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.      

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.     

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.     

Molecular evolution of PISTILLATA-like genes in the dogwood genus Cornus (Cornaceae)

The MADS-box gene family encodes critical regulators determining floral organ development. Understanding evolutionary patterns and processes of MADS-box genes is an important step toward unraveling the molecular basis of floral morphological evolution. In this study, we investigated the evolution of PI-like genes of the MADS-box family in the dogwood genus Cornus (Cornaceae). Cornus is a eudicot lineage in the asterids clade, and is intriguing in evolving petaloid bract morphology in two major lineages within the genus. The gene genealogy reconstructed using genomic DNA and cDNA sequences suggests multiple PI-like gene duplication events in Cornus. An ancient duplication event resulted in two ancient paralogs, CorPI-A and CorPI-B, which have highly diverged intron regions. Duplication of CorPI-A further resulted in two paralogs in one subgroup of Cornus, the BW group that does not produce modified bracts. Most species analyzed were found to contain more than one copy of the PI-like gene with most copies derived recently within species. Estimation and comparison of dN/dS ratios revealed relaxed selection in the PI-like gene in Cornus in comparison with the gene in the closely related outgroups Alangium and Davidia, and in other flowering plants. Selection also differed among major gene copies, CorPI-A and CorPI-B, and among different morphological subgroups of Cornus. Variation in selection pressures may indicate functional changes in PI-like genes after gene duplication and among different lineages. Strong positive selection at three amino acid sites of CorPI was also detected from a region critical for dimerization activity. Total substitution rates of the CorPI gene also differ among lineages of Cornus, showing a trend similar to that found in dN/dS ratios. We also found that the CorPI-A copy contains informative phylogenetic information when compared across species of Cornus.     

Phylogenetic analysis suggests that sociality is associated with reduced effectiveness of selection

The evolution of sociality in spiders is associated with female bias, reproductive skew and an inbreeding mating system, factors that cause a reduction in effective population size and increase effects of genetic drift. These factors act to decrease the effectiveness of selection, thereby increasing the fixation probability of deleterious mutations. Comparative studies of closely related species with contrasting social traits and mating systems provide the opportunity to test consequences of low effective population size on the effectiveness of selection empirically. We used phylogenetic analyses of three inbred social spider species and seven outcrossing subsocial species of the genus Stegodyphus, and compared dN/dS ratios and codon usage bias between social Inbreeding and subsocial outcrossing mating systems to assess the effectiveness of selection. The overall results do not differ significantly between the social inbreeding and outcrossing species, but suggest a tendency for lower codon usage bias and higher dN/dS ratios in the social inbreeding species compared with their outcrossing congeners. The differences in dN/dS ratio and codon usage bias between social and subsocial species are modest but consistent with theoretical expectations of reduced effectiveness of selection in species with relatively low effective population size. The modest differences are consistent with relatively recent evolution of social mating systems. Additionally, the short terminal branches and lack of speciation of the social lineages, together with low genetic diversity lend support for the transient state of permanent sociality in spiders.     

Maximum likelihood estimation of ancestral codon usage bias parameters in Drosophila

We present a likelihood method for estimating codon usage bias parameters along the lineages of a phylogeny. The method is an extension of the classical codon-based models used for estimating dN/dS ratios along the lineages of a phylogeny. However, we add one extra parameter for each lineage: the selection coefficient for optimal codon usage (S), allowing joint maximum likelihood estimation of S and the dN/dS ratio. We apply the method to previously published data from Drosophila melanogaster, Drosophila simulans, and Drosophila yakuba and show, in accordance with previous results, that the D. melanogaster lineage has experienced a reduction in the selection for optimal codon usage. However, the D. melanogaster lineage has also experienced a change in the biological mutation rates relative to D. simulans, in particular, a relative reduction in the mutation rate from A to G and an increase in the mutation rate from C to T. However, neither a reduction in the strength of selection nor a change in the mutational pattern can alone explain all of the data observed in the D. melanogaster lineage. For example, we also confirm previous results showing that the Notch locus has experienced positive selection for previously classified unpreferred mutations.     

Adaptive gamete-recognition divergence in a hybridizing Mytilus population

Gamete-recognition proteins often evolve rapidly, but it is not known if their divergence occurs within species and corresponds with the evolution of reproductive isolation, or if divergence typically accumulates between already isolated lineages. We examined the evolution of a candidate gamete-recognition protein in several sympatric and allopatric populations of Mytilus blue mussels, species that hybridize in nature. Within a single species, Mytilus galloprovincialis, we found adaptive divergence of Lysin-M7, a sperm acrosomal protein that dissolves the egg vitelline envelope during fertilization. Mytilus galloprovincialis Lysin-M7 alleles group into two distinct clades (termed G and G(D)), and individual alleles in these clades are separated from each other by at least three and up to eleven amino-acid substitutions. Maximum-likelihood estimates of selective pressure (dN/dS =omega) implicate selection in the divergence between M. galloprovincialis Lysin-M7 clades, and within the G(D) clade. Exact tests of population differentiation indicate that the relative frequency of G and G(D) Lysin-M7 alleles differs significantly among M. galloprovincialis populations. Compared with allopatric Mediterranean samples, Lysin-M7 alleles in the G(D) clade are found at elevated frequency in samples from the East Atlantic and California, areas of secondary contact and hybridization between Mytilus species, and Australia, an area of unknown species composition. Adaptive divergence between the alleles most common in allopatry and those found at elevated frequency in samples from sympatry suggests that selection pressures acting in hybridizing populations, likely following Pleistocene secondary contact with M. edulis in the East Atlantic, drove the divergence of Lysin-M7 in M. galloprovincialis.     

Selection in coastal Synechococcus (cyanobacteria) populations evaluated from environmental metagenomes

Environmental metagenomics provides snippets of genomic sequences from all organisms in an environmental sample and are an unprecedented resource of information for investigating microbial population genetics. Current analytical methods, however, are poorly equipped to handle metagenomic data, particularly of short, unlinked sequences. A custom analytical pipeline was developed to calculate dN/dS ratios, a common metric to evaluate the role of selection in the evolution of a gene, from environmental metagenomes sequenced using 454 technology of flow-sorted populations of marine Synechococcus, the dominant cyanobacteria in coastal environments. The large majority of genes (98%) have evolved under purifying selection (dN/dS<1). The metagenome sequence coverage of the reference genomes was not uniform and genes that were highly represented in the environment (i.e. high read coverage) tended to be more evolutionarily conserved. Of the genes that may have evolved under positive selection (dN/dS>1), 77 out of 83 (93%) were hypothetical. Notable among annotated genes, ribosomal protein L35 appears to be under positive selection in one Synechococcus population. Other annotated genes, in particular a possible porin, a large-conductance mechanosensitive channel, an ATP binding component of an ABC transporter, and a homologue of a pilus retraction protein had regions of the gene with elevated dN/dS. With the increasing use of next-generation sequencing in metagenomic investigations of microbial diversity and ecology, analytical methods need to accommodate the peculiarities of these data streams. By developing a means to analyze population diversity data from these environmental metagenomes, we have provided the first insight into the role of selection in the evolution of Synechococcus, a globally significant primary producer.     

Uncorrected nucleotide bias in mtDNA can mimic the effects of positive Darwinian selection

The relative rates of nucleotide substitution at synonymous and nonsynonymous sites within protein-coding regions have been widely used to infer the action of natural selection from comparative sequence data. It is known, however, that mutational and repair biases can affect rates of evolution at both synonymous and nonsynonymous sites. More importantly, it is also known that synonymous sites are particularly prone to the effects of nucleotide bias. This means that nucleotide biases may affect the calculated ratio of substitution rates at synonymous and nonsynonymous sites. Using a large data set of animal mitochondrial sequences, we demonstrate that this is, in fact, the case. Highly biased nucleotide sequences are characterized by significantly elevated dN/dS ratios, but only when the nucleotide frequencies are not taken into account. When the analysis is repeated taking the nucleotide frequencies at each codon position into account, such elevated ratios disappear. These results suggest that the recently reported differences in dN/dS ratios between vertebrate and invertebrate mitochondrial sequences could be explained by variations in mitochondrial nucleotide frequencies rather than the effects of positive Darwinian selection.     

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.     

Molecular evolution of hepatitis B virus over 25 years

Determining the longitudinal molecular evolution of hepatitis B virus (HBV) is difficult due to HBV's genomic complexity and the need to study paired samples collected over long periods of time. In this study, serial samples were collected from eight hepatitis B virus e antigen-negative asymptomatic carriers of HBV genotype B in 1979 and 2004, thus providing a 25-year period to document the long-term molecular evolution of HBV. The rate, nature, and distribution of mutations that emerged over 25 years were determined by phylogenetic and linear regression analysis of full-length HBV genome sequences. Nucleotide hypervariability was observed within the polymerase and pre-S/S overlap region and within the core gene. The calculated mean number of nucleotide substitutions/site/year (7.9 x 10(-5)) was slightly higher than published estimates (1.5 x 10(-5) to 5 x 10(-5)). Nucleotide changes in the quasispecies population did not significantly alter the molecular evolutionary rate, based on linear regression analysis of evolutionary distances among serial clone pre-S region sequences. Therefore, the directly amplified or dominant sequence was sufficient to estimate the putative molecular evolutionary rate for these long-term serial samples. On average, the ratio of synonymous (dS) to nonsynonymous (dN) substitutions was highest for the polymerase-coding region and lowest for the core-coding region. The low dS/dN ratios observed within the core suggest that selection occurs within this gene region, possibly as an immune evasion strategy. The results of this study suggest that HBV sequence divergence may occur more rapidly than previously estimated, in a host immune phase-dependent manner.     

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.     

A genetic algorithm approach to detecting lineage-specific variation in selection pressure

The ratio of nonsynonymous (dN) to synonymous (dS) substitution rates, omega, provides a measure of selection at the protein level. Models have been developed that allow omega to vary among lineages. However, these models require the lineages in which differential selection has acted to be specified a priori. We propose a genetic algorithm approach to assign lineages in a phylogeny to a fixed number of different classes of omega, thus allowing variable selection pressure without a priori specification of particular lineages. This approach can identify models with a better fit than a single-ratio model, and with fits that are better than (in an information theoretic sense) a fully local model, in which all lineages are assumed to evolve under different values of omega, but with far fewer parameters. By averaging over models which explain the data reasonably well, we can assess the robustness of our conclusions to uncertainty in model estimation. Our approach can also be used to compare results from models in which branch classes are specified a priori with a wide range of credible models. We illustrate our methods on primate lysozyme sequences and compare them with previous methods applied to the same data sets.     

Apparent Selection Intensity for the Cytochrome Oxidase Subunit I Gene Varies with Mode of Reproduction in Echinoderms

When most amino acid substitutions in protein-coding genes are slightly deleterious rather than selectively neutral, life history differences can potentially modify the effective population size or the selective regime, resulting in altered ratios of non-synonymous to synonymous substitutions among taxa. We studied substitution patterns for the mitochondrial cytochrome oxidase subunit I (COI) gene in a sea star genus (Leptasterias spp.) with an obligate brood-protecting mode of reproduction and small-scale population genetic subdivision, and compared the results to available COI sequences in nine other genera of echinoderms with pelagic larvae: three sea stars, five sea urchins and one brittle star. We predicted that this life history difference would be associated with differences in the ratio of non-synonymous (dN) to synonymous (dS) substitution rates. Leptasterias had a significantly greater dN/dS ratio (both between species and within species), a significantly smaller transition/transversion rate ratio, and a significantly lower average nucleotide diversity within species, than did the non-brooding genera. Other explanations for the results, such as altered mutation rates or selective sweeps, were not supported by the data analysis. These findings highlight the potential influence of reproductive traits and other life history factors on patterns of nucleotide substitution within and between species.     

Evidence for positive selection at the pantophysin (Pan I) locus in walleye pollock, Theragra chalcogramma

Nucleotide polymorphism at the pantophysin (Pan I) locus in walleye pollock, Theragra chalcogramma, was examined using DNA sequence data. Two distinct allelic lineages were detected in pollock, resulting from three amino acid replacement mutations in the first intravesicular domain of the protein. The common Pan I allelic group, comprising 94% of the samples, was less polymorphic (pi = 0.005) than the uncommon group (pi = 0.008), and nucleotide diversity in both was higher than for two allelic lineages in the related Atlantic cod, Gadus morhua. Phylogenetic analyses of Pan I sequences from these two species did not clearly resolve orthology among allelic groups, in part because of recombination that has occurred between the two pollock lineages. Conventional tests of neutrality comparing polymorphisms within and between homologous regions of the Pan I locus in walleye pollock and Atlantic cod did not detect the effects of selection. This result is likely attributed to low levels of synonymous divergence among allelic lineages and a lack of mutation-drift equilibrium inferred from nucleotide mismatch frequency distributions. However, the ratio of nonsynonymous to synonymous substitutions per site (dN/dS) exceeded unity in two intravesicular domains of the protein and the influence of positive selection at multiple codon sites was strongly inferred through the use of maximum-likelihood analyses. In addition, the frequency spectrum of linked neutral variation showed indirect effects of adaptive hitchhiking in pollock resulting from a selective sweep of the common allelic lineage. Recombination between the two allelic classes may have prevented complete loss of the older, more polymorphic lineage. The results suggest that recurrent sweeps driven by positive selection is the principle mode of evolution at the Pan I locus in gadid fishes.     

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.     

Differential rates of evolution for the ZFY-related zinc finger genes,Zfy, Zfx,and Zfa in the mouse genus Mus

A comparative study of the last exon of the zinc finger genes Zfx, Zfy, and Zfa from species of mice in the genus Mus was conducted to assess the extent of gene-specific and chromosome-specific effects on the evolutionary patterns among related X-, Y-, and autosomal-linked genes. Phylogenetic analyses of 29 sequences from Zfx, Zfa, and Zfy from 10 taxa were performed to infer relatedness among the zinc finger loci, and codon-based maximum likelihood analyses were conducted to assess evolutionary pattern among genes. Five models of nucleotide sequence evolution were applied and compared using a likelihood ratio test. Estimates of nonsynonymous to synonymous changes (dN/dS) for these genes suggest that amino acid substitutions are occurring at a more rapid rate across the autosomal- and Y-specific lineages compared to the X-specific lineage, with the Y-specific lineage showing the highest rate under certain models. The data suggest the action of gene-specific effects on evolutionary pattern. In particular, Zfa and Zfy genes, both with presumed restricted expression, appear less functionally constrained relative to ubiquitously expressed Zfx. Slightly elevated dN/dS for Zfy genes in comparison to Zfa also suggest Y-specific effects.     

The evolution of mitochondrial genomes in subterranean caviomorph rodents: adaptation against a background of purifying selection

South American tuco-tucos (Ctenomys) and the related coruro (Spalacopus) are two rodent lineages that have independently colonised the subterranean niche. The energetically demanding lifestyles of these species, coupled with the hypoxic atmospheres characteristic of subterranean environments, may have altered the selective regimes on genes encoding proteins related to cellular respiration. Here, we examined the molecular evolution of 13 protein-coding genes in the mitochondrial genome of seven caviomorph rodents, including these two subterranean genera and their above-ground relatives. Using maximum-likelihood and Bayesian approaches, we estimated rates of synonymous (dS) and nonsynonymous (dN) substitutions. We found a significantly higher ω ratio (dN/dS) in subterranean groups as compared to their non-subterranean counterparts in 11 of 13 genes, although no ω ratio was larger than 1. Additionally, we applied a method based on quantitative physicochemical properties to test for positive selection. Amino acid changes implicated in radical structural or functional shifts in the protein property were found to be ubiquitous across the phylogeny, but concentrated in the subterranean lineages. Convergent changes were also found between the subterranean genera used in this study and other mammals adapted to hypoxia. The results of this study suggest a link between niche shifts and weak directional (or episodic) selection at the molecular level against a background of purifying selection.