The Effect of Copy Number Hemiplasy on Gene Family Evolution.

The evolution of gene families is complex, involving gene-level evolutionary events such as gene duplication, horizontal gene transfer, and gene loss, and other processes such as incomplete lineage sorting (ILS). Because of this, topological differences often exist between gene trees and species trees. A number of models have been recently developed to explain these discrepancies, the most realistic of which attempts to consider both gene-level events and ILS. When unified in a single model, the interaction between ILS and gene-level events can cause polymorphism in gene copy number, which we refer to as copy number hemiplasy (CNH). In this paper, we extend the Wright-Fisher process to include duplications and losses over several species, and show that the probability of CNH for this process can be significant. We study how well two unified models-multilocus multispecies coalescent (MLMSC), which models CNH, and duplication, loss, and coalescence (DLCoal), which does not-approximate the Wright-Fisher process with duplication and loss. We then study the effect of CNH on gene family evolution by comparing MLMSC and DLCoal. We generate comparable gene trees under both models, showing significant differences in various summary statistics; most importantly, CNH reduces the number of gene copies greatly. If this is not taken into account, the traditional method of estimating duplication rates (by counting the number of gene copies) becomes inaccurate. The simulated gene trees are also used for species tree inference with the summary methods ASTRAL and ASTRAL-Pro, demonstrating that their accuracy, based on CNH-unaware simulations calibrated on real data, may have been overestimated.

Purging of deleterious burden in the endangered Iberian lynx.

SignificanceThe dynamics of deleterious variation under contrasting demographic scenarios remain poorly understood in spite of their relevance in evolutionary and conservation terms. Here we apply a genomic approach to study differences in the burden of deleterious alleles between the endangered Iberian lynx (Lynx pardinus) and the widespread Eurasian lynx (Lynx lynx). Our analysis unveils a significantly lower deleterious burden in the former species that should be ascribed to genetic purging, that is, to the increased opportunities of selection against recessive homozygotes due to the inbreeding caused by its smaller population size, as illustrated by our analytical predictions. This research provides theoretical and empirical evidence on the evolutionary relevance of genetic purging under certain demographic conditions.

Haplotype divergence supports long-term asexuality in the oribatid mite Oppiella nova.

Sex strongly impacts genome evolution via recombination and segregation. In the absence of these processes, haplotypes within lineages of diploid organisms are predicted to accumulate mutations independently of each other and diverge over time. This so-called "Meselson effect" is regarded as a strong indicator of the long-term evolution under obligate asexuality. Here, we present genomic and transcriptomic data of three populations of the asexual oribatid mite species Oppiella nova and its sexual relative Oppiella subpectinata We document strikingly different patterns of haplotype divergence between the two species, strongly supporting Meselson effect-like evolution and long-term asexuality in O. nova: I) variation within individuals exceeds variation between populations in O. nova but vice versa in O. subpectinata; II) two O. nova sublineages feature a high proportion of lineage-specific heterozygous single-nucleotide polymorphisms (SNPs), indicating that haplotypes continued to diverge after lineage separation; III) the deepest split in gene trees generally separates the two haplotypes in O. nova, but populations in O. subpectinata; and IV) the topologies of the two haplotype trees match each other. Our findings provide positive evidence for the absence of canonical sex over evolutionary time in O. nova and suggest that asexual oribatid mites can escape the dead-end fate usually associated with asexual lineages.

Detection of F1 Hybrids from Single-genome Data Reveals Frequent Hybridization in Hymenoptera and Particularly Ants.

Hybridization occupies a central role in many fundamental evolutionary processes, such as speciation or adaptation. Yet, despite its pivotal importance in evolution, little is known about the actual prevalence and distribution of current hybridization across the tree of life. Here we develop and implement a new statistical method enabling the detection of F1 hybrids from single-individual genome sequencing data. Using simulations and sequencing data from known hybrid systems, we first demonstrate the specificity of the method, and identify its statistical limits. Next, we showcase the method by applying it to available sequencing data from more than 1,500 species of Arthropods, including Hymenoptera, Hemiptera, Coleoptera, Diptera, and Archnida. Among these taxa, we find Hymenoptera, and especially ants, to display the highest number of candidate F1 hybrids, suggesting higher rates of recent hybridization between previously isolated gene pools in these groups. The prevalence of F1 hybrids was heterogeneously distributed across ants, with taxa including many candidates tending to harbor specific ecological and life-history traits. This work shows how large-scale genomic comparative studies of recent hybridization can be implemented, uncovering the determinants of first-generation hybridization across whole taxa.

Is adaptation limited by mutation? A timescale-dependent effect of genetic diversity on the adaptive substitution rate in animals.

Whether adaptation is limited by the beneficial mutation supply is a long-standing question of evolutionary genetics, which is more generally related to the determination of the adaptive substitution rate and its relationship with species effective population size (Ne) and genetic diversity. Empirical evidence reported so far is equivocal, with some but not all studies supporting a higher adaptive substitution rate in large-Ne than in small-Ne species. We gathered coding sequence polymorphism data and estimated the adaptive amino-acid substitution rate ωa, in 50 species from ten distant groups of animals with markedly different population mutation rate θ. We reveal the existence of a complex, timescale dependent relationship between species adaptive substitution rate and genetic diversity. We find a positive relationship between ωa and θ among closely related species, indicating that adaptation is indeed limited by the mutation supply, but this was only true in relatively low-θ taxa. In contrast, we uncover no significant correlation between ωa and θ at a larger taxonomic scale, suggesting that the proportion of beneficial mutations scales negatively with species' long-term Ne.

The Multilocus Multispecies Coalescent: A Flexible New Model of Gene Family Evolution.

Incomplete lineage sorting (ILS), the interaction between coalescence and speciation, can generate incongruence between gene trees and species trees, as can gene duplication (D), transfer (T), and loss (L). These processes are usually modeled independently, but in reality, ILS can affect gene copy number polymorphism, that is, interfere with DTL. This has been previously recognized, but not treated in a satisfactory way, mainly because DTL events are naturally modeled forward-in-time, while ILS is naturally modeled backward-in-time with the coalescent. Here, we consider the joint action of ILS and DTL on the gene tree/species tree problem in all its complexity. In particular, we show that the interaction between ILS and duplications/transfers (without losses) can result in patterns usually interpreted as resulting from gene loss, and that the realized rate of D, T, and L becomes nonhomogeneous in time when ILS is taken into account. We introduce algorithmic solutions to these problems. Our new model, the multilocus multispecies coalescent, which also accounts for any level of linkage between loci, generalizes the multispecies coalescent (MSC) model and offers a versatile, powerful framework for proper simulation, and inference of gene family evolution. [Gene duplication; gene loss; horizontal gene transfer; incomplete lineage sorting; multispecies coalescent; hemiplasy; recombination.].

Determinants of genetic diversity.

Genetic polymorphism varies among species and within genomes, and has important implications for the evolution and conservation of species. The determinants of this variation have been poorly understood, but population genomic data from a wide range of organisms now make it possible to delineate the underlying evolutionary processes, notably how variation in the effective population size (Ne) governs genetic diversity. Comparative population genomics is on its way to providing a solution to 'Lewontin's paradox' - the discrepancy between the many orders of magnitude of variation in population size and the much narrower distribution of diversity levels. It seems that linked selection plays an important part both in the overall genetic diversity of a species and in the variation in diversity within the genome. Genetic diversity also seems to be predictable from the life history of a species.

Influence of Recombination and GC-biased Gene Conversion on the Adaptive and Nonadaptive Substitution Rate in Mammals versus Birds.

Recombination is expected to affect functional sequence evolution in several ways. On the one hand, recombination is thought to improve the efficiency of multilocus selection by dissipating linkage disequilibrium. On the other hand, natural selection can be counteracted by recombination-associated transmission distorters such as GC-biased gene conversion (gBGC), which tends to promote G and C alleles irrespective of their fitness effect in high-recombining regions. It has been suggested that gBGC might impact coding sequence evolution in vertebrates, and particularly the ratio of nonsynonymous to synonymous substitution rates (dN/dS). However, distinctive gBGC patterns have been reported in mammals and birds, maybe reflecting the documented contrasts in evolutionary dynamics of recombination rate between these two taxa. Here, we explore how recombination and gBGC affect coding sequence evolution in mammals and birds by analyzing proteome-wide data in six species of Galloanserae (fowls) and six species of catarrhine primates. We estimated the dN/dS ratio and rates of adaptive and nonadaptive evolution in bins of genes of increasing recombination rate, separately analyzing AT → GC, GC → AT, and G ↔ C/A ↔ T mutations. We show that in both taxa, recombination and gBGC entail a decrease in dN/dS. Our analysis indicates that recombination enhances the efficiency of purifying selection by lowering Hill-Robertson effects, whereas gBGC leads to an overestimation of the adaptive rate of AT → GC mutations. Finally, we report a mutagenic effect of recombination, which is independent of gBGC.

Codon Usage Bias in Animals: Disentangling the Effects of Natural Selection, Effective Population Size, and GC-Biased Gene Conversion.

Selection on codon usage bias is well documented in a number of microorganisms. Whether codon usage is also generally shaped by natural selection in large organisms, despite their relatively small effective population size (Ne), is unclear. In animals, the population genetics of codon usage bias has only been studied in a handful of model organisms so far, and can be affected by confounding, nonadaptive processes such as GC-biased gene conversion and experimental artefacts. Using population transcriptomics data, we analyzed the relationship between codon usage, gene expression, allele frequency distribution, and recombination rate in 30 nonmodel species of animals, each from a different family, covering a wide range of effective population sizes. We disentangled the effects of translational selection and GC-biased gene conversion on codon usage by separately analyzing GC-conservative and GC-changing mutations. We report evidence for effective translational selection on codon usage in large-Ne species of animals, but not in small-Ne ones, in agreement with the nearly neutral theory of molecular evolution. C- and T-ending codons tend to be preferred over synonymous G- and A-ending ones, for reasons that remain to be determined. In contrast, we uncovered a conspicuous effect of GC-biased gene conversion, which is widespread in animals and the main force determining the fate of AT↔GC mutations. Intriguingly, the strength of its effect was uncorrelated with Ne.

Consequences of Asexuality in Natural Populations: Insights from Stick Insects.

Recombination is a fundamental process with significant impacts on genome evolution. Predicted consequences of the loss of recombination include a reduced effectiveness of selection, changes in the amount of neutral polymorphisms segregating in populations, and an arrest of GC-biased gene conversion. Although these consequences are empirically well documented for nonrecombining genome portions, it remains largely unknown if they extend to the whole genome scale in asexual organisms. We identify the consequences of asexuality using de novo transcriptomes of five independently derived, obligately asexual lineages of stick insects, and their sexual sister-species. We find strong evidence for higher rates of deleterious mutation accumulation, lower levels of segregating polymorphisms and arrested GC-biased gene conversion in asexuals as compared with sexuals. Taken together, our study conclusively shows that predicted consequences of genome evolution under asexuality can indeed be found in natural populations.

Sexually dimorphic gene expression and transcriptome evolution provide mixed evidence for a fast-Z effect in Heliconius.

Sex chromosomes have different evolutionary properties compared to autosomes due to their hemizygous nature. In particular, recessive mutations are more readily exposed to selection, which can lead to faster rates of molecular evolution. Here, we report patterns of gene expression and molecular evolution for a group of butterflies. First, we improve the completeness of the Heliconius melpomene reference annotation, a neotropical butterfly with a ZW sex determination system. Then, we analyse RNA from male and female whole abdomens and sequence female ovary and gut tissue to identify sex- and tissue-specific gene expression profiles in H. melpomene. Using these expression profiles, we compare (a) sequence divergence and polymorphism; (b) the strength of positive and negative selection; and (c) rates of adaptive evolution, for Z and autosomal genes between two species of Heliconius butterflies, H. melpomene and H. erato. We show that the rate of adaptive substitutions is higher for Z than autosomal genes, but contrary to expectation, it is also higher for male-biased than female-biased genes. Additionally, we find no significant increase in the rate of adaptive evolution or purifying selection on genes expressed in ovary tissue, a heterogametic-specific tissue. Our results contribute to a growing body of literature from other ZW systems that also provide mixed evidence for a fast-Z effect where hemizygosity influences the rate of adaptive substitutions.

Shedding Light on the Grey Zone of Speciation along a Continuum of Genomic Divergence.

Speciation results from the progressive accumulation of mutations that decrease the probability of mating between parental populations or reduce the fitness of hybrids-the so-called species barriers. The speciation genomic literature, however, is mainly a collection of case studies, each with its own approach and specificities, such that a global view of the gradual process of evolution from one to two species is currently lacking. Of primary importance is the prevalence of gene flow between diverging entities, which is central in most species concepts and has been widely discussed in recent years. Here, we explore the continuum of speciation thanks to a comparative analysis of genomic data from 61 pairs of populations/species of animals with variable levels of divergence. Gene flow between diverging gene pools is assessed under an approximate Bayesian computation (ABC) framework. We show that the intermediate "grey zone" of speciation, in which taxonomy is often controversial, spans from 0.5% to 2% of net synonymous divergence, irrespective of species life history traits or ecology. Thanks to appropriate modeling of among-locus variation in genetic drift and introgression rate, we clarify the status of the majority of ambiguous cases and uncover a number of cryptic species. Our analysis also reveals the high incidence in animals of semi-isolated species (when some but not all loci are affected by barriers to gene flow) and highlights the intrinsic difficulty, both statistical and conceptual, of delineating species in the grey zone of speciation.

Adaptive Protein Evolution in Animals and the Effective Population Size Hypothesis.

The rate at which genomes adapt to environmental changes and the prevalence of adaptive processes in molecular evolution are two controversial issues in current evolutionary genetics. Previous attempts to quantify the genome-wide rate of adaptation through amino-acid substitution have revealed a surprising diversity of patterns, with some species (e.g. Drosophila) experiencing a very high adaptive rate, while other (e.g. humans) are dominated by nearly-neutral processes. It has been suggested that this discrepancy reflects between-species differences in effective population size. Published studies, however, were mainly focused on model organisms, and relied on disparate data sets and methodologies, so that an overview of the prevalence of adaptive protein evolution in nature is currently lacking. Here we extend existing estimators of the amino-acid adaptive rate by explicitly modelling the effect of favourable mutations on non-synonymous polymorphism patterns, and we apply these methods to a newly-built, homogeneous data set of 44 non-model animal species pairs. Data analysis uncovers a major contribution of adaptive evolution to the amino-acid substitution process across all major metazoan phyla-with the notable exception of humans and primates. The proportion of adaptive amino-acid substitution is found to be positively correlated to species effective population size. This relationship, however, appears to be primarily driven by a decreased rate of nearly-neutral amino-acid substitution because of more efficient purifying selection in large populations. Our results reveal that adaptive processes dominate the evolution of proteins in most animal species, but do not corroborate the hypothesis that adaptive substitutions accumulate at a faster rate in large populations. Implications regarding the factors influencing the rate of adaptive evolution and positive selection detection in humans vs. other organisms are discussed.

Large Variation in the Ratio of Mitochondrial to Nuclear Mutation Rate across Animals: Implications for Genetic Diversity and the Use of Mitochondrial DNA as a Molecular Marker.

It is commonly assumed that mitochondrial DNA (mtDNA) evolves at a faster rate than nuclear DNA (nuDNA) in animals. This has contributed to the popularity of mtDNA as a molecular marker in evolutionary studies. Analyzing 121 multilocus data sets and four phylogenomic data sets encompassing 4,676 species of animals, we demonstrate that the ratio of mitochondrial over nuclear mutation rate is highly variable among animal taxa. In nonvertebrates, such as insects and arachnids, the ratio of mtDNA over nuDNA mutation rate varies between 2 and 6, whereas it is above 20, on average, in vertebrates such as scaled reptiles and birds. Interestingly, this variation is sufficient to explain the previous report of a similar level of mitochondrial polymorphism, on average, between vertebrates and nonvertebrates, which was originally interpreted as reflecting the effect of pervasive positive selection. Our analysis rather indicates that the among-phyla homogeneity in within-species mtDNA diversity is due to a negative correlation between mtDNA per-generation mutation rate and effective population size, irrespective of the action of natural selection. Finally, we explore the variation in the absolute per-year mutation rate of both mtDNA and nuDNA using a reduced data set for which fossil calibration is available, and discuss the potential determinants of mutation rate variation across genomes and taxa. This study has important implications regarding DNA-based identification methods in predicting that mtDNA barcoding should be less reliable in nonvertebrates than in vertebrates.

Avian Genomes Revisited: Hidden Genes Uncovered and the Rates versus Traits Paradox in Birds.

According to current assemblies, avian genomes differ from those of the other lineages of amniotes in 1) containing a lower number of genes; 2) displaying a high stability of karyotype and recombination map; and 3) lacking any correlation between evolutionary rates (dN/dS) and life-history traits, unlike mammals and nonavian reptiles. We question the reality of the bird missing genes and investigate whether insufficient representation of bird gene content might have biased previous evolutionary analyses. Mining RNAseq data, we show that the vast majority of the genes missing from avian genome assemblies are actually present in most species of birds. These mainly correspond to the GC-rich fraction of the bird genome, which is the most difficult to sequence, assemble and annotate. With the inclusion of these genes in a phylogenomic analysis of high-quality alignments, we uncover a positive and significant correlation between the ratio of nonsynonymous to synonymous substitution rate (dN/dS) and life-history traits in Neoaves. We report a strong effect of GC-biased gene conversion on the dN/dS ratio in birds and a peculiar behavior of Palaeognathae (ostrich and allies) and Galloanserae (chickens, ducks and allies). Avian genomes do not contain fewer genes than mammals or nonavian reptiles. Previous analyses have overlooked ∼15% of the bird gene complement. GC-rich regions, which are the most difficult to access, are a key component of amniote genomes. They experience peculiar molecular processes and must be included for unbiased functional and comparative genomic analyses in birds.

Quantification of GC-biased gene conversion in the human genome.

Much evidence indicates that GC-biased gene conversion (gBGC) has a major impact on the evolution of mammalian genomes. However, a detailed quantification of the process is still lacking. The strength of gBGC can be measured from the analysis of derived allele frequency spectra (DAF), but this approach is sensitive to a number of confounding factors. In particular, we show by simulations that the inference is pervasively affected by polymorphism polarization errors and by spatial heterogeneity in gBGC strength. We propose a new general method to quantify gBGC from DAF spectra, incorporating polarization errors, taking spatial heterogeneity into account, and jointly estimating mutation bias. Applying it to human polymorphism data from the 1000 Genomes Project, we show that the strength of gBGC does not differ between hypermutable CpG sites and non-CpG sites, suggesting that in humans gBGC is not caused by the base-excision repair machinery. Genome-wide, the intensity of gBGC is in the nearly neutral area. However, given that recombination occurs primarily within recombination hotspots, 1%-2% of the human genome is subject to strong gBGC. On average, gBGC is stronger in African than in non-African populations, reflecting differences in effective population sizes. However, due to more heterogeneous recombination landscapes, the fraction of the genome affected by strong gBGC is larger in non-African than in African populations. Given that the location of recombination hotspots evolves very rapidly, our analysis predicts that, in the long term, a large fraction of the genome is affected by short episodes of strong gBGC.

Incomplete Lineage Sorting in Mammalian Phylogenomics.

The impact of incomplete lineage sorting (ILS) on phylogenetic conflicts among genes, and the related issue of whether to account for ILS in species tree reconstruction, are matters of intense controversy. Here, focusing on full-genome data in placental mammals, we empirically test two assumptions underlying current usage of tree-building methods that account for ILS. We show that in this data set (i) distinct exons from a common gene do not share a common genealogy, and (ii) ILS is only a minor determinant of the existing phylogenetic conflict. These results shed new light on the relevance and conditions of applicability of ILS-aware methods in phylogenomic analyses of protein coding sequences.

Overestimation of the adaptive substitution rate in fluctuating populations.

Estimating the proportion of adaptive substitutions (α) is of primary importance to uncover the determinants of adaptation in comparative genomic studies. Several methods have been proposed to estimate α from patterns polymorphism and divergence in coding sequences. However, estimators of α can be biased when the underlying assumptions are not met. Here we focus on a potential source of bias, i.e. variation through time in the long-term population size (N) of the considered species. We show via simulations that ancient demographic fluctuations can generate severe overestimations of α, and this is irrespective of the recent population history.

Patterns of cross-contamination in a multispecies population genomic project: detection, quantification, impact, and solutions.

BACKGROUND: Contamination is a well-known but often neglected problem in molecular biology. Here, we investigated the prevalence of cross-contamination among 446 samples from 116 distinct species of animals, which were processed in the same laboratory and subjected to subcontracted transcriptome sequencing. RESULTS: Using cytochrome oxidase 1 as a barcode, we identified a minimum of 782 events of between-species contamination, with approximately 80% of our samples being affected. An analysis of laboratory metadata revealed a strong effect of the sequencing center: nearly all the detected events of between-species contamination involved species that were sent the same day to the same company. We introduce new methods to address the amount of within-species, between-individual contamination, and to correct for this problem when calling genotypes from base read counts. CONCLUSIONS: We report evidence for pervasive within-species contamination in this data set, and show that classical population genomic statistics, such as synonymous diversity, the ratio of non-synonymous to synonymous diversity, inbreeding coefficient FIT, and Tajima's D, are sensitive to this problem to various extents. Control analyses suggest that our published results are probably robust to the problem of contamination. Recommendations on how to prevent or avoid contamination in large-scale population genomics/molecular ecology are provided based on this analysis.

Life History Traits, Protein Evolution, and the Nearly Neutral Theory in Amniotes.

The nearly neutral theory of molecular evolution predicts that small populations should accumulate deleterious mutations at a faster rate than large populations. The analysis of nonsynonymous (dN) versus synonymous (dS) substitution rates in birds versus mammals, however, has provided contradictory results, questioning the generality of the nearly neutral theory. Here we analyzed the impact of life history traits, taken as proxies of the effective population size, on molecular evolutionary and population genetic processes in amniotes, including the so far neglected reptiles. We report a strong effect of species body mass, longevity, and age of sexual maturity on genome-wide patterns of polymorphism and divergence across the major groups of amniotes, in agreement with the nearly neutral theory. Our results indicate that the rate of protein evolution in amniotes is determined in the first place by the efficiency of purifying selection against deleterious mutations-and this is true of both radical and conservative amino acid changes. Interestingly, the among-species distribution of dN/dS in birds did not follow this general trend: dN/dS was not higher in large, long-lived than in small, short-lived species of birds. We show that this unexpected pattern is not due to a more narrow range of life history traits, a lack of correlation between traits and Ne, or a peculiar distribution of fitness effects of mutations in birds. Our analysis therefore highlights the bird dN/dS ratio as a molecular evolutionary paradox and a challenge for future research.