Abstract: The generation of EST information is an essential step in the genomic characterisation of species. In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs in commercial marine mollusk species and more specifically in the less studied but ecologically and commercially important groups, such as mussel and clam genera. Normalized cDNA libraries were constructed for four different relevant bivalves species (Crassostrea gigas, Mytilus edulis, Ruditapes decussatus and Bathymodiolus azoricus), using numerous tissues and physiological conditions. In this paper, we present the analysis of the 13,013 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1300-3000 unique sequences were identified in each species. For the different species, functional categories could be assigned to only about 16 to 27% of ESTs using the GO annotation tool. All sequences have been incorporated into a publicly available database and form the basis for subsequent microarray design, SNP detection and polymorphism analysis, and the placement of novel markers on genetic linkage maps.
Abstract: DNA sequence polymorphism and codon usage bias were investigated in a set of 41 nuclear loci in the Pacific oyster Crassostrea gigas. Our results revealed a very high level of DNA polymorphism in oysters, in the order of magnitude of the highest levels reported in animals to date. A total of 290 single nucleotide polymorphisms (SNPs) were detected, 76 of which being localised in exons and 214 in non-coding regions. Average density of SNPs was estimated to be one SNP every 60 bp in coding regions and one every 40 bp in non-coding regions. Non-synonymous substitutions contributed substantially to the polymorphism observed in coding regions. The non-synonymous to silent diversity ratio was 0.16 on average, which is fairly higher to the ratio reported in other invertebrate species recognised to display large population sizes. Therefore, purifying selection does not appear to be as strong as it could have been expected for a species with a large effective population size. The level of non-synonymous diversity varied greatly from one gene to another, in accordance with varying selective constraints. We examined codon usage bias and its relationship with DNA polymorphism. The table of optimal codons was deduced from the analysis of an EST dataset, using EST counts as a rough assessment of gene expression. As recently observed in some other taxa, we found a strong and significant negative relationship between codon bias and non-synonymous diversity suggesting correlated selective constraints on synonymous and non-synonymous substitutions. Codon bias as measured by the frequency of optimal codons for expression might therefore provide a useful indicator of the level of constraint upon proteins in the oyster genome.
Abstract: Previous surveys of allozyme variation in smooth-shell Mytilus spp. mussels have reported the presence in the Southern Hemisphere of both Mytilus edulis and Mytilus galloprovincialis mussels. In the present study, nuclear DNA markers mac-1 and Glu-5'/Glu-3', both diagnostic for Northern-Hemisphere M. edulis and M. galloprovincialis, were used to further characterize the nuclear genomes of M. edulis from Kerguelen and M. galloprovincialis from Tasmania. Genomic reticulation was observed, with typical M. edulis allelomorphs fixed in both populations at locus mac-1 whereas, at locus Glu-5'/Glu-3', allelomorphs characteristic of M. galloprovincialis were present in Kerguelen and nearly fixed in Tasmania. Kerguelen mussels had a genome of mixed M. edulis and M. galloprovincialis ancestry without evidence of barriers to merging as shown by Hardy–Weinberg and linkage equilibrium. Tasmanian mussels possessed a predominantly M. galloprovincialis genomic background introgressed by M. edulis allelomorphs at locus mac-1. Genetic drift superimposed on ancient hybridization and introgression may explain the genomic reticulation observed in both Kerguelen and Tasmanian mussels. There was no evidence of a recent introduction of Northern-Hemisphere M. galloprovincialis or M. edulis to Kerguelen or Tasmania.
Abstract: We describe a simple protocol to reduce the number of cloning reactions of nuclear DNA sequences in population genetic studies of diploid organisms. Cloning is a necessary step to obtain correct haplotypes in such organisms, and, while traditional methods are efficient at cloning together many genes of a single individual, population geneticists rather need to clone the same locus in many individuals. Our method consists of marking individual sequences during the polymerase chain reaction (PCR) using 5'-tailed primers with small polynucleotide tags. PCR products are mixed together before the cloning reaction and clones are sequenced with universal plasmid primers. The individual from which a sequence comes from is identified by the tag sequences upstream of each initial primer. We called our protocol mark–recapture (MR) cloning. We present results from 57 experiments of MR cloning conducted in four distinct laboratories using nuclear loci of various lengths in different invertebrate species. Rate of capture (proportion of individuals for which one or more sequences were retrieved) and multiple capture (proportion of individuals for which two or more sequences were retrieved) empirically obtained are described. We estimated that MR cloning allowed reducing costs by up to 70% when compared to conventional individual-based cloning. However, we recommend to adjust the mark:recapture ratio in order to obtain multiple sequences from the same individual and circumvent inherent technical artefacts of PCR, cloning and sequencing. We argue that MR cloning is a valid and reliable high-throughput method, providing the number of sequences exceeds the number of individuals initially amplified.
Abstract: A multilocus analysis was initiated in order to infer the general effect of demography and the indirect effect of positive selection on some chromosome segments in Bathymodiolus. Mussels of the genus Bathymodiolus inhabit the very hostile, fragmented and variable environment of deep-sea hydrothermal vents which is thought to cause recurrent population bottlenecks via extinction/colonisation processes and adaptation to new environmental conditions. In the course of this work we discovered that the assumption of neutrality of non-coding polymorphisms usually made in genome scan experiments was likely to be violated at one of the loci we analysed. The direct effect of slight purifying selection on non-coding polymorphisms shares many resemblances with the indirect effect of positive selection through genetic hitchhiking. Combining polymorphism with divergence data for several closely related species allowed us to obtain different expectations for the direct effect of negative selection and the indirect effect of positive selection. We observed a strong excess of rare non-coding polymorphisms at the second intron of the EF1alpha gene in the two species Bathymodiolus azoricus and Bathymodiolus thermophilus, while two other loci, the mitochondrial COI gene and an intron of the Lysozyme gene, did not exhibit such a deviation. In addition, the divergence rate of the EF1alpha intron was estimated to be unexpectedly low when calibrated using the closure of the Panama Isthmus that interrupted gene flow between the two species. The polymorphism to divergence ratio was similar to the one observed for the other two loci, in accordance to the hypothesis of purifying selection. We conclude that slight purifying selection is likely to act on polymorphic intronic mutations of the EF1alpha second intron and discuss the possible relationship with the specific biology of Bathymodiolus mussels.
Abstract: The study of base composition evolution in Drosophila has been achieved mostly through the analysis of coding sequences. Third codon position GC content, however, is influenced by both neutral forces (e.g., mutation bias) and natural selection for codon usage optimization. In this article, large data sets of noncoding DNA sequence polymorphism in D. melanogaster and D. simulans were gathered from public databases to try to disentangle these two factors-noncoding sequences are not affected by selection for codon usage. Allele frequency analyses revealed an asymmetric pattern of AT vs. GC noncoding polymorphisms: AT --> GC mutations are less numerous, and tend to segregate at a higher frequency, than GC --> AT ones, especially at GC-rich loci. This is indicative of nonstationary evolution of base composition and/or of GC-biased allele transmission. Fitting population genetics models to the allele frequency spectra confirmed this result and favored the hypothesis of a biased transmission. These results, together with previous reports, suggest that GC-biased gene conversion has influenced base composition evolution in Drosophila and explain the correlation between intron and exon GC content.
Abstract: We studied the genetic basis of post-zygotic isolation in the marine mussels Mytilus edulis and Mytilus galloprovincialis. Evidence was obtained for a high number of recessive Dobzhansky-Muller substitutions in the genome of these two mussel taxa. We analysed the segregation of unlinked diagnostic markers in the progeny of two backcrosses and an F2 cross, 36 h and 200 days after fertilization. Directional selection favouring M. galloprovincialis genotypes was observed in both kinds of cross. In the F2, epistatic interactions between each pair of chromosome fragments mapped by the markers were identified in addition. Our results imply that homozygous-homozygous interactions are required for breakdown of coadaptation, in accordance with the dominance theory of post-zygotic isolation. Endogenous post-zygotic selection distributed over many loci throughout the genome provides the missing factor explaining the astonishing persistence and strength of barriers to neutral introgression in such a dispersive taxon as Mytilus.
Abstract: A strong negative correlation between the rate of amino-acid substitution and codon usage bias in Drosophila has been attributed to interference between positive selection at nonsynonymous sites and weak selection on codon usage. To further explore this possibility we have investigated polymorphism and divergence at three kinds of sites: synonymous, nonsynonymous and intronic in relation to codon bias in D. melanogaster and D. simulans. We confirmed that protein evolution is one of the main explicative parameters for interlocus codon bias variation (r(2) approximately 40%). However, intron or synonymous diversities, which could have been expected to be good indicators of local interference [here defined as the additional increase of drift due to selection on tightly linked sites, also called 'genetic draft' by Gillespie (2000)] did not covary significantly with codon bias or with protein evolution. Concurrently, levels of polymorphism were reduced in regions of low recombination rates whereas codon bias was not. Finally, while nonsynonymous diversities were very well correlated between species, neither synonymous nor intron diversities observed in D. melanogaster were correlated with those observed in D. simulans. All together, our results suggest that the selective constraint on the protein is a stable component of gene evolution while local interference is not. The pattern of variation in genetic draft along the genome therefore seems to be instable through evolutionary times and should therefore be considered as a minor determinant of codon bias variance. We argue that selective constraints for optimal codon usage are likely to be correlated with selective constraints on the protein, both between codons within a gene, as previously suggested, and also between genes within a genome.
Abstract: The proportion of amino acid substitutions driven by adaptive evolution can potentially be estimated from polymorphism and divergence data by an extension of the McDonald-Kreitman test. We have developed a maximum-likelihood method to do this and have applied our method to several data sets from three Drosophila species: D. melanogaster, D. simulans, and D. yakuba. The estimated number of adaptive substitutions per codon is not uniformly distributed among genes, but follows a leptokurtic distribution. However, the proportion of amino acid substitutions fixed by adaptive evolution seems to be remarkably constant across the genome (i.e., the proportion of amino acid substitutions that are adaptive appears to be the same in fast-evolving and slow-evolving genes; fast-evolving genes have higher numbers of both adaptive and neutral substitutions). Our estimates do not seem to be significantly biased by selection on synonymous codon use or by the assumption of independence among sites. Nevertheless, an accurate estimate is hampered by the existence of slightly deleterious mutations and variations in effective population size. The analysis of several Drosophila data sets suggests that approximately 25% +/- 20% of amino acid substitutions were driven by positive selection in the divergence between D. simulans and D. yakuba.
Abstract: Hybrid zones are fascinating systems to investigate the structure of genetic barriers. Marine hybrid zones deserve more investigation because of the generally high dispersion potential of planktonic larvae which allows migration on scales unrivalled by terrestrial species. Here we analyse the genetic structure of the mosaic hybrid zone between the marine mussels Mytilus edulis and M. galloprovincialis, using three length-polymorphic PCR loci as neutral and diagnostic markers on 32 samples along the Atlantic coast of Europe. Instead of a single genetic gradient from M. galloprovincialis on the Iberian Peninsula to M. edulis populations in the North Sea, three successive transitions were observed in France. From South to North, the frequency of alleles typical of M. galloprovincialis first decreases in the southern Bay of Biscay, remains low in Charente, then increases in South Brittany, remains high in most of Brittany, and finally decreases again in South Normandy. The two enclosed patches observed in the midst of the mosaic hybrid zone in Charente and Brittany, although predominantly M. edulis-like and M. galloprovincialis-like, respectively, are genetically original in two respects. First, considering only the various alleles typical of one species, the patches show differentiated frequencies compared to the reference external populations. Second, each patch is partly introgressed by alleles of the other species. When introgression is taken into account, linkage disequilibria appear close to their maximum possible values, indicating a strong genetic barrier within all transition zones. Some pre- or postzygotic isolation mechanisms (habitat specialization, spawning asynchrony, assortative fertilization and hybrid depression) have been documented in previous studies, although their relative importance remains to be evaluated. We also provided evidence for a recent migratory 'short-cut' connecting M. edulis-like populations of the Charente patch to an external M. edulis population in Normandy and thought to reflect artificial transfer of spat for aquaculture.
Abstract: Most methods for estimating the rate of synonymous and nonsynonymous substitution per site define a site as a mutational opportunity: the proportion of sites that are synonymous is equal to the proportion of mutations that would be synonymous under the model of evolution being considered. Here we demonstrate that this definition of a site can give misleading results and that a physical definition of site should be used in some circumstances. We illustrate our point by reexamining the relationship between codon usage bias and the synonymous substitution rate. It has recently been shown that the rate of synonymous substitution, calculated using the Goldman-Yang method, which encapsulates the mutational-opportunity definition of a site at a high level of sophistication, is either positively correlated or uncorrelated to synonymous codon bias in Drosophila. Using other methods, which account for synonymous codon bias but define a site physically, we show that there is a negative correlation between the synonymous substitution rate and codon bias and that the lack of a negative correlation using the Goldman-Yang method is due to the way in which the number of synonymous sites is counted. We also show that there is a positive correlation between the synonymous substitution rate and third position GC content in mammals, but that the relationship is considerably weaker than that obtained using the Goldman-Yang method. We argue that the Goldman-Yang method is misleading in this context and conclude that methods that rely on a mutational-opportunity definition of a site should be used with caution.
Abstract: Marine organisms challenge the classical theories of local adaptation and speciation because their planktonic larvae have the potential to maintain high gene flow. The marine-speciation paradox is illustrated by contact zones between incipient species that are so large that allopatric divergence seems unlikely. For this reason any mechanism preventing sympatric larvae of two incipient species from coexisting in the same habitats can be a powerful promoter of speciation. The contact zone between two hybridizing taxa of mussel, Mytilus edulis and M. galloprovincialis, in Europe provides an excellent example. Although the zone itself extends over thousands of kilometres, the opportunities for interbreeding are considerably reduced by the small-scale mosaic structure of the zone, where local patches of each taxon alternate at scales of kilometres or less, in response to locally variable ecological factors. Habitat choice by settling larvae would be a less costly mechanism than post-settlement selection to maintain such a mosaic structure. Unfortunately the role of selective settlement has remained hypothetical because larvae could not be scored by classical genetic markers. PCR markers allowed us to study larvae and settlement in ecologically contrasting sites within the zone. We show that only a subset of the genotypes present in the plankton settle in some sites, and that the adults on these sites show the same genetic bias. Genetically based variation in pre-settlement processes therefore accounts for the ecological segregation observed, though it is not the only factor involved in limiting successful interbreeding. The present dataset also supports previous reports of partial spawning asynchrony.
Abstract: Unequal differentiation between two types of loci (allozyme and DNA markers) across a Mytilus hybrid zone has recently been claimed as evidence for direct selection on some allozyme loci. We provide here a counter-example: a noncoding DNA locus that exhibits as much differentiation as the incriminated allozymes do. The levels of genetic differentiation varied widely among both allozymes and noncoding DNA markers and no clear difference emerged between the two types of markers. This suggests that the strong interlocus variance in genetic differentiation has been confounded with a discrepancy between marker types as a result of an insufficient and unbalanced locus sampling. Heterogeneity in differentiation among neutral loci can be created by stochastic variance during the allopatric divergence preceding a secondary contact. In hybrid zones, a further source of variance is differential introgression among chromosomal regions after the secondary contact owing to the local influence of selected genes on more or less distant markers. However, the degree of differentiation alone gives no way to distinguish indirect pseudo-selection (a regular and ubiquitous feature of hybrid zones) from direct selection. More generally, we suggest that comparative neutrality tests based on discrepancies among marker types have to be applied with caution when the presence of semi-permeable genetic barriers to gene exchange is suspected.
Abstract: The aim of this paper is to investigate the effect of deleterious mutations in a hybrid zone maintained by selection against hybrids. In such zones, linkage disequilibria among hybrid depression loci, resulting from a balance between migration and selection, are crucial in maintaining the barrier because they allow each locus, in addition to its own selection coefficient, to cumulate indirect selective effects from other loci. Deleterious alleles produce heterosis and increase by this means the effective migration rate in structured populations. In a hybrid zone, they therefore contribute to decrease linkage disequilibria as well as the barrier to gene flow imposed by hybrid depression. However, deleterious mutations have no effect: (i) when selection against hybrids is weak, because linkage disequilibria are small even without heterosis in this case, or (ii) when selection against hybrids is so strong that it overwhelms heterosis. On the other hand, with moderate selection against hybrids, the decrease in the strength of the barrier due to heterosis may reach detectable levels, although it requires relatively small population sizes and/or migration rates. The effect is expected to be small and only within small genomes where loci are tightly linked can it become strong. Nevertheless, neglecting mutational load may to some extent obscure the estimations of selective parameters based either on artificial F1 crosses or on cline characteristics.
Abstract: Assortative mating (prezygotic isolation) and reduced hybrid fitness (postzygotic isolation) are typically invoked to explain the stability of hybrid zones. In the tension zone model, these factors work in opposition to migration, which promotes genetic homogeneity. Many marine animals migrate over long distances through a planktonic larval stage. Therefore, strong reproductive isolation is needed to maintain stable marine hybrid zones. However, surprisingly little is known about mating preferences and hybrid fitness in marine organisms. Smooth-shelled mussels (Mytilus spp.) form a well-known species complex, with hybridization over extensive areas such as the contact zone of M. edulis and M. galloprovincialis around European Atlantic coasts. This paper reports direct experimental evidence of assortative fertilization, hybrid larval inviability, and early heterosis for growth rate in M. edulis and M. galloprovincialis. Four crosses between pure M. edulis and M. galloprovincialis were analyzed with a new polymerase-chain-reaction-based diagnostic marker. Gamete competition between taxa was allowed in two out of the four crosses. Genotype frequencies observed at an early stage (36 h after fertilization) unambiguously revealed assortative fertilization when gamete competition was allowed. A significant reduction in hybrid viability was subsequently observed during the larval stage. At the same stage an antagonistic effect, heterosis, was observed on growth rate. However, even if heterosis is observed in the F1, it is expected to vanish in subsequent hybrid generations. Although specialization for different habitats and asynchronous spawning have been mentioned as factors contributing to the maintenance of the blue mussel hybrid zone in Europe, we argue that assortative fertilization and reduced hybrid fitness are important factors that also contribute to the stabilization of this zone. These results emphasize that multiple factors may act concomitantly in a barrier to gene flow, especially in complex life cycles. Furthermore, they show that assortative mating through gamete preference, as already demonstrated for sea urchins, may play a role in speciation processes taking place in the sea.
Abstract: Experimental examination of reproductive isolation is the first step in understanding hybridization processes. Here, we studied preferential fertilization between 2 cupped oyster taxa, Crassostrea angulata and Crassostrea gigas, as a potential prezygotic reproductive isolation. Early examination of sperm competition is now possible by molecular analysis of oyster embryos. This avoids the confounding effect of differential mortality during the larval stage. Six hundred embryos were sampled from 2 crosses. Three microsatellite loci were enough to determine without ambiguity the taxa of contributing sires of embryos. No evidence of preferential fertilization between gametes from the same taxa was shown. A significantly higher contribution of the C. gigas males was revealed with the C. angulata females, but not with the C. gigas females, which might suggest early heterosis or interaction differences between gametes. In the light of these results, natural hybridization between both taxa can be expected in cases of their geographical coexistence, as in the Southern European populations in which both taxa are in contact as a result of aquaculture development.
Abstract: Associative overdominance, the fitness difference between heterozygotes and homozygotes at a neutral locus, is classically described using two categories of models: linkage disequilibrium in small populations or identity disequilibrium in infinite, partially selfing populations. In both cases, only equilibrium situations have been considered. In the present study, associative overdominance is related to the distribution of individual inbreeding levels (i.e., genomic autozygosity). Our model integrates the effects of physical linkage and variation in inbreeding history among individual pedigrees. Hence, linkage and identity disequilibrium, traditionally presented as alternatives, are summarized within a single framework. This allows studying nonequilibrium situations in which both occur simultaneously. The model is applied to the case of an infinite population undergoing a sustained population bottleneck. The effects of bottleneck size, mating system, marker gene diversity, deleterious genomic mutation parameters, and physical linkage are evaluated. Bottlenecks transiently generate much larger associative overdominance than observed in equilibrium finite populations and represent a plausible explanation of empirical results obtained, for instance, in marine species. Moreover, the main origin of associative overdominance is random variation in individual inbreeding whereas physical linkage has little effect.
Abstract: This paper reports new experimental evidence on the effect of inbreeding on growth and survival in the early developmental phase of a marine bivalve, the flat oyster Ostrea edulis. Two crosses between full sibs were analyzed using four microsatellite markers. Samples of 96 individuals were taken just after spawning (day 1), at the end of the larval stage before metamorphosis (day 10) and at the postlarval stage (day 70). Significant departure from Mendelian expectation was observed at two loci in the first cross and two loci in the second. Departure from 1:1 segregation occurred in one parent of the first cross at three loci and genotypic selection, which resulted in highly significant heterozygote excesses, was recorded at three out of four loci in cross C1 and at two out of three loci in cross C2. Across the four markers, there were similar significant excesses of multilocus heterozygosity, and significant multilocus heterozygosity-growth correlations were recorded for both crosses at all stages. These results suggest that microsatellite markers, often assumed to be neutral, cosegregated with fitness-associated genes, the number of which is estimated to be between 15 and 38 in the whole genome, and that there is a potentially high genetic load in Ostrea edulis genome. This load provides a genetic basis for heterosis in marine bivalves.
