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Viral nervous necrosis (VNN) is an infectious disease caused by the red-spotted grouper nervous necrosis virus (RGNNV) in European sea bass and is considered a serious concern for the aquaculture industry with fry and juveniles being highly susceptible. To understand the genetic basis for resistance against VNN, a survival phenotype through the challenge test against the RGNNV was recorded in populations from multiple year classes (YC2016 and YC2017). A total of 4,851 individuals from 181 families were tested, and a subset (n∼1,535) belonging to 122 families was genotyped using a ∼57K Affymetrix Axiom array. The survival against the RGNNV showed low to moderate heritability with observed scale estimates of 0.18 and 0.25 obtained using pedigree vs. genomic information, respectively. The genome-wide association analysis showed a strong signal of quantitative trait loci (QTL) at LG12 which explained ∼33% of the genetic variance. The QTL region contained multiple genes (

The association of molecular variants with phenotypic variation is a main issue in biology, often tackled with genome-wide association studies (GWAS). GWAS are challenging, with increasing, but still limited, use in evolutionary biology. We used redundancy analysis (RDA) as a complimentary ordination approach to single- and multitrait GWAS to explore the molecular basis of pigmentation variation in brown trout (Salmo trutta) belonging to wild populations impacted by hatchery fish. Based on 75,684 single nucleotide polymorphic (SNP) markers, RDA, single- and multitrait GWAS allowed the extraction of 337 independent colour patterning loci (CPLs) associated with trout pigmentation traits, such as the number of red and black spots on flanks. Collectively, these CPLs (i) mapped onto 35 out of 40 brown trout linkage groups indicating a polygenic genomic architecture of pigmentation, (ii) were found to be associated with 218 candidate genes, including 197 genes formerly mentioned in the literature associated to skin pigmentation, skin patterning, differentiation or structure notably in a close relative, the rainbow trout (Onchorhynchus mykiss), and (iii) related to functions relevant to pigmentation variation (e.g., calcium- and ion-binding, cell adhesion). Annotated CPLs include genes with well-known pigmentation effects (e.g., PMEL, SLC45A2, SOX10), but also markers associated with genes formerly found expressed in rainbow or brown trout skins. RDA was also shown to be useful to investigate management issues, especially the dynamics of trout pigmentation submitted to several generations of hatchery introgression.

Abstract Background In fish, minimally invasive blood sampling is widely used to monitor physiological stress with blood plasma biomarkers. As fish blood cells are nucleated, they might be a source a potential new markers derived from ‘omics technologies. We modified the epiGBS (epiGenotyping By Sequencing) technique to explore changes in genome-wide cytosine methylation in the red blood cells (RBCs) of challenged European sea bass (Dicentrarchus labrax), a species widely studied in both natural and farmed environments. Results We retrieved 501,108,033 sequencing reads after trimming, with a mean mapping efficiency of 73.0% (unique best hits). Minor changes in RBC methylome appeared to manifest after the challenge test and a family-effect was detected. Only fifty-seven differentially methylated cytosines (DMCs) close to 51 distinct genes distributed on 17 of 24 linkage groups (LGs) were detected between RBCs of pre- and post-challenge individuals. Thirty-seven of these genes were previously reported as differentially expressed in the brain of zebrafish, most of them involved in stress coping differences. While further investigation remains necessary, few DMC-related genes associated to the Brain Derived Neurotrophic Factor, a protein that favors stress adaptation and fear memory, appear relevant to integrate a centrally produced stress response in RBCs. Conclusion Our modified epiGBS protocol was powerful to analyze patterns of cytosine methylation in RBCs of D. labrax and to evaluate the impact of a challenge using minimally invasive blood samples. This study is the first approximation to identify epigenetic biomarkers of exposure to stress in fish.

Background: In fish, minimally invasive blood sampling is widely used to monitor physiological stress with blood plasma biomarkers. As fish blood cells are nucleated, they might be a source a potential new markers derived from ‘omics technologies. We modified the epiGBS (epiGenotyping By Sequencing) technique to explore changes in genome-wide cytosine methylation to a repeated acute stress challenge in the red blood cells (RBCs) of the European sea bass (Dicentrarchus labrax), species is widely studied in both natural and farmed environments.Results: We retrieved 501,108,033 sequencing reads after trimming, with a mean mapping efficiency of 73.0% (unique best hits). Minor changes in RBC methylome appeared to manifest after the challenge test and a family-effect was detected. Fifty-seven differentially methylated cytosines (DMCs) close to 51 distinct genes distributed on 17 of 24 linkage groups (LGs) were detected between RBCs of pre- and post-challenge individuals. Thirty-seven of these genes were previously reported as differentially expressed in the brain of zebrafish, most of them involved in stress coping differences. While further investigation remains necessary, few DMC-related genes associated to the Brain Derived Neurotrophic Factor, a protein that favors stress adaptation and fear memory, appear relevant to integrate a centrally produced stress response in RBCs.Conclusion: Our modified epiGBS protocol was powerful to analyze patterns of cytosine methylation in RBCs of D. labrax and to evaluate the impact of a stress challenge using minimally invasive blood samples. This study is the first approximation to identify epigenetic biomarkers of exposure to stress in fish.

Background: While the stress response inspired genome-wide epigenetic studies in vertebrate models, it remains mostly ignored in fish. We modified the epiGBS (epiGenotyping By sequencing) technique to explore changes in genome-wide cytosine methylation to a repeated acute stress challenge in the nucleated red blood cells (RBCs) of the European sea bass (Dicentrarchus labrax). This species is widely studied in both the natural and farmed environments, including issues regarding health and welfare.Results: We retrieved 501,108,033 sequencing reads after trimming, with a mean mapping efficiency of 73.0% (unique best hits). Minor changes in RBC methylome appear to manifest after the stress challenge. Only, fifty-seven differentially methylated cytosines (DMCs) close to 51 distinct stress-related genes distributed on 17 of 24 linkage groups (LGs) were detected between RBCs of pre- and post-stress individuals. However, literature surveys indicated that 38 of these genes were previously reported as differentially expressed in the brain of zebrafish, most of them involved in stress coping differences. DMC-related genes associated to the Brain Derived Neurotrophic Factor, a protein that favors stress adaptation and fear memory, appear especially relevant to integrate a centrally produced stress response.Conclusion: By putting forward DMCs associated to stress-related genes, we show that minimally invasive RBCs deserve more attention to investigate the epigenetic response to stress and components of the stress response without sacrificing fish. In parallel to blood parameter measurements (e.g. cortisol, glucose levels), and other molecular approaches (e.g. gene expression variation), features of the epigenetic landscape may offer new opportunities for biomonitoring components of the stress response in fish.

Understanding the molecular basis of stress is crucial in biology and of long standing interest in fish science. We tackled this question by modifying the epiGBS (epiGenotyping By sequencing) technique to screen for cytosine methylation and explore the genome-wide epigenomic response to a three months repeated acute stress challenge in the European sea bass (Dicentrarchus labrax). Following a minimally invasive sampling using nucleated red blood cells (RBCs), our modified epiGBS protocol retrieved 501,108,033 sequencing reads after trimming, with a mean mapping efficiency of 73.0% for unique best hits. Sequencing reads were shown to map across all linkage groups (LGs) of sea bass. A total of 47,983 CpG coordinates with a minimum 30X read depth was retained for differential methylation analysis between pre- and post-stress fish. A strong family effect was demonstrated, and fifty-seven distinct differentially methylated cytosines (DMCs) distributed on 17 of 24 LGs were found between RBCs of pre- and post-stress individuals. Twelve of them were located in intergenic regions, one in a repeated element; and forty four in gene bodies. Overall, DMCs were found inside or in the vicinity of 51 distinct genes shown previously to be related to stress. Thirty-eight of these genes were previously reported as differentially expressed in the brain of zebrafish, most of them involved in stress coping differences. Some DMC-related genes appear as good candidates to study the stress response in sea bass, especially a set of them associated to the Brain Derived Neurotrophic Factor (BDNF), a protein that favors stress adaptation and fear memory. Limits to our study and future directions are presented, including the use of RBCs as a surrogate to other target tissues, especially brain, to provide with classical physiological measurements a more complete picture of the stress response in fish.

Variation in body pigmentation attracted fish biologists for a while, but high-throughput genomic studies investigating its molecular basis remain limited to few species and associated conservation issues ignored. Using 75,684 SNPs, we explored the genomic basis of pigmentation pattern variation among individuals of the Atlantic and Mediterranean clades of the brown trout (Salmo trutta), a polytypic species in which Atlantic hatchery individuals are commonly used to supplement local wild populations. Using redundancy analyses and genome-wide association studies, a set of 337 independent “colour patterning loci” (CPLs) significantly associated with pigmentation traits such as the number of red and black spots on flanks, or the presence of a black spot on the pre-opercular bone was identified. CPLs map onto 35 out of 40 brown trout linkage groups indicating a polygenic basis to pigmentation patterns. They are mostly located in coding regions (43.4%) of 223 candidate genes, and correspond to GO-terms known to be involved in pigmentation (e.g. calcium and ion-binding, cell adhesion). Annotated candidates include genes with known pigmentation effects (e.g. SOX10, PEML, SLC45A2), but also the Gap-junction ⊗2 (GJD2) gene already shown differentially expressed in trout skin. Patterns of admixture were found significantly distinct when using either the full SNP data set or the set of CPLs, indicating that pigmentation patterns accessible to practitioners are not a reliable proxy of genome-wide admixture. Consequences for management are discussed.

Understanding the genetic underpinnings of fitness trade-offs across spatially variable environments remains a major challenge in evolutionary biology. In Mediterranean gilthead sea bream, first-year juveniles use various marine and brackish lagoon nursery habitats characterized by a trade-off between food availability and environmental disturbance. Phenotypic differences among juveniles foraging in different habitats rapidly appear after larval settlement, but the relative role of local selection and plasticity in phenotypic variation remains unclear. Here, we combine phenotypic and genetic data to address this question. We first report correlations of opposite signs between growth and condition depending on juvenile habitat type. Then, we use single nucleotide polymorphism (SNP) data obtained by Restriction Associated DNA (RAD) sequencing to search for allele frequency changes caused by a single generation of spatially varying selection between habitats. We found evidence for moderate selection operating at multiple loci showing subtle allele frequency shifts between groups of marine and brackish juveniles. We identified subsets of candidate outlier SNPs that, in interaction with habitat type, additively explain up to 3.8% of the variance in juvenile growth and 8.7% in juvenile condition, these SNPs also explained significant fraction of growth rate in an independent larval sample. Our results indicate that selective mortality across environments during early-life stages involves complex trade-offs between alternative growth strategies.

Understanding the evolutionary consequences of human-mediated introductions of domesticated strains into the wild and their subsequent admixture with natural populations is of major concern in conservation biology. However, the genomic impacts of stocking from distinct sources (locally derived vs. divergent) on the genetic integrity of wild populations remain poorly understood. We designed an approach based on estimating local ancestry along individual chromosomes to provide a detailed picture of genomic admixture in supplemented populations. We used this approach to document admixture consequences in the brown trout Salmo trutta, for which decades of stocking practices have profoundly impacted the genetic make-up of wild populations. In southern France, small local Mediterranean populations have been subject to successive introductions of domestic strains derived from the Atlantic and Mediterranean lineages. To address the impact of stocking, we evaluate the extent of admixture from both domestic strains within populations, using 75,684 mapped SNPs obtained from double-digested restriction site-associated DNA sequencing. Then, the chromosomal ancestry profiles of admixed individuals reveal a wider diversity of hybrid and introgressed genotypes than estimated using classical methods for inferring ancestry and hybrid pedigrees. In addition, the length distribution of introgressed tracts retained different timings of introgression between the two domestic strains. We finally reveal opposite consequences of admixture on the level of polymorphism of the recipient populations between domestic strains. Our study illustrates the potential of using the information contained in the genomic mosaic of ancestry tracts in combination with classical methods based on allele frequencies for analysing multiple-way admixture with population genomic data.

International audience; We examined specimens of the macrostigma trout Salmo macrostigma, which refers to big black spots on the flanks, to assess whether it is an example of taxonomic inflation within the brown trout Salmo trutta complex. Using new specimens, publicly available data and a mitogenomic protocol to amplify the control and cytochrome b regions of the mitochondrial genome from degraded museum samples, including one syntype specimen, the present study shows that the macrostigma trout is not a valid species. Our results suggest the occurrence of a distinct evolutionary lineage of S. trutta in North Africa and Sicily. The name of the North African lineage is proposed for this lineage, which was found to be sister to the Atlantic lineage of brown trout, S. trutta.

Background The brown trout (Salmo trutta) is an economically and ecologically important species for which population genetic monitoring is frequently performed. The most commonly used genetic markers for this species are microsatellites and mitochondrial markers that lack replicability among laboratories, and a large genome coverage. An alternative that may be particularly efficient and universal is the development of small to large panels of Single Nucleotide Polymorphism markers (SNPs). Here, we used Restriction site Associated DNA sequences (RADs) markers to identify a set of 12,204 informative SNPs positioned on the brown trout linkage map and suitable for population genetics studies. Then, we used this novel resource to develop a cost-effective array of 192 SNPs (96 × 2) evenly spread on this map. This array was tested for genotyping success in five independent rivers occupied by two main brown trout evolutionary lineages (Atlantic -AT- and Mediterranean -ME-) on a total of 1862 individuals. Moreover, inference of admixture rate with domestic strains and population differentiation were assessed for a small river system (the Taurion River, 190 individuals) and results were compared to a panel of 13 microsatellites. Results A high genotyping success was observed for all rivers (

A genome-wide assessment of diversity is provided for wild Mediterranean brown trout Salmo trutta populations from headwater tributaries of the Orb River and from Atlantic and Mediterranean hatchery-reared strains that have been used for stocking. Double-digest restriction-site-associated DNA sequencing (dd-RADseq) was performed and the efficiency of de novo and reference-mapping approaches to obtain individual genotypes was compared. Large numbers of single nucleotide polymorphism (SNP) markers with similar genome-wide distributions were discovered using both approaches (196 639 v. 121 016 SNPs, respectively), with c. 80% of the loci detected de novo being also found with reference mapping, using the Atlantic salmon Salmo salar genome as a reference. Lower mapping density but larger nucleotide diversity (π) was generally observed near extremities of linkage groups, consistent with regions of residual tetrasomic inheritance observed in salmonids. Genome-wide diversity estimates revealed reduced polymorphism in hatchery strains (π = 0·0040 and π = 0·0029 in Atlantic and Mediterranean strains, respectively) compared to wild populations (π = 0·0049), a pattern that was congruent with allelic richness estimated from microsatellite markers. Finally, pronounced heterozygote deficiency was found in hatchery strains (Atlantic FIS = 0·18; Mediterranean FIS = 0·42), indicating that stocking practices may affect the genetic diversity in wild populations. These new genomic resources will provide important tools to define better conservation strategies in S. trutta.

International audience; Six candidate gene SNP assays and 12 new microsatellites were developed in the European flounder (Platichthys flesus), in order to provide new tools to (1) further investigate signatures of selection suspected on candidate genes, and (2) better depict the neutral evolutionary background shaping the differentiation of populations. All markers were successfully tested in two populations from contrasted estuaries, and should be helpful to better understand the possible adaptive responses of flounder populations to anthropogenic pressures (e.g. global warming, eutrophication, pollutions), over the whole distribution range of the species.

High-density linkage maps are valuable tools for conservation and eco-evolutionary issues. In salmonids, a complex rediploidization process consecutive to an ancient whole genome duplication event makes linkage maps of prime importance for investigating the evolutionary history of chromosome rearrangements. Here, we developed a high-density consensus linkage map for the brown trout (Salmo trutta), a socio-economically important species heavily impacted by human activities. A total of 3,977 ddRAD markers were mapped and ordered in 40 linkage groups using sex- and lineage-averaged recombination distances obtained from two family crosses. Performing map comparison betweenS. truttaand its sister speciesSalmo salarrevealed extensive chromosomal rearrangements. Strikingly, all of the fusion and fission events that occurred after theS.salar/S.truttaspeciation happened in the Atlantic salmon branch, whereas the brown trout remained closer to the ancestral chromosome structure. Using the strongly conserved synteny within chromosome arms, we aligned the brown trout linkage map to the Atlantic salmon genome sequence to estimate the local recombination rate inS.truttaat 3,721 loci. A significant positive correlation between recombination rate and within-population nucleotide diversity (π) was found, indicating that selection constrains variation at linked neutral sites in brown trout. This new high density linkage map provides a useful genomic resource for future aquaculture, conservation and eco-evolutionary studies in brown trout.

Fasting and refeeding occur naturally in predators but this is largely ignored when dealing with farmed fish. Therefore, the effects of 3-week fasting and re-alimentation (2.5% of the individual body mass) were investigated using two genetically selected populations (F2 generation) of 250 g juvenile sea bass (Dicentrarchus labrax L.). Blood osmolarity, gill and intestinal morphology and expression of the sodium pump (Na+, K+-ATPase, NKA) were studied on two phenotypes showing different degrees of body mass loss during food deprivation: one group losing body mass rapidly during fasting (F +) and the other one limiting body mass loss during the same period (F −). Blood osmotic pressure significantly decreases due to re-alimentation in both groups, but this is compensated in the F + group. In this group, gill ionocytes are smaller and less numerous, but a significantly higher NKA gene expression is noted in the gills in comparison to the F − individuals 48 and 72 h after re-alimentation, and also in the posterior intestine 72 h after re-alimentation. This most probably occurs to compensate for a higher salt intake during nutrient absorption in comparison to the F − group. Furthermore, refed F − fish absorb more lipids along the proximal anterior intestine, and take longer to digest than the F + group, and show enterocyte vacuolization in the posterior intestine. Therefore, the two selected populations have different postprandial digestive strategies: the F − fish optimize feed efficiency first at the cost of optimal hydromineral adjustment, while the F + group invests in osmoregulatory performance at the expense of digestive physiology. Statement of relevance Our paper is highly relevant to the general field of commercial aquaculture. There is an increasing number of research articles dealing with fasting and refeeding in commercial fish and how to improve fish nutrition based on these physiological data and genetic selection.

Ajouter clé UT WOS; Describing and explaining the geographic within-species variation in phenotypes ("phenogeography") in the sea over a species distribution range is central to our understanding of a variety of eco-evolutionary topics. However, phenogeographic studies that have a large potential to investigate adaptive variation are overcome by phylogeographic studies, still mainly focusing on neutral markers. How genotypic and phenotypic data could covary over large geographic scales remains poorly understood in marine species. We crossed 75 noninbred sires (five origins) and 26 dams (two origins; each side of a hybrid zone) in a factorial diallel cross in order to investigate geographic variation for early survival and sex ratio in the metapopulation of the European sea bass (Dicentrarchus labrax), a highly prized marine fish species. Full-sib families (N = 1,950) were produced and reared in a common environment. Parentage assignment of 7,200 individuals was performed with seven microsatellite markers. Generalized linear models showed significant additive effects for both traits and pleiotropy between traits. A significant nonadditive genetic effect was detected. Different expression of traits and distinct relative performances were found for reciprocal crosses involving populations located on each side of the main hybrid zone located at the Almeria-Oran front, illustrating asymmetric reproductive isolation. The poor fitness performance observed for the Western Mediterranean population of sea bass is discussed as it represents the main source of seed hatchery production, but also because it potentially illustrates nonadaptive introgression and maladaptation.

The existence of cryptic salinity-related phenotypes has been hypothesized in the “euryhaline” sea bass ( Dicentrarchus labrax ). How differential osmoregulation costs between freshwater and saltwater environments affect fitness and phenotypic variation is misunderstood in this species. During an experiment lasting around five months, we investigated changes in the whole body mass and in the expression of growth-related genes (insulin-like growth factor 1 [IGF-1]; growth hormone receptor [GHR]) in the intestine and the liver of sea bass thriving in sea water (SSW), successfully acclimated to freshwater (SFW), and unsuccessfully acclimated to freshwater (UFW). Albeit non-significant, a trend toward change in body mass was demonstrated among SSW, UFW and SFW fish, suggesting that SSW fish were a mixture of the other phenotypes. Several mortality peaks were observed during the experiment, with batches of UFW fish showing higher expression in the osmoregulatory intestine due to down-regulation of genes in the liver and significant up-regulation of GHR in the intestine compared with SFW fish. Energy investment toward growth or ion homeostasis hence partly mediates the fitness difference between cryptic SFW and UFW phenotypes. The use of a genetic marker located within the IGF-1 gene showed no genotype-phenotype relationship with levels of gene expression.

Anthropogenic pressures in estuaries may impair not only the fitness of organisms and the evolutionary processes necessary to the survival, but also the management of exploited marine resources. In order to assess these impacts, phenotypic and genotypic data pertaining to young-of-the-year (YOY) sole ( Solea solea ) were screened in differentially contaminated North East Atlantic estuaries ranging from Portugal (Mondego) to the English Channel (Seine). YOY sole inhabiting each estuary was found being phenotypically distinct from each other; each of them being impacted by pollution when comparing observed phenotypic data with published literature. According to the strong pollution in the Seine estuary, the corresponding YOY sole sample showed significantly lower body mass, size and condition factor compared to other samples. Using fifteen genetic markers, the Mondego estuary demonstrated low, but significant levels of genetic differentiation with most of the samples. Using neutrality tests, two loci were found to be putatively under directional selection ( Soso7 and Soso23 ), but only locus Soso7 demonstrated significant positive heterozygosity–fitness correlation (HFC) for body size and body mass, suggesting a possible ongoing adaptive process. The candidate locus MT associated to a detoxifying metallothionein gene did not show significant genetic differentiation, but ( i ) significant deficit in heterozygote in the Seine estuary as already reported in other coastal samples impacted by metallic contaminants, and ( ii ) significant positive HFC with body mass were found. Selective rather than demographic processes better explain an observed genetic structure in YOY sole and are predominantly associated with the heavily contaminated Seine estuary whose nursery function and capacity are degraded.

International audience; Evaluating biodiversity and understanding the processes involved in diversification are noticeable conservation issues in fishes subject to large, sometimes illegal, ornamental trade purposes. Here, the diversity and evolutionary history of the Neotropical dwarf cichlid genus Apistogramma from several South American countries are investigated. Mitochondrial and nuclear markers are used to infer phylogenetic relationships between 31 genetically identified species. The monophyly of Apistogramma is suggested, and Apis-togramma species are distributed into four clades, corresponding to three morphological lineages. Divergence times estimated with the Yule process and an uncorrelated lognor-mal clock dated the Apistogramma origin to the beginning of the Eocene (% 50 Myr) suggesting that diversification might be related to marine incursions. Our molecular dating also suggests that the Quaternary glacial cycles coincide with the phases leading to Apis-togramma speciation. These past events did not influence diversification rates in the spe-ciose genus Apistogramma, since diversification appeared low and constant through time. Further characterization of processes involved in recent Apistogramma diversity will be necessary.

International audience; In marine fishes, the extent to which spatial patterns induced by selection remain stable across generations remains largely unknown. In the gilthead sea bream Sparus aurata, polymorphisms in the growth hormone (GH) and prolactin (Prl) genes can display high levels of differentiation between marine and lagoon habitats. These genotype-environment associations have been attributed to differential selection following larval settlement, but it remains unclear whether selective mortality during later juvenile stages further shapes genetic differences among habitats. We addressed this question by analysing differentiation patterns at GH and Prl markers together with a set of 21 putatively neutral microsatellite loci. We compared genetic variation of spring juveniles that had just settled in 3 ecologically different lagoons against older juveniles sampled from the same sites in autumn, at the onset of winter outmigration. In spring, genetic differentiation among lagoons was greater than expected from neutrality for both candidate gene markers. Surprisingly, this signal disappeared completely in the older juveniles, with no significant differentiation for either locus a few months later in autumn. We searched for signals of haplotype structure within GH and Prl genes using next-generation amplicon deep sequencing. Both genes contained 2 groups of haplotypes, but high similarities among groups indicated that signatures of selection, if any, had largely been erased by recombination. Our results are consistent with the view that differential selection operates during early juvenile life in sea bream and highlight the importance of temporal replication in studies of post-settlement selection in marine fish.

The re-analysis of mtDNA sequence data on sea bass (Dicentrarchus labrax) in the north-east Atlantic revealed the presence of a ‘slippage error’ in the alignment of the mitochondrial DNA (mtDNA) control region sequences. This induced an overestimation of the genetic variability within this species, and hence the inference of a striking multi-clade partitioning of D. labrax populations within this area. After correction, the existence of highly distinct D. labrax haplogroups in Atlantic areas does not hold anymore, but the robust dichotomy between a single Mediterranean and a single Atlantic subgroup is confirmed. Here we present the new results related to the amended mtDNA control region alignment, and also summarize the key message in relation to the microsatellite data, which are unaffected by this revision. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 455–458.

In this study, new DNA markers were explored for the flounder Platichthys flesus. cDNA and genomic sequences of the genes encoding the glyceraldehyde-3-phosphate-deshydrogenase (GAPDH), the cytosolic creatine kinase (CK), the prostaglandin D synthase (PGDS) and the betaine homocysteine methyltransferase (BHMT) were characterized. The tumour suppressor p53 gene structure was already described. A PCR–SSCP (Single Strand Conformation Polymorphism) analysis was finally conducted to study the genetic polymorphism of different populations of flounders collected along the French Atlantic coast. Four highly contaminated French estuaries (Seine, Vilaine, Loire and Gironde) were sampled and compared to a reference estuary (Ster) to explore possible selective effect of the environment on specific allelic frequencies. Our results showed that two loci p53 and PGDS, could be potential markers of chemical stress: p53A allele frequency increased in contaminated systems compared to the reference system. In the Vilaine estuary, PGDS polymorphism could be related to pesticide stress.

The completion of genomic sequences of physiologically important genes frequently reveals non-coding genetic elements such as tandem repeats (micro- and minisatellites) that are often more polymorphic than nearby coding sequences. We obtained the complete genomic sequences of three hormone genes in sea bass Dicentrarchus labrax : growth hormone (dlGH ), somatolactin (dlSL ) and insulin-like growth factor-1 (dlIGF-1 ), including 5′- and 3′-untranslated regions. Mini- and microsatellites were discovered in both flanking and intron regions. Some were partially conserved across Perciformes. To assess the usefulness and relevance of these gene-associated markers for understanding population structure, an investigation was made on genetic diversity and differentiation at four of them in (i ) five wild populations from the North Sea, the Bay of Biscay and the Western Mediterranean, and (ii ) two samples of hatchery-bred individuals from a freshwater-acclimation experiment. Gene and allelic diversities were lower in cultured individuals than in wild ones. Significant genetic differentiation was demonstrated between Bay of Biscay + North Sea and Mediterranean populations (F st > 0.06, p -associated markers. Significant genetic differentiation was also detected among the Atlantic and North Sea samples, but restricted to the locus associated with dlSL . Significant genetic differentiation was also found among experimental individuals before and after a salinity challenge (F st ≈ 0.05, p and dlIGF-1 loci. Gene-associated markers proved to be more efficient than formerly used anonymous microsatellite markers in providing a clear picture of genetic differentiation.

Coastal and estuarine areas in the Bay of Biscay are essential nursery habitats for sole (Solea solea). Using three intronic markers, we investigated patterns of genetic differentiation among cohorts of juveniles (0+, 1+, sub-adults) inhabiting four major nurseries in the Vilaine and Loire river estuaries, the Pertuis d' Antioche strait, and the Gironde estuary. Genetic differentiation was not significant for the age 0- and 1-groups, but was among sub-adult samples (theta(WC) = 0.017; P = 0.031). Hierarchical analyses of genotypic frequencies by nested multivariate ANOVA using redundancy analysis indicated that northern nurseries of Loire and Vilaine rivers had different patterns of genotypic variation with age than southern nurseries of the Pertuis d' Antioche and Gironde estuaries (F = 1.36; P = 0.007). Bay of Biscay nurseries appeared to be spatially structured between a southern and a northern group that exhibit different genotypic distributions. Genetic drift and gene flow explains this pattern poorly, but a statistical test (P < 0.035) suggested that the metallothionein (MT) locus may be significantly impacted by selection in southern estuaries. As metallothionein is involved in heavy-metal detoxification, and southern nurseries are known to be affected by cadmium, this indicates a possible correlation between observed genotypic distributions and some nursery habitat features in sole. Some selective patterns might also be proposed to explain patterns of linkage disequilibrium observed at amylase loci in three of the four nurseries. (C) 2008 Elsevier Ltd. All rights reserved.

Despite extensive knowledge of the genetic structure of sea bass (Dicentrarchus labrax) populations, no studies have investigated genetic structure within early life stages, or compared such structure between such stages and (sub)adults. Using nine newly developed microsatellite loci, we investigated patterns of genetic variation and relatedness among juveniles that settled in two NW Mediterranean nursery grounds in close geographical proximity. There was no evidence for differentiation among samples at settlement (θ = −0.0010), and no significant genetic relatedness. Nevertheless, significant departures from Hardy-Weinberg equilibrium were detected in each sample, which could not be attributed exclusively to the presence of null alleles and to Wahlund effect. These results are compared with and discussed in relation to the genetic structure of adults described in the same area 10 years ago.

dAlthough prolactin has been demonstrated to be the main hormone involved in adaptation to dilute media in several freshwater teleosts, few studies have been conducted in marine teleosts. In the Mediterranean, the sea bass Dicentrarchus labrax inhabits environments ranging from the open sea to coastal lagoons, where salinity varies greatly. We characterised the prolactin (prl) gene and analysed its expression in two organs (gill and intestine) in D. labrax acclimated to either freshwater or seawater. A 2819 bp long sequence encompassing the prl gene and a part (282 bp) of the promoter were identified, and these comprised 5 coding exons separated by 4 introns. Prolactin was similarly expressed in fresh- and seawater adapted fish, although expression in gills was significantly greater than in the intestine. Nonetheless, individuals unable to successfully regulate osmotic balance in freshwater presented overall low expression rates. Results are discussed according to the mechanism of sea bass adaptation in the wild and to their life cycle between open sea and lagoons. Finally, a phylogenetic analysis indicated that teleosts are not branched according to their life-history features (e.g. seawater vs. freshwater habitats), and no signature of positive selection was detected across the phylogeny of the prl gene in teleosts. (c) 2006 Elsevier Inc. All rights reserved.

International audience; Convincing evidence reporting adaptive variation for physiologically and/or ecologically important traits in marine taxa is often derived from studies comparing field and laboratory observations and based on candidate-gene markers, mainly allozymes, responding to habitat selection. Likewise, patterns of genetic differentiation between sea and lagoon populations of the euryhaline European sea bass (Dicentrarchus labrax) were shown to correlate with changes in allele frequencies recorded during experimental salinity challenges, suggesting potential role for local adaptation. However, there has been criticism of these past approaches, causing a need for further investigation. We reinvestigated this correlation in European sea bass using a set of 20 micro- and one minisatellite markers, some of which are associated with candidate genes. We used rigorous experimental settings to demonstrate that only one single gene-associated marker consistently reported significant genetic differentiation between fish raised in seawater compared to fish acclimated to freshwater (locus EIF3E; F ST = 0.060; p = 0.007, n = 268). A few other loci were shown to be false positive and did not respond to selection. Furthermore, a field study of sea–lagoon differentiation in wild European sea bass (n = 144) using the same loci reported marginally significant genetic differentiation at locus EIF3E (F ST = 0.0114, p = 0.068). This locus was identified as putatively under selection. Two other microsatellite loci associated with genes (DLA0070 and DLA0060, respectively) were found to significantly participate to sea–lagoon differentiation and deviate from neutral expectation. The evidence for sea–lagoon local adaptation in sea bass remains faint if not highly elusive.

The euryhaline European sea bass Dicentrarchus labrax L., inhabiting the coasts of the eastern Atlantic Ocean and Mediterranean Sea, has had many opportunities for differentiation throughout its large natural range. However, evidence for this has been incompletely documented geographically and with an insufficient number of markers. Therefore, its full range was sampled at 22 sites and individuals were genotyped with a suite of mapped markers, including 14 microsatellite loci (N = 536) and 46 neutral or gene-linked single nucleotide polymorphisms (SNPs; N = 644). We confirm that the Atlantic and Mediterranean basins harbour two distinct lineages. Within the Atlantic Ocean no pattern was obvious based on the microsatellite and SNP genotypes, except for a subtle difference between South-eastern and North-eastern Atlantic sea bass attributed to limited introgression of alleles of Mediterranean origin. SNP genotypes of the Mediterranean lineage differentiated into three groups, probably under the influence of geographical isolation. The Western Mediterranean group showed genetic homogeneity without evidence for outlier loci. The Adriatic group appeared as a distinct unit. The Eastern Mediterranean group showed a longitudinal gradient of genotypes and most interestingly an outlier locus linked to the somatolactin gene. Overall, the spatial pattern fits those observed with other taxa of between-basin segregation and within-basin connectivity, which concurs well with the swimming capabilities of European sea bass. Evidence from a few outlier loci in this and other studies encourages further exploration of its regional connectivity and adaptive evolution. ispartof: Biological Journal of the Linnean Society vol:116 issue:1 pages:86-105 status: published

Tine, Mbaye ... et. al.-- 10 pages, 5 figures, supplementary information https://doi.org/10.1038/ncomms6770, The European sea bass (Dicentrarchus labrax) is a temperate-zone euryhaline teleost of prime importance for aquaculture and fisheries. This species is subdivided into two naturally hybridizing lineages, one inhabiting the north-eastern Atlantic Ocean and the other the Mediterranean and Black seas. Here we provide a high-quality chromosome-scale assembly of its genome that shows a high degree of synteny with the more highly derived teleosts. We find expansions of gene families specifically associated with ion and water regulation, highlighting adaptation to variation in salinity. We further generate a genome-wide variation map through RAD-sequencing of Atlantic and Mediterranean populations. We show that variation in local recombination rates strongly influences the genomic landscape of diversity within and differentiation between lineages. Comparing predictions of alternative demographic models to the joint allele-frequency spectrum indicates that genomic islands of differentiation between sea bass lineages were generated by varying rates of introgression across the genome following a period of geographical isolation, This work was initiated during the MARINE GENOMICS EUROPE project EU-FP6 505403 and was supported by grants from the Max Planck Society (MPG for genome sequencing, assembly and annotation), LIFECYCLE EU-FP7 222719 and BMBF-01GS0805 to R.R., and the French ANR grants LABRAD-SEQ 11-PDOC-009-01 to P.-A.G. and REGULBASS 09-GENM-003 to B.G. M.T. received a fellowship from the MPG, H.K. was partly financed by LIFECYCLE EU-FP7 222719 and F.K. by the BMBF-01GS0805 grant. L.B. was supported by the grant Progetto 12/MI/2004 from the Veneto Region. R.S.T.M. and B.L. were in receipt of fellowships SFRH/BPD/66742/2009 and SFRH/BPD/89889/2012 from the Foundation for Science and Technology of Portugal

A sharp genetic break separates Atlantic from Indo-Pacific bigeye tuna (Thunnus obesus) populations, as the frequencies of two major mitochondrial (mt) DNA types (α and β) found in this species are different across the tip of southern Africa. The level of nucleotide divergence between mtDNA types α and β is of the same order as that between reproductively isolated taxa. To further investigate the genetic structure of bigeye tuna over its distribution range and in the contact zone off southern Africa, bigeye tuna samples collected between 1992 and 2001 (including samples from a previous mtDNA survey) were characterized for four nuclear DNA loci and for mtDNA. Nuclear markers did not support the hypothesis that α and β mitochondria characterize sibling species. Significant allele-frequency differences at one intronic locus (GH2) and one microsatellite locus (µ208) were found between Atlantic and Indo-Pacific samples, although the level of nuclear genetic differentiation (Weir and Cockerham’s % MathType!Translator!2!1!AMS LaTeX.tdl!TeX -- AMS-LaTeX!

Populations of marine organisms are potentially affected by numerous selective pressures such as temperature and salinity, or anthropogenic pressures such as xenobiotics that may preclude adaptation to particular habitats. Such selective pressures may also affect their demography. Examples include modifications of the population dynamics through shifts in growth rate, and in life history traits affecting fitness such as size or age of first reproduction. However, the documentation of variation in phenotypically plastic traits specific to distinct environments cannot be taken as the ultimate proof that natural selection has occurred. Measurement of the impact of selection and subsequent local adaptation of fish populations based exclusively on morphological or physiological characters is one of the most difficult things to achieve because it depends on the use of phenotypic characters that closely match the genotype. Molecular markers can help to overcome this problem and, under some circumstances, can record the footprints of selection. A combination of polymorphisms that are under selection and those that are not can provide complementary information. In this paper, we review how and why selection can be detected at the molecular level, using genetic markers analysed in a population genetic framework. We then report and discuss case studies in fish.

We demonstrate the effectiveness of a genetic algorithm for discovering multi-locus combinations that provide accurate individual assignment decisions and estimates of mixture composition based on likelihood classification. Using simulated data representing different levels of inter-population differentiation (Fst ∼ 0.01 and 0.10), genetic diversities (four or eight alleles per locus), and population sizes (20, 40, 100 individuals in baseline populations), we show that subsets of loci can be identified that provide comparable levels of accuracy in classification decisions relative to entire multi-locus data sets, where 5, 10, or 20 loci were considered. Microsatellite data sets from hatchery strains of lake trout, Salvelinus namaycush, representing a comparable range of inter-population levels of differentiation in allele frequencies confirmed simulation results. For both simulated and empirical data sets, assignment accuracy was achieved using fewer loci (e.g., three or four loci out of eight for empirical lake trout studies). Simulation results were used to investigate properties of the ‘leave-one-out’ (L1O) method for estimating assignment error rates. Accuracy of population assignments based on L1O methods should be viewed with caution under certain conditions, particularly when baseline population sample sizes are low (

Des methodes statistiques d'evaluation des goulots d'etranglement basees sur les caracteristiques genetiques des populations naturelles ont ete proposees recemment. En absence de toute connaissance directe de la taille des populations actuelles et passees, des evaluations indirectes de tels evenements evolutifs grâce aux donnees genetiques sont importantes pour mener a bien des plans de conservation et de gestion des especes. Nous avons teste ici la methode du rapport M (M-ratio) qui repose sur les profils observes de dispersion des alleles a des locus microsatellites en utilisant trois populations historiques et trois populations contemporaines de la truite des Grands Lacs (salvelinus namaysuch) genotypees a cinq locus microsatellite. Le suivi historique des populations de cette espece dans les Grands Lacs nord-americains montrent des reductions severes des effectifs sur deux generations. Une gamme plausible de parametres caracteristiques des populations naturelles a ete utilisee pour explorer par simulation si chacune de ces populations avait ete affectee ou non par un goulot d'etranglement. Nous montrons que les seules valeurs du rapport M ne sont pas suffisantes pour rendre compte efficacement d'un tel evenement sans connaissance du taux de mutation (μ) et de la taille effective de la population (N e ) a travers le parametre Θ (= 4N e μ). Une meilleure preuve de l'existence d'un goulot d'etranglement recent sera apporte si les etudes employant cette methode sont conduites sur une gamme plausible de valeurs de Θ, ainsi que par des comparaisons qualitatives entre echantillons.

Classification methods used in machine learning (e.g., artificial neural networks, decision trees, and k-nearest neighbor clustering) are rarely used with population genetic data. We compare different nonparametric machine learning techniques with parametric likelihood estimations commonly employed in population genetics for purposes of assigning individuals to their population of origin ("assignment tests"). Classifier accuracy was compared across simulated data sets representing different levels of population differentiation (low and high F(ST)), number of loci surveyed (5 and 10), and allelic diversity (average of three or eight alleles per locus). Empirical data for the lake trout (Salvelinus namaycush) exhibiting levels of population differentiation comparable to those used in simulations were examined to further evaluate and compare classification methods. Classification error rates associated with artificial neural networks and likelihood estimators were lower for simulated data sets compared to k-nearest neighbor and decision tree classifiers over the entire range of parameters considered. Artificial neural networks only marginally outperformed the likelihood method for simulated data (0-2.8% lower error rates). The relative performance of each machine learning classifier improved relative likelihood estimators for empirical data sets, suggesting an ability to "learn" and utilize properties of empirical genotypic arrays intrinsic to each population. Likelihood-based estimation methods provide a more accessible option for reliable assignment of individuals to the population of origin due to the intricacies in development and evaluation of artificial neural networks.

Although food deprivation is a major ecological pressure in fishes, there is wide individual variation in tolerance of fasting, whose mechanistic bases are poorly understood. Two thousand individually tagged juvenile European sea bass were submitted to two 'fasting/feeding' cycles each comprising 3 weeks of food deprivation followed by 3 weeks of ad libitum feeding at 25 degrees C. Rates of mass loss during the two fasting periods were averaged for each individual to calculate a population mean. Extreme fasting tolerant (FT) and sensitive (FS) phenotypes were identified that were at least one and a half standard deviations, on opposing sides, from this mean. Respirometry was used to investigate two main hypotheses: (1) tolerance of food deprivation reflects lower mass-corrected routine metabolic rate (RMR) in FT phenotypes when fasting, and (2) tolerance reflects differences in substrate utilisation; FT phenotypes use relatively less proteins as metabolic fuels during fasting, measured as their ammonia quotient (AQ), the simultaneous ratio of ammonia excretion to RMR. There was no difference in mean RMR between FT and FS over 7 days fasting, being 6.70 +/- 0.24 mmol h(-1) fish(-1) (mean +/- s.e.m., N=18) versus 6.76 +/- 0.22 mmol h(-1) fish(-1) (N=17), respectively, when corrected to a body mass of 130 g. For any given RMR, however, the FT lost mass at a significantly lower rate than FS, overall 7-day average being 0.72 +/- 0.05 versus 0.90 +/- 0.05 g day(-1) fish(-1), respectively (P

International audience; Feed efficiency is a major goal for aquaculture sustainability, and selecting fish to genetically enhance this trait would be highly valuable. However, no selective breeding program specifically targeted to feed efficiency exists for farmed fish, mostly because of the difficulty of measuring individual feed intake. However, a negative phenotypic correlation between feed efficiency and weight loss at fasting has been previously demonstrated in sea bass submitted to feed deprivation (FD). We mated sea bass parents selected for their high (FD+) or low (FD-) weight loss at fasting to produce FD+ and FD- progeny, which were reared in a single tank to avoid common environmental effects. At 8 months of age, 1200 of those fish were submitted to three alternating periods of fasting (3 weeks) and re-feeding (3 weeks). Individuals were weighed at the end of each feeding and fasting period. Their line of origin was identified by genotyping of 12 microsatellite markers, resulting in 1130 unambiguously assigned fish (484 FD-, 686 FD+). FD- offspring lost significantly less weight than FD+ offspring in this feed deprivation trial. After that the feed efficiency of eight groups of 50 FD+ fish and eight groups of 50 FD- fish was evaluated in four successive 20-day periods. At the end of the fourth period, 10 fish per tank were sacrificed to evaluate their carcass yield. The FD- fish had a better overall growth and were fatter, and FD+ fish had a better carcass yield. A better feed efficiency was expected for the FD- fish, but differences between the two groups for this trait, measured either with feed efficiency ratio or with residual feed intake, were not consistently significant Although the two lines were clearly divergent for several traits, demonstration of feed efficiency differences between the FD+ and the FD- lines was not consistently observed in sea bass. A second generation of selection may allow further divergence in the lines and reveal differences in feed efficiency. (C) 2013 Elsevier B.V. All rights reserved.

Numerous methods can be used in intraspecific phylogeographic studies to infer the evolutionary mechanisms that shaped observed genetic variation in populations. However, these methods are scarcely used jointly, and the evolutionary outcomes they could propose are not fully compared. In this study, using a chub ( Leuciscus cephalus ; Cyprinidae) mitochondrial DNA data set (13 populations in Western Greece, 14 haplotypes), we compare three distinct ‘historical’ methods that could possibly infer relative importance of basic evolutionary mechanisms (isolation vs migration) shaping genetic variation: the nested clade analysis, the Ψ -test and the ‘mismatch distributions’. Taking together, interpretations of these analyses allow to draw a picture of the evolutionary history of chub in Western Greece based on isolation and genetic drift for higher clades. However, results issued each method can differ for low differentiated clades. We discuss such differences and suggest that methods should be used jointly in phylogeographic studies for a better evaluation of the evolutionary mechanisms that shaped genetic variation.

Using four allozymic loci, we analysed the genetic structure and differentiation of Hydropsyche exocellata (Trichoptera; Hydropsychidae) larvae in seven sites along the Upper Loire River (France). The genetic differentiation is low for this species despite distribution patchiness, and only due to one locus in one study site. No trend relating genetic differentiation to geographic distance (isolation by distance) was observed. Significant deviations from Hardy-Weinberg equilibrium were observed at most sites and loci. The population genetics analysis of an amphibiotic insect such as H. exocellata in light of current ecological and population genetics knowledge is discussed following two main factors: 1) sampling of partially reproductively isolated, genetically distinct subpopulations, and 2) form of selection. Despite knowledge on several aspects of the ecology of H. exocellata in the River Loire, numerous points limit genetic data interpretation. These points are underlined and discussed with reference to studies on other amphibiotic insects.

Phylogenetic relationships among Greek populations of the chub, Leuciscus cephalus, were investigated using 600 bp of the cytochrome b gene. The aim of this study was to test the assumption that the main difference in ichthyological composition between both sides of the Balkan Peninsula is directly linked to differences in the dispersion mechanisms used by fish in order to extend their distribution range. Phylogenetic and nested clade analyses clearly showed that populations in Greece are significantly differentiated. Greek populations were found to descend from three lineages in three geographical provinces: Western, Central, and Eastern Greece. The chub reached Western Greece at the beginning of the Pleistocene and Eastern Greece during the mid-Pleistocene. Chub dispersion occurred mainly by river confluence due to sea level lowering and river capture in Western Greece and sea dispersal with low-salinity conditions within the Aegean Sea in Eastern Greece. However, in Central Greece, the original mtDNA lineage has presumably been lost owing to a genetic introgression following a second invasion from the Danube during the final stage of the last glaciation. This study provides new elements for a better understanding of the composition of the contemporary ichthyofauna in Greece and highlights possible evolutionary mechanisms responsible for the high endemism rate in the Western Greek biogeographic province.