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A classical understanding of marine fishes is that they have high effective population sizes and high levels of dispersal due to an apparent lack of barriers in the marine environment. The realization of a genetic population structure is therefore thought to be a slow process. To gain insights in this process, it is a challenge to disentangle the interaction between selection, gene flow, population history and genetic drift. The sand goby (Pomatoschistus minutus), which is an important ecological but noncommercial species, was chosen to distinguish between natural and anthropologically induced processes. A spatio-temporal analysis with samples from different localities throughout the species distribution and with two types of genetic markers was performed to assess the neutral genetic population structure. Phylogeographical patterns were studied by sequence cytochrome b (mtDNA). Nine new nuclear microsatellites were developed and used to describe the current genetic diversity and population structure. The genetic structure of the sand goby is best explained by a combination of present and historic factors. Due to its high potential for dispersion and high effective population size, it shows the typical features of a marine fish with a high level of diversity and a limited degree of genetic differentiation. The large genetic distance between the Venetian and all other samples shows that the sand gobies from the Adriatic Sea should be considered as a distinct cryptic species of the genus Pomatoschistus. Low but significant differentiation is observed between Atlantic and western Mediterranean P. minutus. In the Atlantic and Baltic basins, there is evidence for a postglacial range expansion and a weak pattern of isolation-by-distance. Furthermore the results support the hypothesis of a glacial refugium and a fine-scale genetic structure in the southern North Sea. The neutral genetic pattern will be compared with putatively adaptive loci in order to study the genomic characteristics of local adaptation in the marine environment. This should provide a better understanding of how fish respond to changes in the environment.

As a multitude of sequence data are published, discovering polymorphisms bioinformatically becomes a valid option. In silico Single Nucleotide Polymorphism (SNP) detection is based on the analysis of multiple alignments. Each column of an alignment is considered a slice containing one base of every sequence aligned. If a mismatch is detected, the slice is further analysed and the mismatch may be reported as a candidate SNP. Around 30,000 European sea bass (Dicentrarchus labrax) Expressed Sequence Tags (ESTs) have been sequenced and processed at the Max Planck Institute for Molecular Genetics. Since 55.1% (16,117) ESTs are redundant, they provide a resource for in silico SNP discovery. To prevent the detection of sequencing errors, a redundancy of 2 is required for a mismatch to be considered a candidate SNP (Picoult- Newberg et al., 1999). Thus, only contigs containing more than 4 overlapping sequences are analysed. 974 (21.3%) contigs qualify for in silico SNPs discovery, representing 5,548 (19%) ESTs and 478,232 base pairs. Various tools are used to detect candidate SNPs; so far, 2 software packages have been tried. 246 candidate SNPs, of which 56 indels, were proposed by SNPServer using default parameters (Savage et al., 2005). Less stringent parameters lead to the discovery of 1027 candidate SNPs, of which 267 indels. PolyBayes (Marth et al., 1999) selected 772 candidates SNPs, of which 231 indels. The Primer3 software is used to design primers flanking each candidate SNP, which are validated in the laboratory by sequencing. Polymorphisms will be mapped, used for selection in aquaculture and the study of adaptation in natural populations.

The BASSMAP consortium, funded by the European Union, has been set up to improve the understanding of the genome of European sea bass (Dicentrarchus labrax). The aim is to locate genes of known function on the physical map and to compare specific regions among perciforms. We produced an F1 cross of outbred sea bass as mapping panel and a Bacterial Artificial Chromosome library (7× redundancy and 165 kb average insert size). We use a panel of 21 wild sea bass to test for marker polymorphisms. All family genotypes are submitted to a database for pedigrees, markers and phenotypes (www.resspecies.org). These data will construct a first generation map including 162 microsatellite markers and 25 linkage groups (2n=24). A new second generation linkage map including 369 polymorphic microsatellite and AFLP markers, covering 34 linkage groups, will be disseminated via the mapping database (www.thearkdb.org). A set of 14 tissue specific cDNA banks have been constructed; 28,000 Expressed Sequence Tags (ESTs) have been sequenced in a data base (CeBiTec, U. Bielefeld). The sequences are annotated, screened for variation and mapped.

We report the isolation and characterization of 15 (12 di-, 1 tri- and 2 tetranucleotide) microsatellite markers from Hippocampus capensis, the Knysna seahorse. This marker set allows the detection of a genetic bottleneck as shown in a captive population. Furthermore, we test their genotyping potential in eight other seahorse taxa.

TROPHOS is a co-operation between five Belgian institutes (RUG-Marine Biology, KULeuven-Aquatic Ecology, Institute of Nature Conservation, MUMM and VLIZ) and one Dutch institute (NIOO-CEMO) and aims at a better understanding of processes structuring the higher trophic levels in the North Sea. As we will not ignore the link between ecosystem functioning and aquatic biodiversity, our results will also serve in aspects of sustainable management for those areas on the Belgian Continental Shelf (BCS) of the North Sea, which fulfil the criteria for protection status in marine reserves.

Microsatellites represent codominant molecular genetic markers, which are ubiquitously distributed within genomes. Due to their high level of polymorphism, relatively small size and rapid detection protocols, these markers are widely used in a variety of fundamental and applied fields of life and medical sciences. In the field of aquaculture, microsatellites represent workhorse markers, which are useful for the characterization of genetic stocks, broodstock selection, constructing dense linkage maps, mapping economically important quantitative traits, identifying genes responsible for these traits and application to marker-assisted breeding programmes. In this review, genomic distribution, function, evolution and practical applications of microsatellites are considered, with special emphasis on fish genetics and aquaculture.

A genetic linkage map of the European sea bass (Dicentrarchus labrax) was constructed from 174 microsatellite markers, including 145 new markers reported in this study. The mapping panel was derived from farmed sea bass from the North Adriatic Sea and consisted of a single family including both parents and 50 full-sib progeny (biparental diploids). A total of 162 microsatellites were mapped in 25 linkage groups. Eleven loci represent type I (coding) markers; 2 loci are located within the peptide Y (linkage group 1) and cytochrome P450 aromatase (linkage group 6) genes. The sex-averaged map spans 814.5 cM of the sea bass genome. The female map covers 905.9 cM, whereas the male map covers only 567.4 cM. The constructed map represents the first linkage map of European sea bass, one of the most important aquaculture species in Europe.

Eleven polymorphic microsatellites were isolated from the sea bass, Dicentrarchus labrax, using a microsatellite enrichment protocol and selective hybridization with an (AC)12 probe. The loci showed different variation patterns in 21 unrelated sea bass individuals, with a mean number of alleles of 8.6 and a mean observed heterozygosity of 0.68. These microsatellite markers should be useful for population genetic analysis and biodiversity studies of sea bass.

Phylogeographical patterns of the sand goby Pomatoschistus minutus (Gobiidae, Teleostei) were studied by means of sequence and single-stranded conformational polymorphism analysis of a 283-bp fragment of the cytochrome b locus of the mtDNA. A total of 228 individuals sampled at 13 sites throughout the species's distributional range revealed a moderate level of diversity and a low but significant level of overall genetic differentiation at all but one site. The goby sample from the Adriatic Sea differed in sequence by approximately 10% from the Atlantic P. minutus and is thought to belong to a cryptic species of the genus Pomatoschistus. Limited genetic differentiation with a weak pattern of isolation-by-distance was recorded throughout the distributional range of the typical P. minutus. Phylogeographical analysis suggested a contiguous range expansion in the Atlantic and Baltic basins during the Eemian and evidence for a glacial refugium in the southern North Sea during the Weichselian. In P. minutus from the western Mediterranean Sea a high number of endemic haplotypes as well as the most common Atlantic haplotype were recorded in appreciable frequencies. This might be explained by secondary contact between different mitochondrial lineages, which evolved in allopatry.

The Belgian Continental Shelf (BCS) is characterized by numerous subtidal sandbanks. Their close position to the coastline and the shallowness of the area make them unique, both from an ecological and geological point of view. It has recently been shown that the sandbank system in the Belgian coastal area acts as an intemationally important resting and foraging area for various seabird species. The project 'Structural and functional biodiversity of North Sea ecosystems: species and their habitats as indicators for a sustainable development of the Belgian continental shelf aims to assess the factors that determine and influence the marine biodiversity of the Belgian Continental Shelf. Within this project, an emphasis is made to incorporate all existing and new biodiversity information (community, population and genetic level) on all benthic compartments, including demersal fish, their parasites and birds. Most emphasis is given on the description of the structural aspects of benthic biodiversity in order to find a pattern within the relationship between the biological and the geochemical characteristics of the sediment. The question whether the structuring of ecosystem and species diversity and its functioning is reflected in the genetic structuring of marine taxa on the Belgian Continental Shelf is addressed for the first time.

The phylogeographical patterns of a small marine fish, the common goby, Pomatoschistus microps, were assessed at 12 sites along the northeastern Atlantic coasts and the western Mediterranean Sea. A combination of two genetic markers was employed: cellulose acetate allozyme electrophoresis (CAGE) and sequence analysis of a 289 bp fragment of the mitochondrial locus cytochrome b . Both markers were congruent in revealing significant differences between samples (global FST = 0.247 for the allozymes and [Greek symbol]Phi FST = 0.437 for the mitochondrial DNA data) and a pattern of isolation-by-distance. Phylogeographical analyses yielded a shallow branching structure with four groups. Three of those were confined to the Atlantic basin and showed a star-like pattern. The fourth group contained a central haplotype occurring at the edges of the species’ distribution, accompanied by a few more rare variants, which were restricted to the Mediterranean Sea. A genetic break was observed around the British Isles, with distinct haplotypes dominating at either side of the English Channel. A significantly negative correlation between the degree of genetic diversity and latitude was recorded both for mitochondrial DNA (mtDNA) and allozymes in the Atlantic basin. Gene flow analysis suggested that recolonization of the North Sea and the coasts of western Scotland and Ireland may have taken place from a glacial refugium in the Southern Bight of the North Sea. These results are discussed in the perspective of possible postglacial migration routes of marine fish along the northeastern Atlantic coasts.

We assessed genetic differentiation and diversity in 15 populations of sand goby (Pomatoschistus minutus) within the Southern Bight of the North Sea, using seven microsatellite and thirteen allozyme loci. While no significant differentiation was observed with allozymes, microsatellite loci revealed a moderate level of differentiation (overall FST=0.026; overall RST=0.058). The hierarchical analysis of molecular variance revealed a significant differentiation (pST and RST) of differentiation lead us to conclude that this genetic structure was mainly due to an historical event, namely colonisation of the estuary during its establishment, and to restricted actual gene flow. Therefore, using all of these loci, we found that the 15 natural populations of the sand goby can be clustered into two different breeding units, namely the Oosterschelde and coastal populations. Despite this clear distinction, there were indications of an actual complex dynamic local structure that would bear further examination to understand the processes involved in the differentiation of these two breeding units.

The excavations of Roman and Early Byzantine contexts at the town of Sagalassos (Turkey) yielded fish remains belonging to species that do not occur near the site. The modern geographical distribution of the identified fish indicates trade with various regions of Anatolia, the Mediterranean coast, Egypt and/or the Levant. Trade with Levant and Egypt is evident throughout the period by the presence of Clarias, a catfish living amongst others in the Nile and Levant. Mitochondrial DNA analysis was successfully carried out on modern populations of this species from Turkey, Syria, Israel and Egypt. Several variable regions were discovered on the mitochondrial control region containing polymorphisms that distinguish the haplotypes. Primer sets were designed to amplify small fragments of ancient DNA containing these informative regions. Ancient fish DNA could be successfully extracted, amplified and sequenced. The analyses indicate that the catfish bones belong to Clarias gariepinus and that they originated from the lower Nile. In addition, this study sheds light on the understanding of the modern distribution of C. gariepinus in Anatolia.

Although the North Sea is a relatively small basin, its hydrodynamical range varies from fully mixed to stratified, with tidal regimes up to 6 m. Tidally induced currents and wind stress generate a complex pattern of local currents. Marine invertebrates and fish, inhabiting this environment, are characterized by a high potential for dispersal. However, the realized dispersal depends on how they behaviourally deal with the current dynamics they experience. Here we report on how dispersal patterns of a mysid and two fishes, all common inhabitants of the North Sea, are reflected in their life-history and in their genetic variation.

Pleistocene genetic structure of the bullhead, Cottus gobio, was evaluated across the western Palearctic using a 771-bp long fragment of the mitochondrial control region in 123 individuals collected at 35 sites (data set I). In total, 59 haplotypes that differed at 73 positions (9.3%) were detected. Data analysis also included sequences from Englbrecht et al. (2000), thus increasing the sampling to a more comprehensive data set of 529 fish and 63 control region sequences of 482 bp (data set II). A minimum spanning and phylogenetic tree identified a seventh clade (Brittany-Loire) in addition to the previously identified six clades. The geographical range of the North Sea and Lower Rhine clades was considerably larger than thought previously. Haplotype diversity was generally low, and the total fixation index high (F-ST = 0.49). Among-group differentiation accounted for 41.7% (data set I) of the variation. Contiguous range expansions and restricted gene flow combined with isolation by distance, interspersed with past fragmentation characterize bullhead across its range. New is the knowledge that dated interglacial periods correlated with population expansions; river captures, proglacial lake systems and sea level played a significant role in the dispersal and expansion either in northern or southern direction. Hence it became possible to identify and date the colonization routes and putative palaeorefugia, most of which were located in Central and North-west Europe. Glacial periods resulted in distinct fragmentation events and lineage sorting.

The primary structure of the ribosomal DNA internal transcribed spacers (ITS-1 and ITS-2) and 5.8S rRNA gene were used to characterize and identify 2 monogenean species of Gyrodactylus living externally on the threespine stickleback (Gasterosteus aculeatus). The ITS region was amplified by PCR from freshwater, brackish, and marine isolates of Gyrodactylus arcuatus and G. branchicus, and the ends of the coding regions were identified by comparative alignment. No intraspecific and very low interspecific variation were observed in the 5.8S rRNA gene; high inter- and low intraspecific variation were revealed in the ITS-1 and ITS-2 regions. The morphological species identification was in all cases confirmed by the molecular identification. Intraspecifically, samples from 2 locations in the North Sea could be differentiated, but the Baltic sample resembled North Sea genotypes. Our approach offers perspectives for a multimetric genetical, morphometrical, and ecological taxonomy of the genus Gyrodactylus.

Fourteen dinucleotide DNA microsatellite loci were screened in the catfishes #Clarias gariepinus$, #C. batrachus$ and #Pangasius hypophthalmus$. They will be used for the screening of wild and cultured populations. (Résumé d'auteur)