The Population Genetics of dN/dS

Evolutionary pressures on proteins are often quantified by the ratio of substitution rates at non-synonymous and synonymous sites. The dN/dS ratio was originally developed for application to distantly diverged sequences, the differences among which represent substitutions that have fixed along independent lineages. Nevertheless, the dN/dS measure is often applied to sequences sampled from a single population, the differences among which represent segregating polymorphisms. Here, we study the expected dN/dS ratio for samples drawn from a single population under selection, and we find that in this context, dN/dS is relatively insensitive to the selection coefficient. Moreover, the hallmark signature of positive selection over divergent lineages, dN/dS>1, is violated within a population. For population samples, the relationship between selection and dN/dS does not follow a monotonic function, and so it may be impossible to infer selection pressures from dN/dS. These results have significant implications for the interpretation of dN/dS measurements among population-genetic samples.     

Comparisons of dN/dS are time dependent for closely related bacterial genomes

The ratio of non-synonymous (dN) to synonymous (dS) changes between taxa is frequently computed to assay the strength and direction of selection. Here we note that for comparisons between closely related strains and/or species a second parameter needs to be considered, namely the time since divergence of the two sequences under scrutiny. We demonstrate that a simple time lag model provides a general, parsimonious explanation of the extensive variation in the dN/dS ratio seen when comparing closely related bacterial genomes. We explore this model through simulation and comparative genomics, and suggest a role for hitch-hiking in the accumulation of non-synonymous mutations. We also note taxon-specific differences in the change of dN/dS over time, which may indicate variation in selection, or in population genetics parameters such as population size or the rate of recombination. The effect of comparing intra-species polymorphism and inter-species substitution, and the problems associated with these concepts for asexual prokaryotes, are also discussed. We conclude that, because of the critical effect of time since divergence, inter-taxa comparisons are only possible by comparing trajectories of dN/dS over time and it is not valid to compare taxa on the basis of single time points.     

Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution

An excess of nonsynonymous substitutions over synonymous ones is an important indicator of positive selection at the molecular level. A lineage that underwent Darwinian selection may have a nonsynonymous/synonymous rate ratio (dN/dS) that is different from those of other lineages or greater than one. In this paper, several codon-based likelihood models that allow for variable dN/dS ratios among lineages were developed. They were then used to construct likelihood ratio tests to examine whether the dN/dS ratio is variable among evolutionary lineages, whether the ratio for a few lineages of interest is different from the background ratio for other lineages in the phylogeny, and whether the dN/dS ratio for the lineages of interest is greater than one. The tests were applied to the lysozyme genes of 24 primate species. The dN/dS ratios were found to differ significantly among lineages, indicating that the evolution of primate lysozymes is episodic, which is incompatible with the neutral theory. Maximum- likelihood estimates of parameters suggested that about nine nonsynonymous and zero synonymous nucleotide substitutions occurred in the lineage leading to hominoids, and the dN/dS ratio for that lineage is significantly greater than one. The corresponding estimates for the lineage ancestral to colobine monkeys were nine and one, and the dN/dS ratio for the lineage is not significantly greater than one, although it is significantly higher than the background ratio. The likelihood analysis thus confirmed most, but not all, conclusions Messier and Stewart reached using reconstructed ancestral sequences to estimate synonymous and nonsynonymous rates for different lineages.      

Estimating diversifying selection and functional constraint in the presence of recombination

Models of molecular evolution that incorporate the ratio of nonsynonymous to synonymous polymorphism (dN/dS ratio) as a parameter can be used to identify sites that are under diversifying selection or functional constraint in a sample of gene sequences. However, when there has been recombination in the evolutionary history of the sequences, reconstructing a single phylogenetic tree is not appropriate, and inference based on a single tree can give misleading results. In the presence of high levels of recombination, the identification of sites experiencing diversifying selection can suffer from a false-positive rate as high as 90%. We present a model that uses a population genetics approximation to the coalescent with recombination and use reversible-jump MCMC to perform Bayesian inference on both the dN/dS ratio and the recombination rate, allowing each to vary along the sequence. We demonstrate that the method has the power to detect variation in the dN/dS ratio and the recombination rate and does not suffer from a high false-positive rate. We use the method to analyze the porB gene of Neisseria meningitidis and verify the inferences using prior sensitivity analysis and model criticism techniques.     

Dolphin genome provides evidence for adaptive evolution of nervous system genes and a molecular rate slowdown

Cetaceans (dolphins and whales) have undergone a radical transformation from the original mammalian bodyplan. In addition, some cetaceans have evolved large brains and complex cognitive capacities. We compared approximately 10 000 protein-coding genes culled from the bottlenose dolphin genome with nine other genomes to reveal molecular correlates of the remarkable phenotypic features of these aquatic mammals. Evolutionary analyses demonstrated that the overall synonymous substitution rate in dolphins has slowed compared with other studied mammals, and is within the range of primates and elephants. We also discovered 228 genes potentially under positive selection (dN/dS > 1) in the dolphin lineage. Twenty-seven of these genes are associated with the nervous system, including those related to human intellectual disabilities, synaptic plasticity and sleep. In addition, genes expressed in the mitochondrion have a significantly higher mean dN/dS ratio in the dolphin lineage than others examined, indicating evolution in energy metabolism. We encountered selection in other genes potentially related to cetacean adaptations such as glucose and lipid metabolism, dermal and lung development, and the cardiovascular system. This study underlines the parallel molecular trajectory of cetaceans with other mammalian groups possessing large brains.     

Lineage‐specific sequence evolution and exon edge conservation partially explain the relationship between evolutionary rate and expression level in A. thaliana

Rapidly evolving proteins can aid the identification of genes underlying phenotypic adaptation across taxa, but functional and structural elements of genes can also affect evolutionary rates. In plants, the ‘edges’ of exons, flanking intron junctions, are known to contain splice enhancers and to have a higher degree of conservation compared to the remainder of the coding region. However, the extent to which these regions may be masking indicators of positive selection or account for the relationship between dN/dS and other genomic parameters is unclear. We investigate the effects of exon edge conservation on the relationship of dN/dS to various sequence characteristics and gene expression parameters in the model plant Arabidopsis thaliana. We also obtain lineage‐specific dN/dS estimates, making use of the recently sequenced genome of Thellungiella parvula, the second closest sequenced relative after the sister species Arabidopsis lyrata. Overall, we find that the effect of exon edge conservation, as well as the use of lineage‐specific substitution estimates, upon dN/dS ratios partly explains the relationship between the rates of protein evolution and expression level. Furthermore, the removal of exon edges shifts dN/dS estimates upwards, increasing the proportion of genes potentially under adaptive selection. We conclude that lineage‐specific substitutions and exon edge conservation have an important effect on dN/dS ratios and should be considered when assessing their relationship with other genomic parameters.     

Patterns of evolutionary selection pressure in the immune signaling protein TRAF3IP2 in mammals

TRAF3 interacting protein 2 (TRAF3IP2) is important for immune responses to pathogens, inflammatory signals and autoimmunity in mammals. In the present study, we collected 19 mammalian TRAF3IP2 sequences and investigated the various types of selection pressure acting on them. Maximum likelihood estimations of nonsynonymous (dN) to synonymous (dS) substitution (dN/dS) ratios for the aligned coding sequences indicated that, as a whole, TRAF3IP2 has been subject to purifying selection. However, the N-terminus of the protein has been subject to higher selection pressure than the C-terminal domain. While eight amino acid residues within the N-terminus appear to have evolved under positive selection, no evidence for such selection was found in the C-terminus. The positively selected residues, which fall outside the currently known functional sites within TRAF3IP2, may have novel functions. The different selection pressures acting on the N- and C-terminal regions are consistent with their protein structures: the C-terminal structure is an ordered structure, whereas the N-terminus is disordered. Taken together with the results of previous studies, it is plausible that positive selection on the N-terminus of TRAF3IP2 may have occurred by competitive coevolution between mammalian hosts and viruses.     

Identification of fast-evolving genes in the scleractinian coral Acropora using comparative EST analysis

To identify fast-evolving genes in reef-building corals, we performed direct comparative sequence analysis with expressed sequence tag (EST) datasets from two acroporid species: Acropora palmata from the Caribbean Sea and A. millepora from the Great Barrier Reef in Australia. Comparison of 589 independent sequences from 1,421 A. palmata contigs, with 10,247 A. millepora contigs resulted in the identification of 196 putative homologues. Most of the homologous pairs demonstrated high amino acid similarities (over 90%). Comparisons of putative homologues showing low amino acid similarities (under 90%) among the Acropora species to the near complete datasets from two other cnidarians (Hydra magnipapillata and Nematostella vectensis) implied that some were non-orthologous. Within 86 homologous pairs, 39 exhibited dN/dS ratios significantly less than 1, suggesting that these genes are under purifying selection associated with functional constraints. Eight independent genes showed dN/dS ratios exceeding 1, while three deviated significantly from 1, suggesting that these genes may play important roles in the adaptive evolution of Acropora. Our results also indicated that CEL-III lectin was under positive selection, consistent with a possible role in immunity or symbiont recognition. Further studies are needed to clarify the possible functions of the genes under positive selection to provide insight into the evolutionary process of corals.     

A Sliding Window-Based Method to Detect Selective Constraints in Protein-Coding Genes and Its Application to RNA Viruses

Here we present a new sliding window-based method specially designed to detect selective constraints in specific regions of a multiple protein-coding sequence alignment. In contrast to previous window-based procedures, our method is based on a nonarbitrary statistical approach to find the appropriate codon-window size to test deviations of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions from the expectation. The probabilities of dN and dS are obtained from simulated data and used to detect significant deviations of dN and dS in a specific window region of the real sequence alignment. The nonsynonymous-to-synonymous rate ratio (w = dN/dS) was used to highlight selective constraints in any window wherein dS or dN was significantly different from the expectation. In these significant windows, w and its variance [V(w)] were calculated and used to test the neutral hypothesis. Computer simulations showed that the method is accurate even for highly divergent sequences. The main advantages of the new method are that it (i) uses a statistically appropriate window size to detect different selective patterns, (ii) is computationally less intensive than maximum likelihood methods, and (iii) detects saturation of synonymous sites, which can give deviations from neutrality. Hence, it allows the analysis of highly divergent sequences and the test of different alternative hypothesis as well. The application of the method to different human immunodeficiency virus type 1 and to foot-and-mouth disease virus genes confirms the action of positive selection on previously described regions as well as on new regions.     

Differentiation of Xylella fastidiosa strains via multilocus sequence analysis of environmentally mediated genes (MLSA-E)

Isolates of the plant pathogen Xylella fastidiosa are genetically very similar, but studies on their biological traits have indicated differences in virulence and infection symptomatology. Taxonomic analyses have identified several subspecies, and phylogenetic analyses of housekeeping genes have shown broad host-based genetic differences; however, results are still inconclusive for genetic differentiation of isolates within subspecies. This study employs multilocus sequence analysis of environmentally mediated genes (MLSA-E; genes influenced by environmental factors) to investigate X. fastidiosa relationships and differentiate isolates with low genetic variability. Potential environmentally mediated genes, including host colonization and survival genes related to infection establishment, were identified a priori. The ratio of the rate of nonsynonymous substitutions to the rate of synonymous substitutions (dN/dS) was calculated to select genes that may be under increased positive selection compared to previously studied housekeeping genes. Nine genes were sequenced from 54 X. fastidiosa isolates infecting different host plants across the United States. Results of maximum likelihood (ML) and Bayesian phylogenetic (BP) analyses are in agreement with known X. fastidiosa subspecies clades but show novel within-subspecies differentiation, including geographic differentiation, and provide additional information regarding host-based isolate variation and specificity. dN/dS ratios of environmentally mediated genes, though <1 due to high sequence similarity, are significantly greater than housekeeping gene dN/dS ratios and correlate with increased sequence variability. MLSA-E can more precisely resolve relationships between closely related bacterial strains with low genetic variability, such as X. fastidiosa isolates. Discovering the genetic relationships between X. fastidiosa isolates will provide new insights into the epidemiology of populations of X. fastidiosa, allowing improved disease management in economically important crops.     

Two mitochondrial genes under episodic positive selection in subterranean octodontoid rodents

Tuco-tucos (Ctenomys) and related coruros (Spalacopus) are South American subterranean rodents. An energetically demanding lifestyle within the hypoxic, underground atmosphere may change the selective regime on oxidative phosphorylation. We examined whether weak and/or episodic positive directional selection affected the evolution of two mitochondrial genes (COX2, CytB), in a background of purifying selection in these lineages. We estimated rates of synonymous (dS) and non-synonymous (dN) substitutions and found: 1) significantly higher dN/dS ratio in subterranean groups relative to non-subterranean related species, and 2) two codons in each gene under episodic selection: 94 and 277 of COX2 and 269 and 307 of CytB.     

Evolution of duplicate genes in a tetraploid animal, Xenopus laevis

To understand the evolution of duplicate genes, we compared rates of nucleotide substitution between 17 pairs of nonallelic duplicated genes in the tetraploid frog Xenopus laevis with rates between the orthologous loci of human and rodent. For all duplicated X. laevis genes, the number of synonymous substitutions per site (dS) was greater than the number of nonsynonymous substitutions per site (dN), indicating that these genes are subject to purifying selection. There was also a significant positive correlation (r = 0.915) between dN for the X. laevis genes and dN for the mammalian genes, suggesting that, at the amino acid level, the X. laevis genes and the mammalian genes are under similar constraints. Results of relative-rate tests showed nearly equal rates of nonsynonymous substitution in each copy of the X. laevis genes; apparently there are similar constraints on both copies. No correlation was found between dS for the X. laevis genes and dS for the mammalian genes. There was a significant positive correlation both between members of pairs of duplicated X. laevis genes (r = 0.951) and between human and rodent orthologues (r = 0.854) with respect to third- position G+C content but no such relationship between the X. laevis genes and either of their mammalian orthologues. The results indicate that both copies of a duplicate gene can be subject to purifying selection and thus support the hypothesis of selection against all genotypes containing a null allele at either of two duplicate loci.      

Individual genome assembly from complex community short-read metagenomic datasets

Assembling individual genomes from complex community metagenomic data remains a challenging issue for environmental studies. We evaluated the quality of genome assemblies from community short read data (Illumina 100 bp pair-ended sequences) using datasets recovered from freshwater and soil microbial communities as well as in silico simulations. Our analyses revealed that the genome of a single genotype (or species) can be accurately assembled from a complex metagenome when it shows at least about 20 × coverage. At lower coverage, however, the derived assemblies contained a substantial fraction of non-target sequences (chimeras), which explains, at least in part, the higher number of hypothetical genes recovered in metagenomic relative to genomic projects. We also provide examples of how to detect intrapopulation structure in metagenomic datasets and estimate the type and frequency of errors in assembled genes and contigs from datasets of varied species complexity.     

Selection on an antimicrobial peptide defensin in ants

Ants live in crowded nests with interacting individuals, which makes them particularly prone to infectious diseases. The question is, how do ants cope with the increased risk of pathogen transmission due to sociality? We have studied the molecular evolution of defensin, a gene encoding an antimicrobial protein, in ants. Defensin sequences from several ant species were analyzed with maximum likelihood models of codon substitution to infer selection. Positive selection was detected in the mature region of defensin, whereas the signal and pro regions seem to be evolving neutrally. We also found a significantly higher rate of nonsynonymous substitutions in some phylogenetic lineages, as well as dN/dS >1, suggesting varying selection pressures in different lineages. Earlier studies on the molecular evolution of insect antimicrobial peptide genes have focused on termites and dipteran species, and detected positive selection only in duplicated termicin genes in termites. These findings, together with our present results, provide an indication that the immune systems of social insects (ants and termites) and dipteran insects may have responded differently to the selection pressure caused by microbial pathogens.     

Gene-specific signatures of elevated non-synonymous substitution rates correlate poorly across the Plasmodium genus

Background

Comparative genome analyses of parasites allow large scale investigation of selective pressures shaping their evolution. An acute limitation to such analysis of Plasmodium falciparum is that there is only very partial low-coverage genome sequence of the most closely related species, the chimpanzee parasite P. reichenowi. However, if orthologous genes have been under similar selective pressures throughout the Plasmodium genus then positive selection on the P. falciparum lineage might be predicted to some extent by analysis of other lineages.

Principal Findings

Here, three independent pairs of closely related species in different sub-generic clades (P. falciparum and P. reichenowi; P. vivax and P. knowlesi; P. yoelii and P. berghei) were compared for a set of 43 candidate ligand genes considered likely to be under positive directional selection and a set of 102 control genes for which there was no selective hypothesis. The ratios of non-synonymous to synonymous substitutions (dN/dS) were significantly elevated in the candidate ligand genes compared to control genes in each of the three clades. However, the rank order correlation of dN/dS ratios for individual candidate genes was very low, less than the correlation for the control genes.

Significance

The inability to predict positive selection on a gene in one lineage by identifying elevated dN/dS ratios in the orthologue within another lineage needs to be noted, as it reflects that adaptive mutations are generally rare events that lead to fixation in individual lineages. Thus it is essential to complete the genome sequences of particular species of phylogenetic importance, such as P. reichenowi.     

Analysis of insertion sequences in thermophilic cyanobacteria: exploring the mechanisms of establishing, maintaining, and withstanding high insertion sequence abundance

Insertion sequences (ISs) are simple mobile genetic elements capable of relocating within a genome. Through this transposition activity, they are known to create mutations which are mostly deleterious to the cell, although occasionally they are beneficial. Two closely related isolates of thermophilic Synechococcus species from hot spring microbial mats are known to harbor a large number of diverse ISs. To explore the mechanism of IS acquisition within natural populations and survival in the face of high IS abundance, we examined IS content and location in natural populations of Synechococcus by comparing metagenomic data to the genomes of fully sequenced cultured isolates. The observed IS distribution in the metagenome was equivalent to the distribution in the isolates, indicating that the cultured isolates are appropriate models for the environmental population. High sequence conservation between IS families shared between the two isolates suggests that ISs are able to move between individuals within populations and between species via lateral gene transfer, consistent with models for IS family accumulation. Most IS families show evidence of recent activity, and interruption of critical genes in some individuals was observed, demonstrating that transposition is an ongoing mutational force in the populations.     

Molecular Evolution of the Genomic RNA of Apple Stem Grooving Capillovirus

The complete genome of the German isolate AC of Apple stem grooving virus (ASGV) was sequenced. It encodes two overlapping open reading frames (ORFs), similarly to previously described ASGV isolates. Two regions of high variability were detected between the ASGV isolates, variable region 1 (V1, from amino acids (aa) 532 to 570), and variable region 2 (V2, from aa 1,583 to 1,868). The phylogenetic analysis of the V1 and V2 regions suggested that the ASGV diversity was structured by host plant species rather than geographical origin. The dN/dS ratio between nonsynonymous and synonymous nucleotide substitution rates varied greatly along the ASGV genome. Most of ORF1 showed predominant negative selection except for the two regions V1 and V2. V1 showed an elevated dN and an average dS when compared to the ORF1 background but no significant positive selection was detected. The V2 region of ORF1 showed an elevated dN and a low dS when compared to the ORF1 background with an average dN/dS????3.0 indicative of positive selection. However, the V2 area includes overlapping ORFs, making the dN/dS estimate biased. Joint estimates of the selection intensity in the different ORFs by a recent method indicated that this region of ORF1 was in fact evolving close to neutrality. This was convergent with previous results showing that introduction of stop codons in this region of ORF1 did not impair plant infection. These data suggest that the elimination of a stop codon caused the overprinting of a novel coding region over the ancestral ORF.     

Positive selection during the diversification of class I vomeronasal receptor-like (V1RL) genes,putative pheromone receptor genes,in human and primate evolution

Vomeronasal receptors are the major receptors for pheromones in vertebrates, and five putative type 1 vomeronasal receptors (V1RL) have been identified in humans. The evolution of the V1RL1 gene in non-human primates, and patterns of selection on V1RL genes, were investigated. The presumed ortholog of V1RL1 was sequenced from 13 species of nonhuman primate, and in eight of these species V1RL1 was a pseudogene. Phylogenetic reconstructions reveal that V1RL1 pseudogene formation occurred independently in multiple primate lineages. Using maximum likelihood estimates of dN/dS ratios in PAML, we show that V1RL genes have evolved under neutral evolution in lineages in which they became a pseudogene. In contrast, among lineages in which V1RL genes contain an open reading frame, the majority of sites are under purifying selection and a minority are under significant positive selection. These results provide an interesting case where all three categories of selection can be teased apart in the same data set using maximum likelihood methods. The finding of positive selection on V1RL genes during primate evolution provides indirect support for the hypothesis that V1RL genes have a function in species-specific pheromone detection in primates.