Contrasts in riverscape patterns of intraspecific genetic variation in a diverse Neotropical fish community of high conservation value.

Spatial patterns of genetic variation compared across species provide information about the predictability of genetic diversity in natural populations, and areas requiring conservation measures. Due to their remarkable fish diversity, rivers in Neotropical regions are ideal systems to confront theory with observations and would benefit greatly from such approaches given their increasing vulnerability to anthropogenic pressures. We used SNP data from 18 fish species with contrasting life-history traits, co-sampled across 12 sites in the Maroni- a major river system from the Guiana Shield -, to compare patterns of intraspecific genetic variation and identify their underlying drivers. Analyses of covariance revealed a decrease in genetic diversity as distance from the river outlet increased for 5 of the 18 species, illustrating a pattern commonly observed in riverscapes for species with low-to-medium dispersal abilities. However, the mean within-site genetic diversity was lowest in the two easternmost tributaries of the Upper Maroni and around an urbanized location downstream, indicating the need to address the potential influence of local pressures in these areas, such as gold mining or fishing. Finally, the relative influence of isolation by stream distance, isolation by discontinuous river flow, and isolation by spatial heterogeneity in effective size on pairwise genetic differentiation varied across species. Species with similar dispersal and reproductive guilds did not necessarily display shared patterns of population structure. Increasing the knowledge of specific life history traits and ecological requirements of fish species in these remote areas should help further understand factors that influence their current patterns of genetic variation.

A low-density single nucleotide polymorphism panel for brown trout (Salmo trutta L.) suitable for exploring genetic diversity at a range of spatial scales.

The rivers of southern England and northern France which drain into the English Channel contain several genetically unique groups of trout (Salmo trutta L.) that have suffered dramatic declines in numbers over the past 40 years. Knowledge of levels and patterns of genetic diversity is essential for effective management of these vulnerable populations. Using restriction site-associated DNA sequencing (RADseq) data, we describe the development and characterisation of a panel of 95 single nucleotide polymorphism (SNP) loci for trout from this region and investigate their applicability and variability in both target (i.e., southern English) and non-target trout populations from northern Britain and Ireland. In addition, we present three case studies which demonstrate the utility and resolution of these genetic markers at three levels of spatial separation:(a) between closely related populations in nearby rivers, (b) within a catchment and (c) when determining parentage and familial relationships between fish sampled from a single site, using both empirical and simulated data. The SNP loci will be useful for population genetic and assignment studies on brown trout within the UK and beyond.

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.

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.

Molecular detection of the swim bladder parasite Anguillicola crassus (Nematoda) in fecal samples of the endangered European eel Anguilla anguilla.

The European eel Anguilla anguilla is listed as critically endangered by the IUCN. Among many threats, the introduced parasitic nematode Anguillicola crassus is suspected to alter the eels' swim bladder and jeopardize their reproductive oceanic migration. To date, gaining knowledge about the distribution and prevalence of A. crassus requires individual sacrifice (over 50,000 eels were sacrificed for epidemiology studies since 2010). This paper describes a non-lethal molecular protocol for identifying prevalence of A. crassus in A. anguilla, based on searching for A. crassus DNA in the feces of eels. Tests using three DNA microsatellite markers specific to the nematode showed that molecular detection provided similar results to visual examination of the swim bladder in up to 80% of the cases, and allowed for comparison of prevalence among sites. Easy to implement, this non-lethal protocol for detecting A. crassus could be valuable for management plans of this endangered 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.

Leveraging the genetic diversity of trout in the rivers of the British Isles and northern France to understand the movements of sea trout (Salmo trutta L.) around the English Channel.

Populations of anadromous brown trout, also known as sea trout, have suffered recent marked declines in abundance due to multiple factors, including climate change and human activities. While much is known about their freshwater phase, less is known about the species' marine feeding migrations. This situation is hindering the effective management and conservation of anadromous trout in the marine environment. Using a panel of 95 single nucleotide polymorphism markers we developed a genetic baseline, which demonstrated strong regional structuring of genetic diversity in trout populations around the English Channel and adjacent waters. Extensive baseline testing showed this structuring allowed high-confidence assignment of known-origin individuals to region of origin. This study presents new data on the movements of anadromous trout in the English Channel and southern North Sea. Assignment of anadromous trout sampled from 12 marine and estuarine localities highlighted contrasting results for these areas. The majority of these fisheries are composed predominately of stocks local to the sampling location. However, there were multiple cases of long-distance movements of anadromous trout, with several individuals originating from rivers in northeast England being caught in the English Channel and southern North Sea, in some cases more than 1000 km from their natal region. These results have implications for the management of sea trout in inshore waters around the English Channel and southern North Sea.

Estimating resistance surfaces using gradient forest and allelic frequencies.

Understanding landscape connectivity has become a global priority for mitigating the impact of landscape fragmentation on biodiversity. Connectivity methods that use link-based methods traditionally rely on relating pairwise genetic distance between individuals or demes to their landscape distance (e.g., geographic distance, cost distance). In this study, we present an alternative to conventional statistical approaches to refine cost surfaces by adapting the gradient forest approach to produce a resistance surface. Used in community ecology, gradient forest is an extension of random forest, and has been implemented in genomic studies to model species genetic offset under future climatic scenarios. By design, this adapted method, resGF, has the ability to handle multiple environmental predicators and is not subjected to traditional assumptions of linear models such as independence, normality and linearity. Using genetic simulations, resistance Gradient Forest (resGF) performance was compared to other published methods (maximum likelihood population effects model, random forest-based least-cost transect analysis and species distribution model). In univariate scenarios, resGF was able to distinguish the true surface contributing to genetic diversity among competing surfaces better than the compared methods. In multivariate scenarios, the gradient forest approach performed similarly to the other random forest-based approach using least-cost transect analysis but outperformed MLPE-based methods. Additionally, two worked examples are provided using two previously published data sets. This machine learning algorithm has the potential to improve our understanding of landscape connectivity and inform long-term biodiversity conservation strategies.

Role of introduction history and landscape in the range expansion of brown trout (Salmo trutta L.) in the Kerguelen Islands.

Human-mediated biological invasions constitute interesting case studies to understand evolutionary processes, including the role of founder effects. Population expansion of newly introduced species can be highly dependant on barriers caused by landscape features, but identifying these barriers and their impact on genetic structure is a relatively recent concern in population genetics and ecology. Salmonid populations of the Kerguelen Islands archipelago are a favorable model system to address these questions as these populations are characterized by a simple history of introduction, little or no anthropogenic influence, and demographic monitoring since the first introductions. We analyzed genetic variation at 10 microsatellite loci in 19 populations of brown trout (Salmo trutta L.) in the Courbet Peninsula (Kerguelen Islands), where the species, introduced in 3 rivers only, has colonized the whole water system in 40 years. Despite a limited numbers of introductions, trout populations have maintained a genetic diversity comparable with what is found in hatchery or wild populations in Europe, but they are genetically structured. The main factor explaining the observed patterns of genetic diversity is the history of introductions, with each introduced population acting as a source for colonization of nearby rivers. Correlations between environmental and genetic parameters show that within each "source population" group, landscape characteristics (type of coast, accessibility of river mouth, distances between rivers, river length ...) play a role in shaping directions and rates of migration, and thus the genetic structure of the colonizing populations.

Fast sequence-based microsatellite genotyping development workflow.

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20-40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

Reconstructing the demographic history of divergence between European river and brook lampreys using approximate Bayesian computations.

Inferring the history of isolation and gene flow during species divergence is a central question in evolutionary biology. The European river lamprey (Lampetra fluviatilis) and brook lamprey (L. planeri) show a low reproductive isolation but have highly distinct life histories, the former being parasitic-anadromous and the latter non-parasitic and freshwater resident. Here we used microsatellite data from six replicated population pairs to reconstruct their history of divergence using an approximate Bayesian computation framework combined with a random forest model. In most population pairs, scenarios of divergence with recent isolation were outcompeted by scenarios proposing ongoing gene flow, namely the Secondary Contact (SC) and Isolation with Migration (IM) models. The estimation of demographic parameters under the SC model indicated a time of secondary contact close to the time of speciation, explaining why SC and IM models could not be discriminated. In case of an ancient secondary contact, the historical signal of divergence is lost and neutral markers converge to the same equilibrium as under the less parameterized model allowing ongoing gene flow. Our results imply that models of secondary contacts should be systematically compared to models of divergence with gene flow; given the difficulty to discriminate among these models, we suggest that genome-wide data are needed to adequately reconstruct divergence history.