. The greater activity of the Nc intergenic region appears to be due to an expanded enhancer that contains multiple copies of an Sp-1 binding motif. We propose that efficient gene expression by Antarctic fishes results in part from evolutionary restructuring of cis-acting gene regulatory elements.

To assess alternative methods for introducing expressing transgenes into the germ line of zebrafish, transgenic fish that express a nuclear-targeted, enhanced, green fluorescent protein (eGFP) gene were produced using both pseudotyped retroviral vector infection and DNA microinjection of embryos. Germ-line transgenic founders were identified and the embryonic progeny of these founders were evaluated for the extent and pattern of eGFP expression. To compare the two modes of transgenesis, both vectors used the Xenopus translational elongation factor 1-[Alpha] enhancer/promoter regulatory cassette. Several transgenic founder fish which transferred eGFP expression to their progeny were identified. The gene expression patterns are described and compared for the two modes of gene transfer. Transient expression of eGFP was detected 1 day after introducing the transgenes via either DNA microinjection or retroviral vector infection. In both cases of gene transfer, transgenic females produced eGFP-positive progeny even before the zygotic genome was turned on. Therefore, GFP was being provided by the oocyte before fertilization. A transgenic female revealed eGFP expression in her ovarian follicles. The qualitative patterns of gene expression in the transgenic progeny embryos after zygotic induction of gene expression were similar and independent of the mode of transgenesis. The appearance of newly synthesized GFP is detectable within 5-7 h after fertilization. The variability of the extent of eGFP expression from transgenic founder to transgenic founder was wider for the DNA-injection transgenics than for the retroviral vector-produced transgenics. The ability to provide expressing germ-line transgenic progeny via retroviral vector infection provides both an alternative mode of transgenesis for zebrafish work and a possible means of easily assessing the insertional mutagenesis frequency of retroviral vector infection of zebrafish embryos. However, because of the transfer of GFP from oocyte to embryo, the stability of GFP may create problems of analysis in embryos which develop as quickly as those of zebrafish. Key Words: zebrafish; pseudotyped retroviral vector; transgenes.