A multi-population approach supports common patterns in marine growth and maturation decision in Atlantic salmon (Salmo salar L.) from southern Europe.

This study provides a regional picture of long-term changes in Atlantic salmon growth at the southern edge of their distribution, using a multi-population approach spanning 49 years and five populations. We provide empirical evidence of salmon life history being influenced by a combination of common signals in the marine environment and population-specific signals. We identified an abrupt decline in growth from 1976 and a more recent decline after 2005. As these declines have also been recorded in northern European populations, our study significantly expands a pattern of declining marine growth to include southern European populations, thereby revealing a large-scale synchrony in marine growth patterns for almost five decades. Growth increments during their sea sojourn were characterized by distinct temporal dynamics. At a coarse temporal resolution, growth during the first winter at sea seemed to gradually improve over the study period. However, the analysis of finer seasonal growth patterns revealed ecological bottlenecks of salmon life histories at sea in time and space. Our study reinforces existing evidence of an impact of early marine growth on maturation decision, with small-sized individuals at the end of the first summer at sea being more likely to delay maturation. However, each population was characterized by a specific probabilistic maturation reaction norm, and a local component of growth at sea in which some populations have better growth in some years might further amplify differences in maturation rate. Differences between populations were smaller than those between sexes, suggesting that the sex-specific growth threshold for maturation is a well-conserved evolutionary phenomenon in salmon. Finally, our results illustrate that although most of the gain in length occurs during the first summer at sea, the temporal variability in body length at return is buffered against the decrease in post-smolt growth conditions. The intricate combination of growth over successive seasons, and its interplay with the maturation decision, could be regulating body length by maintaining diversity in early growth trajectories, life histories, and the composition of salmon populations.

A SNP marker to discriminate the european brook lamprey (Lampetra planeri), river lamprey (L. fluviatilis) and their hybrids.

BACKGROUND: The European River lamprey Lampetra fluviatilis and the brook lamprey L. planeri are two closely related species that are also considered as partially reproductively isolated ecotypes. At the larval stage, they cannot be distinguished morphologically or genetically by mitochondrial DNA. We aimed at developing a molecular tool to identify early life stages of L. fluviatilis and L. planeri. METHODS AND RESULTS: We first identified Single Nucleotide Polymorphism (SNP) markers with distinct alleles between L. fluviatilis and L. planeri based on RADseq data from 186 individuals collected in France. Then, we developed a quantitative PCR protocol to genotype a species-specific SNP, which was tested on 270 samples including larvae and adults from both species and 17 sites from Western Europe. Results were consistent with morphological identification in all cases except for samples from the Rhône drainage and the Loch Lomond in Scotland. CONCLUSIONS: This marker allows the identification of larval stages of L. fluviatilis, L. planeri as well as hybrids. The two cases where the marker was not diagnostic might be explained by an isolated and distinct L. planeri lineage in the Rhône drainage, and high levels of admixture among L. fluviatilis, L. planeri and the lake-parasitic form in Loch Lomond.

Population genetics reveals divergent lineages and ongoing hybridization in a declining migratory fish species complex.

Deciphering the effects of historical and recent demographic processes responsible for the spatial patterns of genetic diversity and structure is a key objective in evolutionary and conservation biology. Using population genetic analyses, we investigated the demographic history, the contemporary genetic diversity and structure, and the occurrence of hybridization and introgression of two species of anadromous fish with contrasting life history strategies and which have undergone recent demographic declines, the allis shad (Alosa alosa) and the twaite shad (Alosa fallax). We genotyped 706 individuals from 20 rivers and 5 sites at sea in Southern Europe at thirteen microsatellite markers. Genetic structure between populations was lower for the nearly semelparous species A. alosa, which disperses greater distances compared to the iteroparous species, A. fallax. Individuals caught at sea were assigned at the river level for A. fallax and at the region level for A. alosa. Using an approximate Bayesian computation framework, we inferred that the most likely long term historical divergence scenario between both species and lineages involved historical separation followed by secondary contact accompanied by strong population size decline. Accordingly, we found evidence for contemporary hybridization and bidirectional introgression due to gene flow between both species and lineages. Moreover, our results support the existence of at least one distinct species in the Mediterrannean sea: A. agone in Golfe du Lion area, and another divergent lineage in Corsica. Overall, our results shed light on the interplay between historical and recent demographic processes and life history strategies in shaping population genetic diversity and structure of closely related species. The recent demographic decline of these species' populations and their hybridization should be carefully considered while implementing conservation programs.

Genetic diversity in a long-lived mammal is explained by the past's demographic shadow and current connectivity.

Within-species genetic diversity is crucial for the persistence and integrity of populations and ecosystems. Conservation actions require an understanding of factors influencing genetic diversity, especially in the context of global change. Both population size and connectivity are factors greatly influencing genetic diversity; the relative importance of these factors can, however, change through time. Hence, quantifying the degree to which population size or genetic connectivity are shaping genetic diversity, and at which ecological time scale (past or present), is challenging, yet essential for the development of efficient conservation strategies. In this study, we estimated the genetic diversity of 42 colonies of Rhinolophus hipposideros, a long-lived mammal vulnerable to global change, sampling locations spanning its continental northern range. Here, we present an integrative approach that disentangles and quantifies the contribution of different connectivity measures in addition to contemporary colony size and historic bottlenecks in shaping genetic diversity. In our study, the best model explained 64% of the variation in genetic diversity. It included historic bottlenecks, contemporary colony size, connectivity and a negative interaction between the latter two. Contemporary connectivity explained most genetic diversity when considering a 65 km radius around the focal colonies, emphasizing the large geographic scale at which the positive impact of connectivity on genetic diversity is most profound and hence, the minimum scale at which conservation should be planned. Our results highlight that the relative importance of the two main factors shaping genetic diversity varies through time, emphasizing the relevance of disentangling them to ensure appropriate conservation strategies.

Riverscape genetics in brook lamprey: genetic diversity is less influenced by river fragmentation than by gene flow with the anadromous ecotype.

Understanding the effect of human-induced landscape fragmentation on gene flow and evolutionary potential of wild populations has become a major concern. Here, we investigated the effect of riverscape fragmentation on patterns of genetic diversity in the freshwater resident European brook lamprey (Lampetra planeri) that has a low ability to pass obstacles to migration. We tested the hypotheses of (i) asymmetric gene flow following water current and (ii) an effect of gene flow with the closely related anadromous river lamprey (L. fluviatilis) ecotype on L. planeri genetic diversity. We genotyped 2472 individuals, including 225 L. fluviatilis, sampled from 81 sites upstream and downstream barriers to migration, in 29 western European rivers. Linear modelling revealed a strong positive relationship between genetic diversity and the distance from the river source, consistent with expected patterns of decreased gene flow into upstream populations. However, the presence of anthropogenic barriers had a moderate effect on spatial genetic structure. Accordingly, we found evidence for downstream-directed gene flow, supporting the hypothesis that barriers do not limit dispersal mediated by water flow. Downstream L. planeri populations in sympatry with L. fluviatilis displayed consistently higher genetic diversity. We conclude that genetic drift and slight downstream gene flow drive the genetic make-up of upstream L. planeri populations whereas gene flow between ecotypes maintains higher levels of genetic diversity in L. planeri populations sympatric with L. fluviatilis. We discuss the implications of these results for the design of conservation strategies of lamprey, and other freshwater organisms with several ecotypes, in fragmented dendritic river networks.

Genetic drift during the spread phase of a biological invasion.

Recent theoretical and experimental models have revealed the role played by evolution during species spread, and in particular have questioned the influence of genetic drift at range edges. By investigating the spread of an aquatic invader in patchy habitats, we quantified genetic drift and explored its consequences for genetic diversity and fitness. We examined the interplay of gene flow and genetic drift in 36 populations of the red swamp crayfish, Procambarus clarkii, in a relatively recently invaded wetland area (30 years, Brière, northwest France). Despite the small spatial scale of our study (15 km2 ), populations were highly structured according to the strong barrier of land surfaces and revealed a clear pattern of colonization through watercourses. Isolated populations exhibited small effective sizes and low dispersal rates that depended on water connectivity, suggesting that genetic drift dominated in the evolution of allele frequencies in these populations. We also observed a significant decrease in the genetic diversity of isolated populations over only a 2-year period, but failed to demonstrate an associated fitness cost using fluctuating asymmetry. This study documents the possible strong influence of genetic drift during the spread of a species, and such findings provide critical insights into the current context of profound rearrangements in species distributions due to global change.

Combining noninvasive genetics and a new mammalian sex-linked marker provides new tools to investigate population size, structure and individual behaviour: An application to bats.

Monitoring wild populations is crucial for their effective management. Noninvasive genetic methods provide robust data from individual free-ranging animals, which can be used in capture-mark-recapture (CMR) models to estimate demographic parameters without capturing or disturbing them. However, sex- and status-specific behaviour, which may lead to differences in detection probabilities, is rarely considered in monitoring. Here, we investigated population size, sex ratio, sex- and status-related behaviour in 19 Rhinolophus hipposideros maternity colonies (Northern France) with a noninvasive genetic CMR approach (using faeces) combined with parentage assignments. The use of the DDX3X/Y-Mam sexual marker designed in this study, which shows inter- and intrachromosomal length polymorphism across placental mammals, together with eight polymorphic microsatellite markers, produced high-quality genetic data with limited genotyping errors and allowed us to reliably distinguish different categories of individuals (males, reproductive and nonreproductive females) and to estimate population sizes. We showed that visual counts represent well-adult female numbers and that population composition in maternity colonies changes dynamically during the summer. Before parturition, colonies mainly harbour pregnant and nonpregnant females with a few visiting males, whereas after parturition, colonies are mainly composed of mothers and their offspring with a few visiting nonmothers and males. Our approach gives deeper insight into sex- and status-specific behaviour, a prerequisite for understanding population dynamics and developing effective monitoring and management strategies. Provided sufficient samples can be obtained, this approach can be readily applied to a wide range of species.

Inferring the demographic history underlying parallel genomic divergence among pairs of parasitic and nonparasitic lamprey ecotypes.

Understanding the evolutionary mechanisms generating parallel genomic divergence patterns among replicate ecotype pairs remains an important challenge in speciation research. We investigated the genomic divergence between the anadromous parasitic river lamprey (Lampetra fluviatilis) and the freshwater-resident nonparasitic brook lamprey (Lampetra planeri) in nine population pairs displaying variable levels of geographic connectivity. We genotyped 338 individuals with RAD sequencing and inferred the demographic divergence history of each population pair using a diffusion approximation method. Divergence patterns in geographically connected population pairs were better explained by introgression after secondary contact, whereas disconnected population pairs have retained a signal of ancient migration. In all ecotype pairs, models accounting for differential introgression among loci outperformed homogeneous migration models. Generating neutral predictions from the inferred divergence scenarios to detect highly differentiated markers identified greater proportions of outliers in disconnected population pairs than in connected pairs. However, increased similarity in the most divergent genomic regions was found among connected ecotype pairs, indicating that gene flow was instrumental in generating parallelism at the molecular level. These results suggest that heterogeneous genomic differentiation and parallelism among replicate ecotype pairs have partly emerged through restricted introgression in genomic islands.

Genetic variation of Lymnaea stagnalis tolerance to copper: A test of selection hypotheses and its relevance for ecological risk assessment.

The use of standardized monospecific testing to assess the ecological risk of chemicals implicitly relies on the strong assumption that intraspecific variation in sensitivity is negligible or irrelevant in this context. In this study, we investigated genetic variation in copper sensitivity of the freshwater snail Lymnaea stagnalis, using lineages stemming from eight natural populations or strains found to be genetically differentiated at neutral markers. Copper-induced mortality varied widely among populations, as did the estimated daily death rate and time to 50% mortality (LT50). Population genetic divergence in copper sensitivity was compared to neutral differentiation using the QST-FST approach. No evidence for homogenizing selection could be detected. This result demonstrates that species-level extrapolations from single population studies are highly unreliable. The study provides a simple example of how evolutionary principles could be incorporated into ecotoxicity testing in order to refine ecological risk assessment.

Genetic and phenotypic changes in an Atlantic salmon population supplemented with non-local individuals: a longitudinal study over 21 years.

While introductions and supplementations using non-native and potentially domesticated individuals may have dramatic evolutionary effects on wild populations, few studies documented the evolution of genetic diversity and life-history traits in supplemented populations. Here, we investigated year-to-year changes from 1989 to 2009 in genetic admixture at 15 microsatellite loci and in phenotypic traits in an Atlantic salmon (Salmo salar) population stocked during the first decade of this period with two genetically and phenotypically distinct source populations. We detected a pattern of temporally increasing introgressive hybridization between the stocked population and both source populations. The proportion of fish returning to the river after a single winter at sea (versus several ones) was higher in fish assigned to the main source population than in local individuals. Moreover, during the first decade of the study, both single-sea-winter and multi-sea-winter (MSW) fish assigned to the main source population were smaller than local fish. During the second decade of the study, MSW fish defined as hybrids were lighter and smaller than fish from parental populations, suggesting outbreeding depression. Overall, this study suggests that supplementation with non-local individuals may alter not only the genetic diversity of wild populations but also life-history traits of adaptive significance.

Environmental versus anthropogenic effects on population adaptive divergence in the freshwater snail Lymnaea stagnalis.

Repeated pesticide contaminations of lentic freshwater systems located within agricultural landscapes may affect population evolution in non-target organisms, especially in species with a fully aquatic life cycle and low dispersal ability. The issue of evolutionary impact of pollutants is therefore conceptually important for ecotoxicologists. The impact of historical exposure to pesticides on genetic divergence was investigated in the freshwater gastropod Lymnaea stagnalis, using a set of 14 populations from contrasted environments in terms of pesticide and other anthropogenic pressures. The hypothesis of population adaptive divergence was tested on 11 life-history traits, using Q(ST)-F(ST) comparisons. Despite strong neutral differentiation (mean F(ST) = 0.291), five adult traits or parameters were found to be under divergent selection. Conversely, two early expressed traits showed a pattern consistent with uniform selection or trait canalization, and four adult traits appeared to evolve neutrally. Divergent selection patterns were mostly consistent with a habitat effect, opposing pond to ditch and channel populations. Comparatively, pesticide and other human pressures had little correspondence with evolutionary patterns, despite hatching rate impairment associated with global anthropogenic pressure. Globally, analyses revealed high genetic variation both at neutral markers and fitness-related traits in a species used as model in ecotoxicology, providing empirical support for the need to account for genetic and evolutionary components of population response in ecological risk assessment.

Population genetics of Lymnaea stagnalis experimentally exposed to cocktails of pesticides.

Freshwater invertebrates may be regularly exposed to pesticides in agricultural landscapes, as water bodies such as ditches and ponds are the final recipient of many chemicals, through various transfer pathways. Local evolutionary impacts may hence be expected on populations, especially for species with a completely aquatic life cycle. We tested the hypothesis that exposure to combinations of pesticides used in crop protection programmes could increase the effect of random genetic drift in a non-target species, via demographic impacts. To do so, experimental populations of the freshwater snail Lymnaea stagnalis were created from a common genetic pool and exposed for three successive generations to treatments corresponding to two different crop protection plans (conventional and low pesticide input). Population genetic parameters were estimated in each generation on the basis of ten polymorphic microsatellite loci. Effects consistent with increased random genetic drift were observed for one sampling campaign performed in the third generation, i.e., decreased genetic variability and increased population differentiation in the group of populations exposed to the treatment programme whose demographic impact was the most effective on L. stagnalis. Otherwise, no clear pattern emerged and even opposed effects could be observed. All populations were found significantly inbred, mostly due to biparental inbreeding. Conversely, selfing was generally not significant, and did not express preferentially under high pesticide pressure. We conclude from this study that population genetics should be used very cautiously in the context of ecological risk assessment, especially when applied to natural populations.

Impact of the redox-cycling herbicide diquat on transcript expression and antioxidant enzymatic activities of the freshwater snail Lymnaea stagnalis.

The presence of pesticides in the environment results in potential unwanted effects on non-target species. Freshwater organisms inhabiting water bodies adjacent to agricultural areas, such as ditches, ponds and marshes, are good models to test such effects as various pesticides may reach these habitats through several ways, including aerial drift, run-off, and drainage. Diquat is a non-selective herbicide used for crop protection or for weed control in such water bodies. In this study, we investigated the effects of diquat on a widely spread aquatic invertebrate, the holarctic freshwater snail Lymnaea stagnalis. Due to the known redox-cycling properties of diquat, we studied transcript expression and enzymatic activities relative to oxidative and general stress in the haemolymph and gonado-digestive complex (GDC). As diquat is not persistent, snails were exposed for short times (5, 24, and 48 h) to ecologically relevant concentrations (22.2, 44.4, and 222.2 µg l(-1)) of diquat dibromide. RT-qPCR was used to quantify the transcription of genes encoding catalase (cat), a cytosolic superoxide dismutase (Cu/Zn-sod), a selenium-dependent glutathione peroxidase (gpx), a glutathione reductase (gred), the retinoid X receptor (rxr), two heat shock proteins (hsp40 and hsp70), cortactin (cor) and the two ribosomal genes r18S and r28s. Enzymatic activities of SOD, Gpx, Gred and glutathione S-transferase (GST) were investigated in the GDC using spectrophoto/fluorometric methods. Opposite trends were obtained in the haemolymph depending on the herbicide concentration. At the lowest concentration, effects were mainly observed after 24 h of exposure, with over-transcription of cor, hsp40, rxr, and sod, whereas higher concentrations down-regulated the expression of most of the studied transcripts, especially after 48 h of exposure. In the GDC, earlier responses were observed and the fold-change magnitude was generally much higher: transcription of all target genes increased significantly (or non-significantly for cat) after 5 h of exposure, and went back to control levels afterwards, suggesting the onset of an early response to oxidative stress associated to the unbalance of reactive oxygen species (ROS) in hepatocytes. Although increases obtained for Gred and SOD activities were globally consistent with their respective transcript expressions, up-regulation of transcription was not always correlated with increase of enzymatic activity, indicating that diquat might affect steps downstream of transcription. However, constitutive levels of enzymatic activities were at least maintained. In conclusion, diquat was shown to affect expression of the whole set of studied transcripts, reflecting their suitability as markers of early response to oxidative stress in L. stagnalis.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2012 - 30 September 2012.

This article documents the addition of 83 microsatellite marker loci and 96 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bembidion lampros, Inimicus japonicus, Lymnaea stagnalis, Panopea abbreviata, Pentadesma butyracea, Sycoscapter hirticola and Thanatephorus cucumeris (anamorph: Rhizoctonia solani). These loci were cross-tested on the following species: Pentadesma grandifolia and Pentadesma reyndersii. This article also documents the addition of 96 sequencing primer pairs and 88 allele-specific primers or probes for Plutella xylostella.

Pyrosequencing-based transcriptomic resources in the pond snail Lymnaea stagnalis, with a focus on genes involved in molecular response to diquat-induced stress.

Due to their ability to explore whole genome response to drugs and stressors, omics-based approaches are widely used in toxicology and ecotoxicology, and identified as powerful tools for future ecological risk assessment and environmental monitoring programs. Understanding the long-term effects of contaminants may indeed benefit from the coupling of genomics and eco-evolutionary hypotheses. Next-generation sequencing provides a new way to investigate pollutants impact, by targeting early responses, screening chemicals, and directly quantifying gene expression, even in organisms without reference genome. Lymnaea stagnalis is a freshwater mollusk in which access to genomic resources is critical for many scientific issues, especially in ecotoxicology. We used 454-pyrosequencing to obtain new transcriptomic resources in L. stagnalis and to preliminarily explore gene expression response to a redox-cycling pesticide, diquat. We obtained 151,967 and 128,945 high-quality reads from control and diquat-exposed individuals, respectively. Sequence assembly provided 141,999 contigs, of which 124,387 were singletons. BlastX search revealed significant match for 34.6 % of the contigs (21.2 % protein hits). KEGG annotation showed a predominance of hits with genes involved in energy metabolism and circulatory system, and revealed more than 400 putative genes involved in oxidative stress, cellular/molecular stress and signaling pathways, apoptosis, and metabolism of xenobiotics. Results also suggest that diquat may have a great diversity of molecular effects. Moreover, new genetic markers (putative SNPs) were discovered. We also created a Ensembl-like web-tool for data-mining ( http://genotoul-contigbrowser.toulouse.inra.fr:9095/Lymnaea_stagnalis/index.html ). This resource is expected to be relevant for any genomic approach aimed at understanding the molecular basis of physiological and evolutionary responses to environmental stress in L. stagnalis.

Quantitative resistance increases the durability of qualitative resistance to Leptosphaeria maculans in Brassica napus.

It has frequently been hypothesized that quantitative resistance increases the durability of qualitative (R-gene mediated) resistance but supporting experimental evidence is rare. To test this hypothesis, near-isogenic lines with/without the R-gene Rlm6 introduced into two Brassica napus cultivars differing in quantitative resistance to Leptosphaeria maculans were used in a 5-yr field experiment. Recurrent selection of natural fungal populations was done annually on each of the four plant genotypes, using crop residues from each genotype to inoculate separately the four series of field trials for five consecutive cropping seasons. Severity of phoma stem canker was measured on each genotype and frequencies of avirulence alleles in L. maculans populations were estimated. Recurrent selection of virulent isolates by Rlm6 in a susceptible background rendered the resistance ineffective by the third cropping season. By contrast, the resistance was still effective after 5 yr of selection by the genotype combining this gene with quantitative resistance. No significant variation in the performance of quantitative resistance alone was noted over the course of the experiment. We conclude that quantitative resistance can increase the durability of Rlm6. We recommend combining quantitative resistance with R-gene mediated resistance to enhance disease control and crop production.

Repeat-induced point mutation (RIP) as an alternative mechanism of evolution toward virulence in Leptosphaeria maculans.

Three avirulence genes, AvrLm1, AvrLm6, and AvrLm4-7, were recently identified in Leptosphaeria maculans and found to be localized as solo genes within large noncoding, heterochromatin-like regions mainly composed of retrotransposons, truncated and degenerated by repeat-induced point mutation (RIP). The Rlm6 resistance gene has been overcome within 3 years in outdoor experiments in France and, here, we investigate the molecular basis of evolution toward virulence at the AvrLm6 locus. A region of 235 kb was sequenced in a virulent isolate and showed the deletion of AvrLm6 and three divergent mosaics of retrotransposons. AvrLm6 was found to be absent from 66% of 70 virulent isolates, with multiple events of deletion. The sequencing of virulent alleles in 24 isolates revealed a few cases of point mutations that had created stop codons in the sequence. The most frequent mutation events, however, were RIP, leading to the modification of 4 to 9% of the bases compared with the avirulent allele and generating 2 to 4 stop codons. Thus, RIP is described for the first time as an efficient mechanism leading to virulence and the multiple patterns of mutation observed suggest that multiple RIP events could occur independently in a single field population during 1 year.