Marion Ballenghien

Comparative population genomics in animals uncovers the determinants of genetic diversity

Genetic diversity is the amount of variation observed between DNA sequences from distinct individuals of a given species. This pivotal concept of population genetics has implications for species health, domestication, management and conservation. Levels of genetic diversity seem to vary greatly in natural populations and species, but the determinants of this variation, and particularly the relative influences of species biology and ecology versus population history, are still largely mysterious. Here we show that the diversity of a species is predictable, and is determined in the first place by its ecological strategy. We investigated the genome-wide diversity of 76 non-model animal species by sequencing the transcriptome of two to ten individuals in each species. The distribution of genetic diversity between species revealed no detectable influence of geographic range or invasive status but was accurately predicted by key species traits related to parental investment: long-lived or low-fecundity species with brooding ability were genetically less diverse than short-lived or highly fecund ones. Our analysis demonstrates the influence of long-term life-history strategies on species response to short-term environmental perturbations, a result with immediate implications for conservation policies.

Reference-free transcriptome assembly in non-model animals from next-generation sequencing data.

Next‐generation sequencing (NGS) technologies offer the opportunity for population genomic study of non‐model organisms sampled in the wild. The transcriptome is a convenient and popular target for such purposes. However, designing genetic markers from NGS transcriptome data requires assembling gene‐coding sequences out of short reads. This is a complex task owing to gene duplications, genetic polymorphism, alternative splicing and transcription noise. Typical assembling programmes return thousands of predicted contigs, whose connection to the species true gene content is unclear, and from which SNP definition is uneasy. Here, the transcriptomes of five diverse non‐model animal species (hare, turtle, ant, oyster and tunicate) were assembled from newly generated 454 and Illumina sequence reads. In two species for which a reference genome is available, a new procedure was introduced to annotate each predicted contig as either a full‐length cDNA, fragment, chimera, allele, paralogue, genomic sequence or other, based on the number of, and overlap between, blast hits to the appropriate reference. Analyses showed that (i) the highest quality assemblies are obtained when 454 and Illumina data are combined, (ii) typical de novo assemblies include a majority of irrelevant cDNA predictions and (iii) assemblies can be appropriately cleaned by filtering contigs based on length and coverage. We conclude that robust, reference‐free assembly of thousands of genes from transcriptomic NGS data is possible, opening promising perspectives for transcriptome‐based population genomics in animals. A Galaxy pipeline implementing our best‐performing assembling strategy is provided.

Reference-free transcriptome assembly in non-model animals from next generation sequencing data

Next‐generation sequencing (NGS) technologies offer the opportunity for population genomic study of non‐model organisms sampled in the wild. The transcriptome is a convenient and popular target for such purposes. However, designing genetic markers from NGS transcriptome data requires assembling gene‐coding sequences out of short reads. This is a complex task owing to gene duplications, genetic polymorphism, alternative splicing and transcription noise. Typical assembling programmes return thousands of predicted contigs, whose connection to the species true gene content is unclear, and from which SNP definition is uneasy. Here, the transcriptomes of five diverse non‐model animal species (hare, turtle, ant, oyster and tunicate) were assembled from newly generated 454 and Illumina sequence reads. In two species for which a reference genome is available, a new procedure was introduced to annotate each predicted contig as either a full‐length cDNA, fragment, chimera, allele, paralogue, genomic sequence or other, based on the number of, and overlap between, blast hits to the appropriate reference. Analyses showed that (i) the highest quality assemblies are obtained when 454 and Illumina data are combined, (ii) typical de novo assemblies include a majority of irrelevant cDNA predictions and (iii) assemblies can be appropriately cleaned by filtering contigs based on length and coverage. We conclude that robust, reference‐free assembly of thousands of genes from transcriptomic NGS data is possible, opening promising perspectives for transcriptome‐based population genomics in animals. A Galaxy pipeline implementing our best‐performing assembling strategy is provided.

Reference-Free Population Genomics from Next-Generation Transcriptome Data and the Vertebrate–Invertebrate Gap

In animals, the population genomic literature is dominated by two taxa, namely mammals and drosophilids, in which fully sequenced, well-annotated genomes have been available for years. Data from other metazoan phyla are scarce, probably because the vast majority of living species still lack a closely related reference genome. Here we achieve de novo, reference-free population genomic analysis from wild samples in five non-model animal species, based on next-generation sequencing transcriptome data. We introduce a pipe-line for cDNA assembly, read mapping, SNP/genotype calling, and data cleaning, with specific focus on the issue of hidden paralogy detection. In two species for which a reference genome is available, similar results were obtained whether the reference was used or not, demonstrating the robustness of our de novo inferences. The population genomic profile of a hare, a turtle, an oyster, a tunicate, and a termite were found to be intermediate between those of human and Drosophila, indicating that the discordant genomic diversity patterns that have been reported between these two species do not reflect a generalized vertebrate versus invertebrate gap. The genomic average diversity was generally higher in invertebrates than in vertebrates (with the notable exception of termite), in agreement with the notion that population size tends to be larger in the former than in the latter. The non-synonymous to synonymous ratio, however, did not differ significantly between vertebrates and invertebrates, even though it was negatively correlated with genetic diversity within each of the two groups. This study opens promising perspective regarding genome-wide population analyses of non-model organisms and the influence of population size on non-synonymous versus synonymous diversity.

Next-generation sequencing of transcriptomes: a guide to RNA isolation in nonmodel animals

Next Generation Sequencing technologies (NGS) are rapidly invading many evolutionary and ecological fields, such as phylogenomics, molecular evolution, population genomics and molecular ecology. Among the potential targets of NGS is transcriptome sequencing, a fast and relatively cheap way to generate massive amounts of coding sequence data, offering promising perspectives for the analysis of molecular diversity in the wild. A number of molecular ecology research groups therefore may switch from DNA‐based to RNA‐based typing in the near future. Sample preparation from natural populations, however, requires specific care and protocols when RNA is the target. Furthermore, NGS sequencing of transcriptome requires high amount of good‐quality RNA. Here we present the results of RNA extraction experiments from various samples of 39 animal species caught in the wild. We compared tissue preparation and storage conditions, evaluated and improved standard RNA extraction protocols, and achieved RNA yield and quality suitable for NGS in all cases. We derive general guidelines for the production of ready‐to‐sequence RNA in nonmodel animals sampled in the field.

Population genomics of eusocial insects: the costs of a vertebrate-like effective population size

The evolution of reproductive division of labour and social life in social insects has lead to the emergence of several life‐history traits and adaptations typical of larger organisms: social insect colonies can reach masses of several kilograms, they start reproducing only when they are several years old, and can live for decades. These features and the monopolization of reproduction by only one or few individuals in a colony should affect molecular evolution by reducing the effective population size. We tested this prediction by analysing genome‐wide patterns of coding sequence polymorphism and divergence in eusocial vs. noneusocial insects based on newly generated RNA‐seq data. We report very low amounts of genetic polymorphism and an elevated ratio of nonsynonymous to synonymous changes – a marker of the effective population size – in four distinct species of eusocial insects, which were more similar to vertebrates than to solitary insects regarding molecular evolutionary processes. Moreover, the ratio of nonsynonymous to synonymous substitutions was positively correlated with the level of social complexity across ant species. These results are fully consistent with the hypothesis of a reduced effective population size and an increased genetic load in eusocial insects, indicating that the evolution of social life has important consequences at both the genomic and population levels.

Contrasting patterns of genome-wide polymorphism in the native and invasive range of the marine mollusc Crepidula fornicata.

Selection processes are believed to be an important evolutionary driver behind the successful establishment of nonindigenous species, for instance through adaptation for invasiveness (e.g. dispersal mechanisms and reproductive allocation). However, evidence supporting this assumption is still scarce. Genome scans have often identified loci with atypical patterns of genetic differentiation (i.e. outliers) indicative of selection processes. Using microsatellite‐ and AFLP‐based genome scans, we looked for evidence of selection following the introduction of the mollusc Crepidula fornicata. Native to the northwestern Atlantic, this gastropod has become an emblematic invader since its introduction during the 19th and 20th centuries in the northeastern Atlantic and northeastern Pacific. We examined 683 individuals from seven native and 15 introduced populations spanning the latitudinal introduction and native ranges of the species. Our results confirmed the previously documented high genetic diversity in native and introduced populations with little genetic structure between the two ranges, a pattern typical of marine invaders. Analysing 344 loci, no outliers were detected between the introduced and native populations or in the introduced range. The genomic sampling may have been insufficient to reveal selection especially if it acts on traits determined by a few genes. Eight outliers were, however, identified within the native range, underlining a genetic singularity congruent with a well‐known biogeographical break along the Florida. Our results call into question the relevance of AFLP genome scans in detecting adaptation on the timescale of biological invasions: genome scans often reveal long‐term adaptation involving numerous genes throughout the genome but seem less effective in detecting recent adaptation from pre‐existing variation on polygenic traits. This study advocates other methods to detect selection effects during biological invasions—for example on phenotypic traits, although genome scans may remain useful for elucidating introduction histories.

Molecular evolution of freshwater snails with contrasting mating systems

Because mating systems affect population genetics and ecology, they are expected to impact the molecular evolution of species. Self-fertilizing species experience reduced effective population size, recombination rates, and heterozygosity, which in turn should decrease the efficacy of natural selection, both adaptive and purifying, and the strength of meiotic drive processes such as GC-biased gene conversion. The empirical evidence is only partly congruent with these predictions, depending on the analyzed species, some, but not all, of the expected effects have been observed. One possible reason is that self-fertilization is an evolutionary dead-end, so that most current selfers recently evolved self-fertilization, and their genome has not yet been strongly impacted by selfing. Here, we investigate the molecular evolution of two groups of freshwater snails in which mating systems have likely been stable for several millions of years. Analyzing coding sequence polymorphism, divergence, and expression levels, we report a strongly reduced genetic diversity, decreased efficacy of purifying selection, slower rate of adaptive evolution, and weakened codon usage bias/GC-biased gene conversion in the selfer Galba compared with the outcrosser Physa, in full agreement with theoretical expectations. Our results demonstrate that self-fertilization, when effective in the long run, is a major driver of population genomic and molecular evolutionary processes. Despite the genomic effects of selfing, Galba truncatula seems to escape the demographic consequences of the genetic load. We suggest that the particular ecology of the species may buffer the negative consequences of selfing, shedding new light on the dead-end hypothesis.

Development of SNP-genotyping arrays in two shellfish species

Use of SNPs has been favoured due to their abundance in plant and animal genomes, accompanied by the falling cost and rising throughput capacity for detection and genotyping. Here, we present in vitro (obtained from targeted sequencing) and in silico discovery of SNPs, and the design of medium‐throughput genotyping arrays for two oyster species, the Pacific oyster, Crassostrea gigas, and European flat oyster, Ostrea edulis. Two sets of 384 SNP markers were designed for two Illumina GoldenGate arrays and genotyped on more than 1000 samples for each species. In each case, oyster samples were obtained from wild and selected populations and from three‐generation families segregating for traits of interest in aquaculture. The rate of successfully genotyped polymorphic SNPs was about 60% for each species. Effects of SNP origin and quality on genotyping success (Illumina functionality Score) were analysed and compared with other model and nonmodel species. Furthermore, a simulation was made based on a subset of the C. gigas SNP array with a minor allele frequency of 0.3 and typical crosses used in shellfish hatcheries. This simulation indicated that at least 150 markers were needed to perform an accurate parental assignment. Such panels might provide valuable tools to improve our understanding of the connectivity between wild (and selected) populations and could contribute to future selective breeding programmes.

Biased gene conversion and GC-content evolution in the coding sequences of reptiles and vertebrates.

Mammalian and avian genomes are characterized by a substantial spatial heterogeneity of GC-content, which is often interpreted as reflecting the effect of local GC-biased gene conversion (gBGC), a meiotic repair bias that favors G and C over A and T alleles in high-recombining genomic regions. Surprisingly, the first fully sequenced nonavian sauropsid (i.e., reptile), the green anole Anolis carolinensis, revealed a highly homogeneous genomic GC-content landscape, suggesting the possibility that gBGC might not be at work in this lineage. Here, we analyze GC-content evolution at third-codon positions (GC3) in 44 vertebrates species, including eight newly sequenced transcriptomes, with a specific focus on nonavian sauropsids. We report that reptiles, including the green anole, have a genome-wide distribution of GC3 similar to that of mammals and birds, and we infer a strong GC3-heterogeneity to be already present in the tetrapod ancestor. We further show that the dynamic of coding sequence GC-content is largely governed by karyotypic features in vertebrates, notably in the green anole, in agreement with the gBGC hypothesis. The discrepancy between third-codon positions and noncoding DNA regarding GC-content dynamics in the green anole could not be explained by the activity of transposable elements or selection on codon usage. This analysis highlights the unique value of third-codon positions as an insertion/deletion-free marker of nucleotide substitution biases that ultimately affect the evolution of proteins.