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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.

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.

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.

In the Mediterranean flatfish are valuable fishery resources, with potential for aquaculture production. The assessment and monitoring of the stocks status are crucial to achieve environment-friendly management. However, the coexistence of two cryptic Solea species, S. solea and S. aegyptiaca, might lead to misidentification of specimens during the collection of fishery data, especially in areas where they live in sympatry. Using recently developed molecular markers, which have enabled their reliable identification, we provide new insights on the evolutionary history of the Mediterranean soles by analyzing species distribution, population structure, demography and inter-specific reproductive interactions with multiple molecular datasets. The analyses reveal a wider distribution of S. aegyptiaca in the Mediterranean Sea than previously described, hence pointing to a possible range expansion. In both species all markers consistently revealed significant population structuring according to a longitudinal gradient and demographic expansion. Despite their sympatric distribution in various regions, nuclear data confirmed the lack of inter-specific hybridization or introgression. Further multidisciplinarity in research (combining morphology, reproductive biology, life history traits analysis with genetics) are needed to unravel the possible range expansion of S. aegyptiaca according to a general hypothesis of Mediterranean tropicalization and its ecological interactions with the sympatric S. solea.

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.

Marine exploited populations exhibit various degrees of discreteness, ranging from historical evolutionary to contemporary ecological levels of differentiation. An integrated approach combining complementary population discrimination tools such as otolith microchemistry and genetics may capitalize on these different time scales to improve the discrimination and traceability power in management applications. Here, I discuss the most recent integrated results obtained from genetic markers (microsatellites and SNPs) and otoliths (microchemistry and shape data) on juvenile/adult sole (Solea solea) populations in the North-East Atlantic Ocean. While genetic markers provide a clear regional differentiation and local adaptation pattern, otoliths data often provide an improved small scale geographical resolution. Our results highlight the power of a multi-marker approach depending on the required spatial resolution scale in future management scenarios. Monitoring efficiently the resilience of marine harvested populations hence requires interdisciplinarity in research priorities, covering both the evolutionary and ecological components of population connectivity. Such information is pivotal to reliably develop, validate and later apply integrated traceability tools for fisheries enforcement.

In the Mediterranean, the common (Solea solea) and Egyptian (S. aegyptiaca) soles are two of the most valuable flatfish fishery resources. In the past, S. aegyptiaca was erroneously synonymised with Solea solea because of the great similarity in external morphology. Recently molecular markers have enabled their reliable identification, showing that the two species seem to co-occur in several areas of the Mediterranean, forming sympatric demes. The sympatric distribution and the close phylogenetic relationship allow a scenario for potential ecological and evolutionary interactions. Here, we aimed at providing new insights on the taxonomy, phylogeography and evolutionary interactions of the Mediterranean soles by i) developing PCR tests for the rapid screening of the two cryptic species, ii) analysing the species composition at several geographical locations, iii) assessing the phylogeographic pattern and genetic structure of populations and iv) the possible occurrence of interspecific hybridization in sympatric populations. In total 435 sole individuals were sequenced for a mitochondrial fragment (cytB gene) and 217 genotyped at two nuclear marker classes (ITS rDNA gene and 8 microsatellite loci). The analysis of species composition revealed a wider distribution of S. aegyptiaca in the Mediterranean than previously described. The observed frequent sympatry with S. solea could have lead to their frequent mis-identification. All nuclear markers however consistently revealed a clear genetic separation of the two species. Both mitochondrial and nuclear data showed the structuring of populations according to a longitudinal gradient, between the Western and Eastern basin, with different degree of differentiation in the two sole species. Higher genetic diversity was observed in S. solea for all markers. Further multidisciplinary data (combining data from morphology, reproductive biology and life history with genetic) are needed to unravel distribution and ecology of S. aegyptiaca in the Mediterranean and its ecological interactions with the cryptic species S. solea. This assessment will have important implications for sustainable management and conservation of fishery resources, because different species can differently respond to environmental pressures and changes.