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Species in a common landscape often face similar selective environments. The capacity of organisms to adapt to these environments may be largely species specific. Quantifying shared and unique adaptive responses across species within landscapes may thus improve our understanding of landscape-moderated biodiversity patterns. Here we test to what extent populations of two coexisting and phylogenetically related fishes—three-spined and nine-spined stickleback—differ in the strength and nature of neutral and adaptive divergence along a salinity gradient. Phenotypic differentiation, neutral genetic differentiation and genomic signatures of adaptation are stronger in the three-spined stickleback. Yet, both species show substantial phenotypic parallelism. In contrast, genomic signatures of adaptation involve different genomic regions, and are thus non-parallel. The relative contribution of spatial and environmental drivers of population divergence in each species reflects different strategies for persistence in the same landscape. These results provide insight in the mechanisms underlying variation in evolutionary versatility and ecological success among species within landscapes.

International audience; Background: European eel is a panmictic species, whose decline has been recorded since the last 20 years. Amonghuman-induced environmental factors of decline, the impact of water dams during species migration is questioned. The main issue of this study was to pinpoint phenotypic traits that predisposed glass eels to successful passage by water barriers. The approach of the study was individual-centred and without any a priori hypothesis on traits involved in the putative obstacles selective pressure. We analyzed the transcription level of 14,913 genes.Results: Transcriptome analysis of three tissues (brain, liver and muscle) from individuals sampled on three successive forebays separated by water obstacles indicated different gene transcription profiles in brain between the two upstream forebays. No differences in gene transcription levels were observed in liver and muscle samples among segments. A total of 26 genes were differentially transcribed in brain. These genes encode for, among others, keratins, cytokeratins, calcium binding proteins (S100 family), cofilin, calmodulin, claudin and thy-1 membrane glycoprotein. The functional analysis of these genes highlighted a putative role of cytoskeletal dynamics and synaptic plasticity in fish upstream migration.Conclusion: Synaptic connections in brain are solicited while eels are climbing the obstacles with poorly designedfishways. Successful passage by such barriers can be related to spatial learning and spatial orientation abilities when fish is out of the water.

Next-generation sequencing and the collection of genome-wide data allow identifying adaptive variation and footprints of directional selection. Using a large SNP data set from 259 RAD-sequenced European eel individuals (glass eels) from eight locations between 34 and 64oN, we examined the patterns of genome-wide genetic diversity across locations. We tested for local selection by searching for increased population differentiation using FST-based outlier tests and by testing for significant associations between allele frequencies and environmental variables. The overall low genetic differentiation found (FST = 0.0007) indicates that most of the genome is homogenized by gene flow, providing further evidence for genomic panmixia in the European eel. The lack of genetic substructuring was consistent at both nuclear and mitochondrial SNPs. Using an extensive number of diagnostic SNPs, results showed a low occurrence of hybrids between European and American eel, mainly limited to Iceland (5.9%), although individuals with signatures of introgression several generations back in time were found in mainland Europe. Despite panmixia, a small set of SNPs showed high genetic differentiation consistent with single-generation signatures of spatially varying selection acting on glass eels. After screening 50 354 SNPs, a total of 754 potentially locally selected SNPs were identified. Candidate genes for local selection constituted a wide array of functions, including calcium signalling, neuroactive ligand–receptor interaction and circadian rhythm. Remarkably, one of the candidate genes identified is PERIOD, possibly related to differences in local photoperiod associated with the >30° difference in latitude between locations. Genes under selection were spread across the genome, and there were no large regions of increased differentiation as expected when selection occurs within just a single generation due to panmixia. This supports the conclusion that most of the genome is homogenized by gene flow that removes any effects of diversifying selection from each new generation.

Unravelling the factors shaping the genetic structure of mobile marine species is challenging due to the high potential for gene flow. However, genetic inference can be greatly enhanced by increasing the genomic, geographical or environmental resolution of population genetic studies. Here, we investigated the population structure of turbot (Scophthalmus maximus) by screening 17 random and gene-linked markers in 999 individuals at 290 geographical locations throughout the northeast Atlantic Ocean. A seascape genetics approach with the inclusion of high-resolution oceanographical data was used to quantify the association of genetic variation with spatial, temporal and environmental parameters. Neutral loci identified three subgroups: an Atlantic group, a Baltic Sea group and one on the Irish Shelf. The inclusion of loci putatively under selection suggested an additional break in the North Sea, subdividing southern from northern Atlantic individuals. Environmental and spatial seascape variables correlated marginally with neutral genetic variation, but explained significant proportions (respectively, 8.7% and 10.3%) of adaptive genetic variation. Environmental variables associated with outlier allele frequencies included salinity, temperature, bottom shear stress, dissolved oxygen concentration and depth of the pycnocline. Furthermore, levels of explained adaptive genetic variation differed markedly between basins (3% vs. 12% in the North and Baltic Sea, respectively). We suggest that stable environmental selection pressure contributes to relatively strong local adaptation in the Baltic Sea. Our seascape genetic approach using a large number of sampling locations and associated oceanographical data proved useful for the identification of population units as the basis of management decisions.

Genomic methodologies applied in evolutionary and fisheries research have been of great benefit to understand the marine ecosystem and the management of natural resources. Although single nucleotide polymorphisms (SNPs) are attractive for the study of local adaptation, spatial stock management and traceability, and investigating the effects of fisheries-induced selection, they have rarely been exploited in non-model organisms. This is partly due to difficulties in finding and validating SNPs in species with limited or no genomic resources. Complementary to random genome-scan approaches, a targeted candidate gene approach has the potential to unveil pre-selected functional diversity and provides more in depth information on the action of selection at specific genes. For example genes can be under selective pressure due to climate change and sustained periods of heavy fishing pressure. In this study, we applied a candidate gene approach in sole (Solea solea L.), an important member of the demersal ecosystem. As consumption flatfish it is heavy exploited and has experienced associated life-history changes over the last 60 years. To discover novel genetic polymorphisms in or around genes linked to important life history traits in sole, we screened a total of 76 candidate genes related to growth and maturation using a targeted resequencing approach. We identified in total 86 putative SNPs in 22 genes and validated 29 SNPs using a multiplex single-base extension genotyping assay. We found 22 informative SNPs, of which two represent non-synonymous mutations, potentially of functional relevance. These novel markers should be rapidly and broadly applicable in analyses of natural sole populations, as a measure of the evolutionary signature of overfishing and for initiatives on marker assisted selection.

Laser ablation ICPMS (inductively coupled plasma mass spectrometry) analysis is a powerful tool for studies of fish ecology, based on measurement of the chemical composition of otoliths (ear stones). A key trade-off for this analysis is selecting the size of the ablation area to maximize the resolution for discrete temporal intervals during the life of an individual fish, vs the amount of otolith material required to produce reliable data. Three different widths of ablation lines were used to analyze the otoliths of European hake (Merluccius merluccius). The best temporal resolution was produced by ablation lines of 10 mu m width, corresponding to less than 2 weeks in the fish's life, but the data from this configuration were variable and often below the detection limit for many elements. Ablation lines of 20 and 30 mu m width produced accurate and precise data corresponding to approximately 20 and 30 days in terms of temporal resolution. When tested on hake otoliths, the measured element concentrations differed significantly between the 20 and 30 mu m lines. The 30 mu m ablation line resulted in a better multivariate model for discrimination between populations, with higher classification success and higher probability of individual assignment to source location.

This article documents the addition of 299 microsatellite marker loci and nine pairs of single-nucleotide polymorphism (SNP) EPIC primers to the Molecular Ecology Resources (MER) Database. Loci were developed for the following species: Alosa pseudoharengus, Alosa aestivalis, Aphis spiraecola, Argopecten purpuratus, Coreoleuciscus splendidus, Garra gotyla, Hippodamia convergens, Linnaea borealis,Menippe mercenaria,Menippe adina, Parus major, Pinus densiflora, Portunus trituberculatus, Procontarinia mangiferae, Rhynchophorus ferrugineus, Schizothorax richardsonii, Scophthalmus rhombus, Tetraponera aethiops, Thaumetopoea pityocampa, Tuta absoluta and Ugni molinae. These loci were cross-tested on the following species: Barilius bendelisis, Chiromantes haematocheir, Eriocheir sinensis, Eucalyptus camaldulensis, Eucalyptus cladocalix, Eucalyptus globulus, Garra litaninsis vishwanath, Garra para lissorhynchus, Guindilla trinervis, Hemigrapsus sanguineus, Luma chequen. Guayaba, Myrceugenia colchagu¨ensis, Myrceugenia correifolia, Myrceugenia exsucca, Parasesarma plicatum, Parus major, Portunus pelagicus, Psidium guayaba, Schizothorax richardsonii, Scophthalmus maximus, Tetraponera latifrons, Thaumetopoea bonjeani, Thaumetopoea ispartensis, Thaumetopoea libanotica, Thaumetopoea pinivora, Thaumetopoea pityocampa ena clade, Thaumetopoea solitaria, Thaumetopoea wilkinsoni and Tor putitora. This article also documents the addition of nine EPIC primer pairs for Euphaea decorata, Euphaea formosa, Euphaea ornata and Euphaea yayeyamana.

A thorough knowledge on the genetic connectivity of marine populations is important for fisheries management and conservation. Using a dense population sampling design and two types of neutral molecular markers (10 nuclear microsatellite loci and a mtDNA cytochrome b fragment), we inferred the genetic connectivity among the main known spawning grounds of sole (Solea solea L.) in the North-East Atlantic Ocean. The results revealed a clear genetic structure for sole in the North-East Atlantic Ocean with at least three different populations, namely the Kattegat/Skagerrak region, the North Sea and the Bay of Biscay, and with indications for a fourth population, namely the Irish/Celtic Sea. The lack of genetically meaningful differences between biological populations within the southern North Sea is likely due to a large effective population size and sufficient connection (gene flow) between populations. Nevertheless, an isolation-by-distance pattern was found based on microsatellite genotyping, while no such pattern was observed with the cytochrome b marker, indicating an historical pattern prevailing in the latter marker. Our results demonstrate the importance of a combined multi-marker approach to understand the connectivity among marine populations at region scales.

Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93–100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

Many marine pelagic fish species are characterized by subtle but complex genetic structures and dynamics, depending on the balance between current-mediated larval dispersal and adult active homing behavior. The circumantarctic continuous hydrodynamics of the Southern Ocean is a prime example of a system with a potentially great homogenizing effect among distant populations. We tested this hypothesis by analyzing the contemporary genetic relatedness among populations of a common and endemic mesopelagic fish of the Southern Ocean, Electrona antarctica. Seven newly developed species-specific microsatellite markers were used to investigate patterns of neutral genetic variation in 11 geographically widespread samples (n=400) collected between 2006 and 2007. We detected a very high level of genetic diversity, but a striking lack of genetic differentiation on a circumantarctic scale, indicating large effective population sizes complemented with high levels of admixture. These findings underscore the large scale homogenizing effect of the Southern Coastal Current, leading to a high level of connectivity of our model species in the Southern Ocean, which is congruent with its huge biomass and central role in marine food webs. As an important Antarctic marine living resource this species may as such be managed on a circumantarctic level, although the demographic stability of this stock should be estimated urgently.

Heavy fishing and other anthropogenic influences can have profound impact on a species’ resilience to harvesting. Besides the decrease in the census and effective population size, strong declines in mature adults and recruiting individuals may lead to almost irreversible genetic changes in life-history traits. Here, we investigated the evolution of genetic diversity and effective population size in the heavily exploited sole (Solea solea), through the analysis of historical DNA from a collection of 1379 sole otoliths dating back from 1957. Despite documented shifts in life-history traits, neutral genetic diversity inferred from 11 microsatellite markers showed a remarkable stability over a period of 50 years of heavy fishing. Using simulations and corrections for fisheries induced demographic variation, both single-sample estimates and temporal estimates of effective population size (Ne) were always higher than 1000, suggesting that despite the severe census size decrease over a 50-year period of harvesting, genetic drift is probably not strong enough to significantly decrease the neutral diversity of this species in the North Sea. However, the inferred ratio of effective population size to the census size (Ne/Nc) appears very small (10-5), suggesting that overall only a low proportion of adults contribute to the next generation. The high Ne level together with the low Ne/Nc ratio is probably caused by a combination of an equalized reproductive output of younger cohorts, a decrease in generation time and a large variance in reproductive success typical for marine species. Because strong evolutionary changes in age and size at first maturation have been observed for sole, changes in adaptive genetic variation should be further monitored to detect the evolutionary consequences of human-induced selection.

Recent improvements in the speed, cost and accuracy of next generation sequencing are revolutionising the discovery of single nucleotide polymorphisms (SNPs). SNPs are increasingly being used as an addition to the molecular ecology toolkit in non-model organisms, but their efficient use remains challenging. Here we discuss common issues when employing SNP markers, including the high numbers of markers typically employed, the effects of ascertainment bias, and the inclusion of non-neutral loci in a marker panel. We provide a critique of considerations specifically associated with the application and population genetic analysis of SNPs in non-model taxa, focusing specifically on some of the most commonly applied methods

4 pages; International audience; This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross-tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.

BackgroundOnce highly abundant, the European eel (Anguilla anguilla L.; Anguillidae; Teleostei) is considered to be critically endangered and on the verge of extinction, as the stock has declined by 90-99% since the 1980s. Yet, the species is poorly characterized at molecular level with little sequence information available in public databases.ResultsThe first European eel transcriptome was obtained by 454 FLX Titanium sequencing of a normalized cDNA library, produced from a pool of 18 glass eels (juveniles) from the French Atlantic coast and two sites in the Mediterranean coast. Over 310,000 reads were assembled in a total of 19,631 transcribed contigs, with an average length of 531 nucleotides. Overall 36% of the contigs were annotated to known protein/nucleotide sequences and 35 putative miRNA identified.ConclusionsThis study represents the first transcriptome analysis for a critically endangered species. EeelBase, a dedicated database of annotated transcriptome sequences of the European eel is freely available at http://compgen.bio.unipd.it/eeelbase webcite. Considering the multiple factors potentially involved in the decline of the European eel, including anthropogenic factors such as pollution and human-introduced diseases, our results will provide a rich source of data to discover and identify new genes, characterize gene expression, as well as for identification of genetic markers scattered across the genome to be used in various applications.

The evolutionary enlightened management of species with complex life cycles often requires the development of mathematical models integrating demographic and genetic data. The genetic structure of the endangered European eel (Anguilla anguilla L.) has been thoroughly analyzed in several studies in the past years. However, the interpretation of the key demographic and biologic processes that determine the observed spatio-temporal genetic structure has been very challenging owing to the complex life cycle of this catadromous species. Here, we present the first integrated demographic-genetic model applied to the European eel that explicitly accounts for different levels of larval and adult mixing during oceanic migrations and allows us to explore alternative hypotheses on genetic differentiation. Our analyses show that (i) very low levels of mixing occurring during larval dispersal or adult migration are sufficient to erase entirely any genetic differences among sub-populations; (ii) small-scale temporal differentiation in recruitment can arise if the spawning stock is subdivided in distinct reproductive groups; and (iii) the geographic differentiation component might be overestimated if a limited number of temporal recruits are analyzed. Our study can inspire the scientific debate on the interpretation of genetic structure in other species characterized by complex life cycle and long-range migrations.

Anguillid freshwater eels show remarkable life histories. In the Atlantic, the European eel (Anguilla anguilla) and American eel (Anguilla rostrata) undertake extensive migrations to spawn in the oceanic Sargasso Sea, and subsequently the offspring drift to foraging areas in Europe and North America, first as leaf-like leptocephali larvae that later metamorphose into glass eels. Since recruitment of European and American glass eels has declined drastically during past decades, there is a strong demand for further understanding of the early, oceanic phase of their life cycle. Consequently, during a field expedition to the eel spawning sites in the Sargasso Sea, we carried out a wide range of dedicated bio-physical studies across areas of eel larval distribution. Our findings suggest a key role of oceanic frontal processes, retaining eel larvae within a zone of enhanced feeding conditions and steering their drift. The majority of the more westerly distributed American eel larvae are likely to follow a westerly/northerly drift route entrained in the Antilles/Florida Currents. European eel larvae are generally believed to initially follow the same route, but their more easterly distribution close to the eastward flowing Subtropical Counter Current indicates that these larvae could follow a shorter, eastward route towards the Azores and Europe. The findings emphasize the significance of oceanic physical–biological linkages in the life-cycle completion of Atlantic eels.

European eels (Anguilla anguilla) undertake spawning migrations of more than 5000 km from continental Europe and North Africa to frontal zones in the Sargasso Sea. Subsequently, the larval offspring are advected by large-scale eastward ocean currents towards continental waters. However, the Sargasso Sea is oligotrophic, with generally low plankton biomass, and the feeding biology of eel larvae has so far remained a mystery, hampering understanding of this peculiar life history. DNA barcoding of gut contents of 61 genetically identified A. anguilla larvae caught in the Sargasso Sea showed that even the smallest larvae feed on a striking variety of plankton organisms, and that gelatinous zooplankton is of fundamental dietary importance. Hence, the specific plankton composition seems essential for eel larval feeding and growth, suggesting a linkage between eel survival and regional plankton productivity. These novel insights into the prey of Atlantic eels may furthermore facilitate eel larval rearing in aquaculture, which ultimately may replace the unsustainable use of wild-caught glass eels.

The fine scale morphological and genetic dynamics of successive waves of Anguilla anguilla glass eel recruitment was studied over a 2 year period at a southern European Mediterranean location (Camargue, France) with continuous recruitment. Using morphometric [total length (LT), mass (M), condition (K) and pigmentation stage] as well as genetic (allozyme) markers, the aim was to test for the existence of temporally separated spawning groups and explore the relation between genetic variability and morphological heterogeneity of recruits. The results showed that LT, M and K varied over time, being highest from the end of summer to winter (peaking in December) and lowest in spring (lowest in April). The pigmentation stages within monthly samples were highly diverse with a heterogeneous seasonal pattern. Allozyme data showed high genetic variability values within samples, but low genetic differentiation among samples (FST = 0.003, P < 0.05). Pairwise comparisons between samples indicated a positive correlation between genetic differentiation and difference in recruitment time (days), with a marked increase in genetic differentiation around 250 days between monthly recruitment samples. Furthermore, genetic diversity increased with the number of pigmentation stages per sample and was negatively correlated with the North Atlantic Oscillation (NAO) index during the putative year of trans-oceanic migration. No correlation, however, was found between the level of multilocus heterozygosity (MLH) and growth variables. A situation of genetic patchiness with fluctuating parental contribution can thus best explain the patterns observed, although the existence of two separate spawning periods cannot be excluded. More discriminatory and sensitive genetic markers, such as (neutral and adaptive) microsatellites, could probably provide additional insights into the most probable hypothesis explaining the population structure and recruitment heterogeneity of A. anguilla.

Marine organisms experience a broad range of intrinsic and extrinsic influences during their lives, which impact their population dynamics and genetic structure. Subtle interpopulation differences reflect the continuity of the marine environment, but also pose challenges to those wishing to define management units. The catadromous European eel (Anguilla anguilla) is no exception. Its spawning habitat in the Sargasso Sea and long migration across the North Atlantic qualify it as marine. However, the synergy between hydrographic variability, changing climate, and the impacts of habitat degradation and overfishing in continental waters has negatively affected stock sizes. Its protracted spawning period, variance in age-at-maturity, parental contribution and reproductive success, and the difficulty in sampling the spawning region together may mask a weak geographical genetic differentiation. Recent genetic data report evidence for spatial as well as temporal differences between populations, with the temporal heterogeneity between intra-annual recruitment and annual cohorts exceeding the spatial differences. Despite its common name of ‘fresh-water eel’, the European eel should really be managed on a North Atlantic scale. The fishery may have to be curtailed, migration routes kept open and water quality restored if it is to survive. Eel aquaculture has to focus on efficient rearing in the short term and controlled breeding in the long term. Future research on eel genetics should focus on (i) sampling and analysing spawning populations and recruitment waves to detect spatio-temporally discrete groups, and establishing a biological baseline from pre-decline historical collections for critical long-term monitoring and modelling of its genetic composition; (ii) the analysis of adaptive genetic polymorphism (genes under selection) to detect adaptive divergence between populations, perhaps requiring separate management strategies; and (iii) improving artificial reproduction to protect natural stocks from heavy exploitation, especially now the species has been categorized as endangered (Maes and Volckaert, 2007).

Otolith microchemistry, the study of the minor and trace elemental composition of otoliths (earbones), has been developing rapidly with a wide range of applications in fishery science. This is because otoliths (1) grow continuously, resulting in daily and seasonal structures, (2) have an elemental composition that reflects the environmental concentrations and conditions, (3) are metabolically inert. All of these characteristics make trace element uptake useful for reconstructing environmental histories. The elemental composition is therefore a powerful tool in stock To study connectivity among North Sea sole (Solea solea) populations and to improve our understanding of the relationship between its spawning grounds and nursery areas, genetic markers and otolith microchemistry will be used as complementary discrimination tools. We performed a pilot study to test for differences in sole otolith elemental fingerprints among three sampling locations, using LA-ICPMS. This technique makes it possible to determine composition at discrete points across the otolith (corresponding with different events in the fish’s lifetime). A hierarchical design (Basin, Sea, within Sea) enables us to assess the power of this method for the simultaneous large and small scale discrimination of populations.Preliminary results of this study will be shown and discussed in the light of current European wide research objectives. size and increasing the number of sampling locations.

The recognition of the vulnerability of marine species to environmental conditions throughout their life-cycle has broadened the scope of investigations on factors influencing their population dynamics and demographics. The European eel Anguilla anguilla (Linnaeus, 1758) stock is declining rapidly due to overfishing, pollution, habitat degradation, diseases, and oceanic conditions. We analyzed arrival waves of glass eels collected in the Bay of Biscay and the western Mediterranean area during the period 2001-2003. We observed significant differences in length, weight, and condition between Atlantic and Mediterranean samples, and among arrival waves within sites. All samples were screened for genetic variation using ten allozyme and six microsatellite loci. We observed a pattern of genetic patchiness among arrival waves, namely a highly significant genetic differentiation without a temporal grouping of samples. Although natural selection and gene flow could also play a role, we suggest that the pattern observed results from the high variance in reproductive success in each spawning season. A low effective population size might have contributed to the current decline in the abundance of European eel. A precautionary approach to fisheries should be implemented in order to preserve maximal genetic potential to cope with changing anthropogenic and environment pressures.

Marine organisms experience a broad range of intrinsic and extrinsic influences during their lives, which impact their population dynamics and genetic structure. Subtle interpopulation differences reflect the continuity of the marine environment, but also pose challenges to those wishing to define management units. The catadromous European eel (Anguilla anguilla) is no exception. Its spawning habitat in the Sargasso Sea and long migration across the North Atlantic qualify it as marine. However, the synergy between hydrographic variability, changing climate, and the impacts of habitat degradation and overfishing in continental waters has negatively affected stock sizes. Its protracted spawning period, variance in age-at-maturity, parental contribution and reproductive success, and the difficulty in sampling the spawning region together may mask a weak geographical genetic differentiation. Recent molecular data report evidence for spatial as well as temporal differences between populations, with the temporal heterogeneity between intra-annual recruitment and annual cohorts exceeding the spatial differences. Despite its common name of "fresh-water eel", the European eel should really be managed on a North Atlantic scale. The fishery may have to be curtailed, migration routes kept open and water quality restored if it is to survive. Eel aquaculture has to focus on efficient rearing in the short term and controlled breeding in the long term. Future research on eel genetics should focus on (i) sampling and analysing spawning populations and recruitment waves to detect spatio-temporally discrete groups, and establishing a biological baseline from pre-decline historical collections for critical long-term monitoring and modelling of its genetic composition; (ii) the analysis of adaptive genetic polymorphism (genes under selection) to detect adaptive divergence between populations, perhaps requiring separate management strategies; and (iii) improving artificial reproduction to protect natural stocks from heavy exploitation, especially now the species has been categorized as endangered.

Several commercial marine fish stocks show signs of collapse due to the synergy between climate change and adverse human impacts, such as habitat degradation and overfishing. Besides the reduction in abundance of the exploited species, intense fishing activities have an array of additional ecological impacts. Fisheries may cause irreversible evolutionary changes in life history traits and the genetic structure of populations. One of the species for which such phenotypic-genetic changes are likely to occur is common sole (Solea solea). This project aims at studying the connectivity and demographic stability of sole populations in the North Sea and adjacent waters before and after heavy (beam trawl) fishing. First, we will characterize the spatial and temporal connectivity of the different life stages of Solea solea. Are the spawning populations of Solea solea separated in time and space or are they well mixed? Do adult fish spawn near the nursery ground where they grew up? Do they return to the same spawning ground year after year? Through the combined analysis of otolith microchemistry and neutral genetic markers, we will answer these questions and investigate the extent to which spawning grounds, nursery areas and feeding grounds are linked. The level of gene flow between different populations will be estimated and adult fish from the feeding grounds will be assigned to their nursery origin. As such, we will evaluate the relative importance of different North Sea nurseries to adult populations. Secondly, we will analyze the long-term demographic stability of the sole population in the North Sea. Using DNA extracted from historical otolith collections (1960’s-1990’s), we will investigate possible fluctuations in genetic variability through time, in relation to fishery pressure and methods. The evolution of the effective number of breeding fish will be followed through time, providing a good indicator for the ‘genetic health status’ of the North Sea stock(s). Results will improve our understanding of the population genetic structure and the connectivity of spawning grounds, nurseries and feeding grounds of common sole. This knowledge is relevant for fisheries management, in order to identify appropriate management units and to evaluate the benefits of marine protected areas.

Heterogeneity in genetic composition among recruits of marine species is mostly due to a large variance in reproductive success mediated by oceanographic processes. Temporal genetic variation in a population of the European eel was quantified over 2 time scales among glass eel (1) interannual samples (cohorts), and (2) intra-annual samples within cohorts ('arrival waves'). A total of 789 glass eels comprising 11 different arrival waves were collected at Den Oever in The Netherlands over the period 2001 to 2003. All samples were screened for genetic variation using 10 allozyme and 6 microsatellite loci. The main result from this study is the highly significant genetic differentiation among arrival waves, despite the low FST values (FST = 0.0036). Heterogeneity in genetic composition was observed both among cohorts and among samples within cohorts. Genetic differentiation partitioned within cohorts was more than 10-fold the differences among cohorts. Genetic heterogeneity is likely to result from a large variance in the contribution of individuals to each cohort determined by genetic drift. Although natural selection and gene flow could also play a role in the observed genetic pattern, we suggest that large variances in reproductive success are a contributing factor to the recruit differentiation. If only a subset of the adults contribute to the new recruits, effective population size in European eel might be much lower than the census size. A low effective population size combined with fluctuating oceanic conditions might have contributed to the current dramatic decline in abundance of European eel.

Understanding the effects of pollutants on the genome is of crucial importance to preserve the evolutionary potential of endangered natural populations. The highly vagile European eel (Anguilla anguilla L.) has suffered a dramatic decline in recruitment since two decades, urging for a better understanding of the genetic impact of pollution. Its catadromous life history constitutes a model to assess local selection of pollutants on condition and genetic variability, as juveniles recruit in European rivers without appreciable pollution load or interfering genetic background. Because of its high fat content and local benthic feeding behaviour, the feeding stage is considered extremely prone to the bioaccumulation of pollutants. We studied the relationship between heavy metal bioaccumulation, fitness (condition) and genetic variability in the European eel. The muscle tissues of 78 sub-adult eels, originating from three Belgian river basins (Scheldt, Meuse and Yser), were examined for nine heavy metal pollutants (Hg, Cd, Pb, Cu, Zn, Ni, Cr, As and Se), while in total 123 individuals were genotyped at 12 allozyme and 8 microsatellite loci. A significant negative correlation between heavy metal pollution load and condition was observed, suggesting an impact of pollution on the health of sub-adult eels. In general, we observed a reduced genetic variability in strongly polluted eels, as well as a negative correlation between level of bioaccumulation and allozymatic multi-locus heterozygosity (MLH). Microsatellite genetic variability did not show any pollution related differences, suggesting a differential response at metabolic enzymes and possibly direct overdominance of heterozygous individuals.

Heterozygosity-fitness correlations (HFCs) have been reported in populations of many species. We provide evidence for a positive correlation between genetic variability and growth rate at 12 allozyme loci in a catadromous marine fish species, the European eel (Anguilla anguilla L.). More heterozygous individuals show a significantly higher length and weight increase and an above average condition index in comparison with more homozygous individuals. To a lesser extent, six microsatellite loci show a similar pattern, with positive but not significant correlations between heterozygosity and growth rate. The HFCs observed could be explained by an effect of either direct allozyme over-dominance or associative overdominance. Selection affecting some of the allozyme loci would explain the greater strength of the HFCs found at allozymes in comparison with microsatellites and the lack of correlation between MLH at allozymes and MLH at microsatellites. Associative overdominance (where allozyme loci are merely acting as neutral markers of closely linked fitness loci) might provide an explanation for the HFCs if we consider that allozyme loci have a higher chance than microsatellites to be in linkage disequilibrium with fitness loci.

Marine organisms usually exhibit a high genetic diversity, a subtle population structure and a low level of genetic differentiation, compared to freshwater organisms. The subtle genetic differences in time and space reflect the continuity of the marine environment. Marine organisms experience a wide range of intrinsic and extrinsic influences during their life cycle, which considerably impact their biological population size and genetic population structure. Furthermore, genetic variability is crucial for the survival of organisms as it enables evolution while maintaining fitness. Marine species however have a high genetic load, affecting the population even more during a population decline or bottleneck. The European eel Anguilla anguilla (Anguillidae; Teleostei), although inhabiting fresh- and saltwater, represents no exception. Its spawning habitat in the Sargasso Sea and extensive migrations across the North Atlantic Ocean qualify it fully as a marine species. This thesis describes the multiple evolutionary consequences of the catadromous life-strategy on the genetic structure of the European eel.Recent data based on microsatellite markers show a subtle genetic structure in the European eel following an Isolation-by-Distance (IBD) pattern. But since genetic introgression from the American eel into the European eel has been suggested in the North Atlantic Ocean, reliable tests were developed to define the species status of the European eel. In this first part the interspecific conservation of a set of microsatellites was tested on other Anguilla taxa and the power of species discrimination was assessed. We then applied this knowledge by screening Icelandic and European samples for introgression of American eel. Indications of unequal but restricted hybridisation were detected, likely maintained through selection against hybrids and the preservation of migrational cues.In the second part, the genetic variability and differentiation between various glass and silver eel populations was compared over a broad geographical range (Iceland to Morocco; Spain to Turkey) with temporal replications. In the first instance a pattern of Isolation-by- Distance was detected in adult populations using allozyme markers. Following a more extended geographical sampling, the temporal stability of this pattern could not be confirmed; the temporal differentiation between populations clearly exceeded the geographical component. By sampling recruiting glass eels over a three-year period, a stronger genetic differentiation was found between temporally separated cohorts. Inter-annual differentiation was much higher than the geographical differentiation. The population genetic structure of eel is likely determined by a double process: (I) a large scale pattern of Isolation-by-Time (IBT) among spawning cohorts, and (2) a smaller scale variance in adult reproductive success (genetic patchiness) among seasonally separated cohorts, most likely originating from oceanic and climatic influences.In the third part, the relation between multi-locus heterozygosity (MLH) and fitness components was studied. If an association exists between genetic variability and fitness traits, it is even more important to maintain the population size of European eel. The catastrophic decline of the European eel might be the consequence of an accelerated loss of genetic diversity, with extinction as possible outcome. This hypothesis was tested in a polluted natural environment and in an eel farm. Eel from three Belgian drainage basins were screened for fitness, heavy metal bioaccumulation and genetic variation. There was a strong negative correlation between MLH and bioaccumulation in highly polluted eels. In a second study, aquacultured eel were screened for fitness and genetic variation; MLH was correlated to growth rate. In both studies, this effect was mainly attributed to metabolical enzymes important in the energy cycle, which points to the importance of

The European eel (Anguilla anguilla, Linnaeus 1758) is a species typical for waters of Western Europe. Thanks to early expeditions on the Atlantic Ocean by the Danish biologist Johannes Schmidt who found small (< 10mm) leptocephali larvae in the Sargasso Sea about 100 years ago, we have now a strong indication where the spawning site for this species is located. The American eel (Anguilla rostrata, LeSueur) also spawns in the Sargasso Sea. The spawning time and location of both species have been supported and refined in recent analyses of the available historical data. Subsequent ichthyoplankton surveys conducted by McCleave (USA) and Tesch (Germany) in the 1980s indicated an increase in the number of leptocephali < 10 mm , confirming and refining the Sargasso Sea theory of Johannes Schmidt. Distinctions between the European and American eel are based on morphological characteristics (number of vertebrae) as well as molecular markers (allozymes, mitochondrial DNA and anonymous genomic-DNA. Although recognised as two distinct species, it remains unclear which mechanisms play a role in species separation during larval drift, and what orientation mechanism eels use during migration in the open sea. The current status of knowledge on these issues will be presented. The hypothesis that all European eel migrate to the Sargasso Sea for reproduction and comprise a single randomly mating population, the so called panmixia theory, was until recently broadly accepted. However, based on field observations, morphological parameters and molecular studies there are some indications that Schmidt's claim of complete homogeneity of the European eel population and a unique spawning location may be an overstatement. Recent molecular work on European eel indicated a genetic mosaic consisting of several isolated groups, leading to a rejection of the panmixia theory. Nevertheless, the latest extensive genetic survey indicated that the geographical component of genetic structure lacked temporal stability, emphasising the need for temporal replication in the study of highly vagile marine species. Induced spawning of hormone treated eels in the aquarium was collective and simultaneous. In this work for the first time group spawning behaviour has ever been observed and recorded in eels. Studies in swim-tunnels indicate that eels can swim four to six times more efficiently than non-anguilliform fish such as trout. After a laboratory swim trial of eels over 5,500 km, the body composition did not change and fat, protein and carbohydrate were used in the same proportion. This study demonstrated for the first time that European eel are physiologically able of reaching the Sargasso Sea without feeding. Based on catches of newly hatched larvae, temperature preference tests and telemetry tracking of mature hormone treated animals, it can be hypothesised that spawning in the Sargasso Sea is collective and simultaneous, while presumably taking place in the upper 200 m of the ocean. Successful satellite tracking of longfin female eels in New Zealand has been performed to monitor migration pathways. Implementation of this new technology is possible in this species because it is three times larger than the European eel. In the future, miniaturisation of tagging technology may allow European eels to be tracked in time by satellite. The most interesting potential contribution of telemetry tracking of silver eels is additional knowledge about migration routes, rates, and depths. In combination with catches of larvae in the Sargasso Sea, it may elucidate the precise spawninglocations of different eel species or groups. Only then, we will be able to define sustainable management issues by integrating this novel knowledge into spawners escapement and juvenile fishing quota.

The anadromous-freshwater three-spined stickleback (Gasterosteus aculeatus) system allows for inferring the role of adaptation in speciation with a high level of accuracy because the freshwater ecotype has evolved multiple times from a uniform anadromous ancestor. A cause for concern is that independent evolution among drainages is not guaranteed in areas with a poorly resolved glacial history. This is the case for the west European great rivers, whose downstream valleys flanked the southern limit of the late Pleistocene ice sheet. We tested for independent and postglacial colonization of these valleys hypothesizing that the relationships among anadromous and freshwater sticklebacks correspond to a raceme structure. We compared the reduction in plate number accompanying this colonization to the genetic differentiation using 13 allozyme and five microsatellite loci in 350 individuals. Overall microsatellite differentiation (FST = 0.147) was twice as large as allozyme differentiation (FST = 0.066). Although habitat-specific gene flow may mask the ancestral relationships among both ecotypes, levels of microsatellite differentiation supported the hypothesis of raceme-like divergence, reflecting independent colonizations rather than the presence of two distinct evolutionary clades. Under an infinite alleles model and in the absence of gene flow, the observed freshwater divergence might be reached after 440 (microsatellites) to 4500 (allozymes) generations. Hence, the anadromous-freshwater stickleback system most likely diverged postglacially. We conclude that the reduction in plate number in two freshwater basins probably occurred independently, and that its considerable variation among populations is not in agreement with the time since divergence.

The catfishes Brachyplatystoma flavicans (n=49) and Pseudoplatystoma fasciatum (n=69)showed comparable low allozyme diversities (He=0.012 and 0.009-0.028, respectively), butcontrasting PCR-RFLP restriction site mitochondrial DNA diversities (three haplotypes:p=0.034-0.092 and five haplotypes: p=0.001-0.023, respectively) in the Rio Ichilo andBeni (Bolivia). Genetic homogeneity between samples was high for B. flavicans and lower forP. fasciatum. Based on mitochondrial diversity, both species probably experienced a historicpopulation reduction but at different time scales.

Marine fish seem to experience evolutionary processes that are expected to produce genetically homogeneous populations. We have assessed genetic diversity and differentiation in 15 samples of the sand goby Pomatoschistus minutus (Pallas, 1770) (Gobiidae, Teleostei) from four major habitats within the Southern Bight of the North Sea, using seven microsatellite and 13 allozyme loci. Despite its high dispersal potential, microsatellite loci revealed a moderate level of differentiation (overall FST=0.026; overall RST=0.058). Both hierarchical analysis of molecular variance and multivariate analysis revealed significant differentiation (P

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 genetic variability and structure of the European eel (Anguilla anguilla L.) in populations throughout Europe was reassessed using 15 allozymic loci, seven of which were polymorphic. Seven sites were sampled on a latitudinal gradient across the natural continental range, extending from southern France to southern Norway. Heterozygosity (He = 0.05) and level of polymorphism (P = 0.43) were comparable to other marine fish. Populations were poorly differentiated (GST = 0.014, FST = 0.002), which is not surprising considering the high dispersal capability of the European eel. However, a significant geographical cline was detected at two alleles (IDh-1*100 and GPI-1*110), andgenetie distances (DCE) were concordant with geographical coastal distances. Mantel tests, pairwise FST's and multidimensional scaling analyses identify three distinct groups: Northern Europe, Western Europe and the Mediterranean Sea. We propose that the clinal genetic structure in the European eel may be due to (1) isolation by distance (as recently detected with microsatellites), (2) temporal reproductive separation, (3) post-larval selective forces, (4) contact between formerly separated groups or (5) some combination thereof.