Divergence and gene flow history at two large chromosomal inversions underlying ecotype differentiation in the long-snouted seahorse.

Chromosomal inversions can play an important role in divergence and reproductive isolation by building and maintaining distinct allelic combinations between evolutionary lineages. Alternatively, they can take the form of balanced polymorphisms that segregate within populations until one arrangement becomes fixed. Many questions remain about how inversion polymorphisms arise, how they are maintained over the long term, and ultimately, whether and how they contribute to speciation. The long-snouted seahorse (Hippocampus guttulatus) is genetically subdivided into geographic lineages and marine-lagoon ecotypes, with shared structural variation underlying lineage and ecotype divergence. Here, we aim to characterize structural variants and to reconstruct their history and suspected role in ecotype formation. We generated a near chromosome-level genome assembly and described genome-wide patterns of diversity and divergence through the analysis of 112 whole-genome sequences from Atlantic, Mediterranean, and Black Sea populations. By also analysing linked-read sequencing data, we found evidence for two chromosomal inversions that were several megabases in length and showed contrasting allele frequency patterns between lineages and ecotypes across the species range. We reveal that these inversions represent ancient intraspecific polymorphisms, one likely being maintained by divergent selection and the other by pseudo-overdominance. A possible selective coupling between the two inversions was further supported by the absence of specific haplotype combinations and a putative functional interaction between the two inversions in reproduction. Lastly, we detected gene flux eroding divergence between inverted alleles at varying levels for the two inversions, with a likely impact on their dynamics and contribution to divergence and speciation.

Following the Dynamics of Structural Variants in Experimentally Evolved Populations.

Structural variants (SVs) (i.e., deletions, insertions, duplications, and inversions) are now known to play an important role in phenotypic variation, and consequently in processes such as disease determination or adaptation to a new environment. However, single-nucleotide variants receive much more attention than SVs, probably because they are easier to detect, and their phenotypic effects are easier to predict. The development of short- and long-read deep sequencing technologies have strongly improved the detection of SVs, but the quantification of their frequency from pooled sequencing (poolseq) data is still technically complex and expensive. Here, we present a rather simple and inexpensive method, which allows researchers to follow the dynamics of SV allele frequency. As an example of application, we follow the frequency of an insertion sequence (IS) insertion in experimental evolution populations of bacteria. This method is based on the design of triplets of primers around the structural variant borders, such that the amplicons produced by amplification of the wild-type (WT) and derived alleles differ in size by at least 5%, and that their amplification efficiency is similar. The quantity of each amplicon is then determined by parallel capillary electrophoresis and normalized to a calibration curve. This method can be easily extended to the quantification of the frequency of other structural variants (deletions, duplications, and inversions) and to pool-seq approaches of natural populations, including within-patient pathogen populations.

Redundancy analysis, genome-wide association studies and the pigmentation of brown trout (Salmo trutta L.).

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.

Family-effects in the epigenomic response of red blood cells to a challenge test in the European sea bass (Dicentrarchus labrax, L.).

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.

Genomic consequences of a recent three-way admixture in supplemented wild brown trout populations revealed by local ancestry tracts.

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.

A Dense Brown Trout (Salmo trutta) Linkage Map Reveals Recent Chromosomal Rearrangements in the Salmo Genus and the Impact of Selection on Linked Neutral Diversity.

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 socioeconomically important species heavily impacted by human activities. A total of 3977 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 between S. trutta and its sister species, S. salar, revealed extensive chromosomal rearrangements. Strikingly, all of the fusion and fission events that occurred after the S. salar/S. trutta speciation 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 in S. trutta at 3721 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.

European sea bass genome and its variation provide insights into adaptation to euryhalinity and speciation.

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.

Physiological mechanisms underlying individual variation in tolerance of food deprivation in juvenile European sea bass, Dicentrarchus labrax.

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°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<0.01, t-test). At 20 h after receiving a ration equivalent to 2% body mass as food pellets, ammonia excretion and simultaneous RMR were elevated and similar in FT and FS, with AQs of 0.105±0.009 and 0.089±0.007, respectively. At the end of the period of fasting, ammonia excretion and RMR had fallen in both phenotypes, but AQ was significantly lower in FT than FS, being 0.038±0.004 versus 0.061±0.005, respectively (P<0.001, t-test). There was a direct linear relationship between individual fasted AQ and rate of mass loss, with FT and FS individuals distributed at opposing lower and upper extremities, respectively. Thus the difference between the phenotypes in their tolerance of food deprivation did not depend upon their routine energy use when fasting. Rather, it depended upon their relative use of tissue proteins as metabolic fuels when fasting, which was significantly lower in FT phenotypes.

Gene flow at major transitional areas in sea bass (Dicentrarchus labrax) and the possible emergence of a hybrid swarm.

The population genetic structure of sea bass (Dicentrarchus labrax) along a transect from the Atlantic Ocean (AO) to the Eastern Mediterranean (EM) Sea differs from that of most other marine taxa in this area. Three populations (AO, Western Mediterranean [WM], EM) are recognized today, which were originally two allopatric populations. How two ancestral genetic units have evolved into three distinct units has not been addressed yet. Therefore, to investigate mechanisms that lead to the emergence of the central WM population, its current status, and its connectivity with the two parental populations, we applied 20 nuclear loci that were either gene associated or gene independent. Results confirmed the existence of three distinct gene pools, with higher differentiation at two transitional areas, the Almeria-Oran Front (AOF) and of the Siculo-Tunisian Strait (STS), than within any population. Significant linkage disequilibrium and heterozygote excess indicated that the STS is probably another tension zone, as already described for the AOF. Neutrality tests fail to reveal marker loci that could be driven by selection within or among metapopulations, except for locus DLA0068. Collectively, results support that the central WM population arose by trapping two tensions zones at distinct geographic locations of limited connectivity. Population assignment further revealed that WM individuals were more introgressed than individuals from the other two metapopulations. This suggests that this population might result from hybrid swarming, and was or is still seeded by genes received through the filter of each tension zone.

SESAME (SEquence Sorter & AMplicon Explorer): genotyping based on high-throughput multiplex amplicon sequencing.

SUMMARY: Characterizing genetic diversity through genotyping short amplicons is central to evolutionary biology. Next-generation sequencing (NGS) technologies changed the scale at which these type of data are acquired. SESAME is a web application package that assists genotyping of multiplexed individuals for several markers based on NGS amplicon sequencing. It automatically assigns reads to loci and individuals, corrects reads if standard samples are available and provides an intuitive graphical user interface (GUI) for allele validation based on the sequences and associated decision-making tools. The aim of SESAME is to help allele identification among a large number of sequences. AVAILABILITY: SESAME and its documentation are freely available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported Licence for Windows and Linux from http://www1.montpellier.inra.fr/CBGP/NGS/ or http://tinyurl.com/ngs-sesame.

Molecular diversity of antimicrobial effectors in the oyster Crassostrea gigas.

BACKGROUND: To gain insight into the molecular diversity of antimicrobial peptides and proteins in the oyster Crassostrea gigas, we characterized and compared the sequence polymorphism of the antimicrobial peptides (AMPs), Cg-Defensins (Cg-Defs) and Cg-Proline Rich peptide (Cg-Prp), and of the bactericidal permeability increasing protein, Cg-BPI. For that, we analyzed genomic and transcript sequences obtained by specific PCR amplification and in silico searches. RESULTS: High diversification among the three antimicrobial effectors was evidenced by this polymorphism survey. On the basis of sequence phylogenies, each AMP aggregates into clearly defined groups of variants and is the product of a multigenic family displaying a variety of gene structures. In contrast, Cg-bpi forms a single group and is encoded by a single gene copy. Moreover, we identified for both AMPs several genetic mechanisms of diversification such as recombination, parallel mutations leading to phylogenetic homoplasy and indel events. In addition, the non synonymous to synonymous substitutions ratio by codon (dN/dS) revealed several negatively and positively selected sites for both AMPs, suggesting that directional selection pressures have shaped their sequence variations. CONCLUSIONS: This study shows for the first time in a mollusc that antimicrobial peptides and proteins have been subject to distinct patterns of diversification and we evidence the existence of different evolutionary routes leading to such sequence variability.

Evidence from population genetics that the ectomycorrhizal basidiomycete Laccaria amethystina is an actual multihost symbiont.

It is commonly assumed that ectomycorrhizal (ECM) fungi associated with temperate forest tree roots are not host-specific. Because this assumption relies on species delineations based on fruitbodies morphology or ribosomal DNA sequences, host-specific, cryptic biological species cannot be ruled out. To demonstrate that Laccaria amethystina has true generalist abilities, we sampled 510 fruitbodies on three French sites situated 150-450 km away from each other. At each site, populations from monospecific stands (Abies alba, Castanea europea and Fagus sylvatica) or mixed stands (F. sylvatica + Quercus robur or Q. robur +Carpinus betulus) were sampled. Three different sets of markers were used for genotyping: (i) five microsatellite loci plus the ribosomal DNA intergenic spacer, (ii) the mitochondrial large ribosomal DNA subunit, and (iii) direct amplification of length polymorphism (DALP), a new method for fungi providing dominant markers. Evidence for allogamous populations (with possible inbreeding at local scale) and possibly for biparental mitochondrial inheritance was found. All markers congruently demonstrated that L. amethystina populations show little structure at this geographical scale, indicating high gene flow (as many as 50% of founding spores in all populations being of external origin). Our results also showed that host species contributed even less to population differentiation, and there was no evidence for cryptic biological species. This first in situ demonstration of a true multihost ability in an ECM species is discussed in terms of ecology and evolutionary biology.

Local mutagenic impact of insertions of LTR retrotransposons on the mouse genome.

Solitary LTR loci are the predominant form of LTR retrotransposons in most eukaryotic genomes. They originate from recombination between the two LTRs of an ancestral retrovirus and are therefore incapable of transposition. Despite this inactivity, they appear to have a substantial impact on the host genome. Here we use the murine RMER10 LTR family as an example to describe how such elements can reshape regions of the genome through multiple mutations on an evolutionary time scale. Specifically, we use phylogenetic analysis of multiple copies of RMER10 in rodent species, as well as comparisons of orthologous pairs in mouse and rat, to argue that insertions of members of this family have locally induced the emergence of tandem repeat loci as well as many indels. Analysis of structural aspects of these sequences (secondary structures and transcription factors signals) may explain why RMER10 can become endogenous "mutagenic" factors through induction of replication fork blockages and/or error-prone repair of aberrant DNA structures. This hypothesis is also consistent with features of other interspersed repeated elements.

Phylogenetic relationships among monogenean gill parasites (Dactylogyridea, Ancyrocephalidae) infesting tilapiine hosts (Cichlidae): systematic and evolutionary implications.

We studied the systematics of 14 species of monogenean (Ancyrocephalidae) gill parasites from West African tilapiine hosts (Cichlidae) using both morphological and genetic data. With these tools, we were able to: (i) confirm the validity of the previously described morphological parasite species and of the genus Scutogyrus; (ii) propose that some stenoxenous species (i.e., parasite species with more than one host) may be composed of sister species (e.g., Cichlidogyrus tilapiae); (iii) state that the use of the morphology of the haptoral sclerites is more suitable to infer phylogenetic relationships than the morphology of the genitalia (which seems to be more useful to resolve species-level identifications, presumably because of its faster rate of change). These results imply that: (i) the specificity of these monogenean parasites is greater than initially supposed (what were thought to be stenoxenous species may be assemblages of oïoxenous sister species); (ii) related species groups (i.e., "tilapiae," "halli," and "tiberianus") have to be, as genus Scutogyrus, validated within the 54 ancyrocephalid species described from 18 species of tilapiine hosts in West Africa, (iii) the group "tilapiae," due to its morphology and host range, have to be considered as being the most primitive; (iv) the occurrence of lateral transfers and parallel speciation processes are necessary to describe the repartition of the newly described parasite groups on the three host genera studied (Tilapia, Oreochromis, and Sarotherodon).

Characterization of a centromeric marker on mouse chromosome 11 and its introgression in a domesticus/musculus hybrid zone.

It has been proposed that the distribution of Robertsonian chromosome fusions and the Chromosome 11 Nucleolar Organizer Region (NOR) in the Danish hybrid zone between M. m. musculus and M. m. domesticus stems from centromeric incompatibilities between the two subspecies. To test this hypothesis, we identified and characterized a diagnostic subspecific marker closely linked to the centromere on mouse Chromosome 11. Using an allele-specific PCR assay, we investigated the introgression pattern of this centromere in a large sample of mice from a North-South transect of the hybrid zone in Jutland. Domesticus alleles were found to introgress far away from the center of the zone on the musculus side. These results suggest there is no incompatibility between the domesticus centromere of Chromosome 11 in the musculus genomic background.