Inferring Weak Selection from Patterns of Polymorphism and Divergence at ``silent' Sites in Drosophila DNA

Patterns of codon usage and ``silent'''' DNA divergence suggest that natural selection discriminates among synonymous codons in Drosophila. ``Preferred'''' codons are consistently found in higher frequencies within their synonymous families in Drosophila melanogaster genes. This suggests a simple model of silent DNA evolution where natural selection favors mutations from unpreferred to preferred codons (preferred changes). Changes in the opposite direction, from preferred to unpreferred synonymous codons (unpreferred changes), are selected against. Here, selection on synonymous DNA mutations is investigated by comparing the evolutionary dynamics of these two categories of silent DNA changes. Sequences from outgroups are used to determine the direction of synonymous DNA changes within and between D. melanogaster and Drosophila simulans for five genes. Population genetics theory shows that differences in the fitness effect of mutations can be inferred from the comparison of ratios of polymorphism to divergence. Unpreferred changes show a significantly higher ratio of polymorphism to divergence than preferred changes in the D. simulans lineage, confirming the action of selection at silent sites. An excess of unpreferred fixations in 28 genes suggests a relaxation of selection on synonymous mutations in D. melanogaster. Estimates of selection coefficients for synonymous mutations (3.6 <|N(e)s| < 1.3) in D. simulans are consistent with the reduced efficacy of natural selection (|N(e)s| < 1) in the three- to sixfold smaller effective population size of D. melanogaster. Synonymous DNA changes appear to be a prevalent class of weakly selected mutations in Drosophila.     

Selective pressures at a codon-level predict deleterious mutations in human disease genes

Deleterious mutations affecting biological function of proteins are constantly being rejected by purifying selection from the gene pool. The non-synonymous/synonymous substitution rate ratio (omega) is a measure of selective pressure on amino acid replacement mutations for protein-coding genes. Different methods have been developed in order to predict non-synonymous changes affecting gene function. However, none has considered the estimation of selective constraints acting on protein residues. Here, we have used codon-based maximum likelihood models in order to estimate the selective pressures on the individual amino acid residues of a well-known model protein: p53. We demonstrate that the number of residues under strong purifying selection in p53 is much higher than those that are strictly conserved during the evolution of the species. In agreement with theoretical expectations, residues that have been noted to be of structural relevance, or in direct association with DNA, were among those showing the highest signals of purifying selection. Conversely, those changing according to a neutral, or nearly neutral mode of evolution, were observed to be irrelevant for protein function. Finally, using more than 40 human disease genes, we demonstrate that residues evolving under strong selective pressures (omega<0.1) are significantly associated (p<0.01) with human disease. We hypothesize that non-synonymous change on amino acids showing omega<0.1 will most likely affect protein function. The application of this evolutionary prediction at a genomic scale will provide an a priori hypothesis of the phenotypic effect of non-synonymous coding single nucleotide polymorphisms (SNPs) in the human genome.     

Fast, accurate and simulation-free stochastic mapping

Mapping evolutionary trajectories of discrete traits onto phylogenies receives considerable attention in evolutionary biology. Given the trait observations at the tips of a phylogenetic tree, researchers are often interested where on the tree the trait changes its state and whether some changes are preferential in certain parts of the tree. In a model-based phylogenetic framework, such questions translate into characterizing probabilistic properties of evolutionary trajectories. Current methods of assessing these properties rely on computationally expensive simulations. In this paper, we present an efficient, simulation-free algorithm for computing two important and ubiquitous evolutionary trajectory properties. The first is the mean number of trait changes, where changes can be divided into classes of interest (e.g. synonymous/non-synonymous mutations). The mean evolutionary reward, accrued proportionally to the time a trait occupies each of its states, is the second property. To illustrate the usefulness of our results, we first employ our simulation-free stochastic mapping to execute a posterior predictive test of correlation between two evolutionary traits. We conclude by mapping synonymous and non-synonymous mutations onto branches of an HIV intrahost phylogenetic tree and comparing selection pressure on terminal and internal tree branches.     

Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process

The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution.     

Increased constraints on MC4R during primate and human evolution

The melanocortin 4 receptor (MC4R) is routinely investigated for the role it plays in human obesity, as mutations in MC4R are the most common dominantly inherited form of the disease. As little is known about the evolutionary history of this locus, we investigated patterns of variation at MC4R in a worldwide sample of 1,015 humans from 51 populations, and in 8 central chimpanzees. There is a significant paucity of diversity at MC4R in humans, but not in chimpanzees. The spectrum of mutations in humans, combined with the overall low level of diversity, suggests that most (if not all) of the observed non-synonymous polymorphisms are likely to be transient deleterious mutations. The MC4R coding region was resequenced in 12 primate species and sequences from an additional 29 vertebrates were included in molecular evolutionary analyses. MC4R is highly conserved throughout vertebrate evolution, and has apparently been subject to high levels of continuous purifying selection that increased approximately threefold during primate evolution. Furthermore, the strong selection extends to codon usage bias, where most silent mutations are expected to be either quickly fixed or removed from the population, which may help explain the unusually low levels of silent polymorphisms in humans. Finally, there is a significant tendency for non-synonymous mutations that impact MC4R function to occur preferentially at sites that are identified by evolutionary analyses as being subject to very strong purifying selection. The information from this study should help inform future epidemiological investigations of MC4R. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence for purifying selection acting on silent sites in BRCA1

In mammals, it is usually assumed that selection cannot be strong enough to act on nucleotide mutations that do not cause a change at the protein level (i.e. 'silent' or 'synonymous' mutations). Here we report the results of a molecular evolutionary analysis of BRCA1. We find a repeatable pronounced peak in the ratio of nonsynonymous to synonymous substitutions between codons 200-300. Unusually, this peak is caused by a plummet in the silent-site rate of evolution. The most parsimonious interpretation of these data is that purifying selection is acting on silent sites.     

Estimate of within population incremental selection through branch imbalance in lineage trees

Incremental selection within a population, defined as limited fitness changes following mutation, is an important aspect of many evolutionary processes. Strongly advantageous or deleterious mutations are detected using the synonymous to non-synonymous mutations ratio. However, there are currently no precise methods to estimate incremental selection. We here provide for the first time such a detailed method and show its precision in multiple cases of micro-evolution. The proposed method is a novel mixed lineage tree/sequence based method to detect within population selection as defined by the effect of mutations on the average number of offspring. Specifically, we propose to measure the log of the ratio between the number of leaves in lineage trees branches following synonymous and non-synonymous mutations. The method requires a high enough number of sequences, and a large enough number of independent mutations. It assumes that all mutations are independent events. It does not require of a baseline model and is practically not affected by sampling biases. We show the method''s wide applicability by testing it on multiple cases of micro-evolution. We show that it can detect genes and inter-genic regions using the selection rate and detect selection pressures in viral proteins and in the immune response to pathogens.     

Quantifying the adaptive potential of an antibiotic resistance enzyme

For a quantitative understanding of the process of adaptation, we need to understand its "raw material," that is, the frequency and fitness effects of beneficial mutations. At present, most empirical evidence suggests an exponential distribution of fitness effects of beneficial mutations, as predicted for Gumbel-domain distributions by extreme value theory. Here, we study the distribution of mutation effects on cefotaxime (Ctx) resistance and fitness of 48 unique beneficial mutations in the bacterial enzyme TEM-1 β-lactamase, which were obtained by screening the products of random mutagenesis for increased Ctx resistance. Our contributions are threefold. First, based on the frequency of unique mutations among more than 300 sequenced isolates and correcting for mutation bias, we conservatively estimate that the total number of first-step mutations that increase Ctx resistance in this enzyme is 87 [95% CI 75-189], or 3.4% of all 2,583 possible base-pair substitutions. Of the 48 mutations, 10 are synonymous and the majority of the 38 non-synonymous mutations occur in the pocket surrounding the catalytic site. Second, we estimate the effects of the mutations on Ctx resistance by determining survival at various Ctx concentrations, and we derive their fitness effects by modeling reproduction and survival as a branching process. Third, we find that the distribution of both measures follows a Fréchet-type distribution characterized by a broad tail of a few exceptionally fit mutants. Such distributions have fundamental evolutionary implications, including an increased predictability of evolution, and may provide a partial explanation for recent observations of striking parallel evolution of antibiotic resistance.     

Phylogenetic Patterns of Codon Evolution in the ACTIN-DEPOLYMERIZING FACTOR/COFILIN (ADF/CFL) Gene Family

The actin-depolymerizing factor/cofilin (ADF/CFL) gene family encodes a diverse group of relatively small proteins. Once known strictly as modulators of actin filament dynamics, recent research has demonstrated that these proteins are involved in a variety of cellular processes, from signal transduction to the cytonuclear trafficking of actin. In both plant and animal lineages, expression patterns of paralogs in the ADF/CFL gene family vary among tissue types and developmental stages. In this study we use computational approaches to investigate the evolutionary forces responsible for the diversification of the ADF/CFL gene family. Estimating the rate of non-synonymous to synonymous mutations (dN/dS) across phylogenetic lineages revealed that the majority of ADF/CFL codon positions were under strong purifying selection, with rare episodic events of accelerated protein evolution. In both plants and animals these instances of accelerated evolution were ADF/CFL subclass specific, and all of the sites under selection were located in regions of the protein that could serve in new functional roles. We suggest these sites may have been important in the functional diversification of ADF/CFL proteins.     

Evolution of HIV-1 in a patient population failing multiple-drug therapy

To understand the evolutionary pathway of the multi-drug-resistant virus HIV-1 under drug-induced selection pressure, plasma from seven patients from baseline to different intervals post-treatment failure were used in RT-PCR protocols. Multiple clones were sequenced for each time point. Drug-resistant mutations were detected in five patients. Phylogenetic analysis showed that at different time points, viral sequences clustered separately and formed independent lineages. Genetic diversity decreased from 1.59 to 0.55, whereas non-synonymous/synonymous mutation ratios increased from 0.067 to 0.118, respectively. These data suggest that the virus population changed dynamically and clustered in a time point-specific manner whereas genetic diversity decreased consistently.     

Population size and molecular evolution on islands

The nearly neutral theory predicts that the rate and pattern of molecular evolution will be influenced by effective population size (Ne), because in small populations more slightly deleterious mutations are expected to drift to fixation. This important prediction has not been widely empirically tested, largely because of the difficulty of comparing rates of molecular evolution in sufficient numbers of independent lineages which differ only in Ne. Island endemic species provide an ideal test of the effect of Ne on molecular evolution because species restricted to islands frequently have smaller Ne than closely related mainland species, and island endemics have arisen from mainland lineages many times in a wide range of taxa. We collated a dataset of 70 phylogenetically independent comparisons between island and mainland taxa, including vertebrates, invertebrates and plants, from 19 different island groups. The rate of molecular evolution in these lineages was estimated by maximum likelihood using two measures: overall substitution rate and the ratio of non-synonymous to synonymous substitution rates. We show that island lineages have significantly higher ratios of non-synonymous to synonymous substitution rates than mainland lineages, as predicted by the nearly neutral theory, although overall substitution rates do not differ significantly.     

SnIPRE: Selection Inference Using a Poisson Random Effects Model

We present an approach for identifying genes under natural selection using polymorphism and divergence data from synonymous and non-synonymous sites within genes. A generalized linear mixed model is used to model the genome-wide variability among categories of mutations and estimate its functional consequence. We demonstrate how the model''s estimated fixed and random effects can be used to identify genes under selection. The parameter estimates from our generalized linear model can be transformed to yield population genetic parameter estimates for quantities including the average selection coefficient for new mutations at a locus, the synonymous and non-synynomous mutation rates, and species divergence times. Furthermore, our approach incorporates stochastic variation due to the evolutionary process and can be fit using standard statistical software. The model is fit in both the empirical Bayes and Bayesian settings using the lme4 package in R, and Markov chain Monte Carlo methods in WinBUGS. Using simulated data we compare our method to existing approaches for detecting genes under selection: the McDonald-Kreitman test, and two versions of the Poisson random field based method MKprf. Overall, we find our method universally outperforms existing methods for detecting genes subject to selection using polymorphism and divergence data.     

Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity

Messenger RNA is a key component of an intricate regulatory network of its own. It accommodates numerous nucleotide signals that overlap protein coding sequences and are responsible for multiple levels of regulation and generation of biological complexity. A wealth of structural and regulatory information, which mRNA carries in addition to the encoded amino acid sequence, raises the question of how these signals and overlapping codes are delineated along non-synonymous and synonymous positions in protein coding regions, especially in eukaryotes. Silent or synonymous codon positions, which do not determine amino acid sequences of the encoded proteins, define mRNA secondary structure and stability and affect the rate of translation, folding and post-translational modifications of nascent polypeptides. The RNA level selection is acting on synonymous sites in both prokaryotes and eukaryotes and is more common than previously thought. Selection pressure on the coding gene regions follows three-nucleotide periodic pattern of nucleotide base-pairing in mRNA, which is imposed by the genetic code. Synonymous positions of the coding regions have a higher level of hybridization potential relative to non-synonymous positions, and are multifunctional in their regulatory and structural roles. Recent experimental evidence and analysis of mRNA structure and interspecies conservation suggest that there is an evolutionary tradeoff between selective pressure acting at the RNA and protein levels. Here we provide a comprehensive overview of the studies that define the role of silent positions in regulating RNA structure and processing that exert downstream effects on proteins and their functions.     

Gene duplication,allelic diversity,selection processes and adaptive value of MHC class II <Emphasis Type="Italic">DRB</Emphasis> genes of the bank vole, <Emphasis Type="Italic">Clethrionomys glareolus</Emphasis>

The generation and maintenance of allelic polymorphism in genes of the major histocompatibility complex (MHC) is a central issue in evolutionary genetics. Recently, the focus has changed from ex situ to in situ populations to understand the mechanisms that determine adaptive MHC polymorphism under natural selection. Birth-and-death evolution and gene conversion events are considered to generate sequence diversity in MHC genes, which subsequently is maintained by balancing selection through parasites. The ongoing arms race between the host and parasites leads to an adaptive selection pressure upon the MHC, evident in high rates of non-synonymous vs synonymous substitution rates. We characterised the MHC class II DRB exon 2 of free living bank voles, Clethrionomys glareolus by single-strand conformation polymorphism and direct sequencing. Unlike other arvicolid species, the DRB locus of the bank vole is at least quadruplicated. No evidence for gene conversion events in the Clgl-DRB sequences was observed. We found not only high allelic polymorphism with 26 alleles in 36 individuals but also high rates of silent polymorphism. Exceptional for MHC class II genes is a purifying selection pressure upon the majority of MHC-DRB sequences. Further, we analysed the association between certain DRB alleles and the parasite burden with gastrointestinal trichostrongyle nematodes Heligmosomum mixtum and Heligmosomoides glareoli and found significant quality differences between specific alleles with respect to infection intensity. Our findings suggest a snapshot in an evolutionary process of ongoing birth-and-death evolution. One allele cluster has lost its function and is already silenced, another is loosing its adaptive value in terms of gastrointestinal nematode resistance, while a third group of alleles indicates all signs of classical functional MHC alleles.     

Natural Selection and the Frequency Distributions of ``silent' DNA Polymorphism in Drosophila

In Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster, codon bias may be maintained by a balance among mutation pressure, genetic drift, and natural selection favoring translationally superior codons. Under such an evolutionary model, silent mutations fall into two fitness categories: preferred mutations that increase codon bias and unpreferred changes in the opposite direction. This prediction can be tested by comparing the frequency spectra of synonymous changes segregating within populations; natural selection will elevate the frequencies of advantageous mutations relative to that of deleterious changes. The frequency distributions of preferred and unpreferred mutations differ in the predicted direction among 99 alleles of two D. pseudoobscura genes and five alleles of eight D. simulans genes. This result confirms the existence of fitness classes of silent mutations. Maximum likelihood estimates suggest that selection intensity at silent sites is, on average, very weak in both D. pseudoobscura and D. simulans (|N(e)s| & 1). Inference of evolutionary processes from within-species sequence variation is often hindered by the assumption of a stationary frequency distribution. This assumption can be avoided when identifying the action of selection and tested when estimating selection intensity.     

Adaptive molecular evolution of virulence genes of avian influenza - A virus subtype H5N1: An analysis of host radiation

The phenomenon of host radiation is strongly influenced by the rates of mutation of their virulence genes. We have studied the molecular evolution of virulence genes (HA, NS, PB2) of the Avian Influenza Virus H5N1 from avian to human hosts. We used a site-specific comparison of synonymous (silent) and non-synonymous (amino acid altering) nucleotide substitutions for the three chosen genes in parasite populations from different hosts. Analyses were made using Maximum Likelihood (ML) genealogies for the null and alternate hypothesis based on differential gamma distribution rates. The null hypothesis had a higher rate of substitution and was found to be more suitable for all the studied genes by Likelihood Ratio Test (LRT). The study showed the NS gene to be having the fastest rate of evolution.     

Characterization of coding synonymous and non-synonymous variants in ADAMTS13 using ex vivo and in silico approaches

Synonymous variations, which are defined as codon substitutions that do not change the encoded amino acid, were previously thought to have no effect on the properties of the synthesized protein(s). However, mounting evidence shows that these "silent" variations can have a significant impact on protein expression and function and should no longer be considered "silent". Here, the effects of six synonymous and six non-synonymous variations, previously found in the gene of ADAMTS13, the von Willebrand Factor (VWF) cleaving hemostatic protease, have been investigated using a variety of approaches. The ADAMTS13 mRNA and protein expression levels, as well as the conformation and activity of the variants have been compared to that of wild-type ADAMTS13. Interestingly, not only the non-synonymous variants but also the synonymous variants have been found to change the protein expression levels, conformation and function. Bioinformatic analysis of ADAMTS13 mRNA structure, amino acid conservation and codon usage allowed us to establish correlations between mRNA stability, RSCU, and intracellular protein expression. This study demonstrates that variants and more specifically, synonymous variants can have a substantial and definite effect on ADAMTS13 function and that bioinformatic analysis may allow development of predictive tools to identify variants that will have significant effects on the encoded protein.     

Mitogenomic analysis of a 50-generation chicken pedigree reveals a rapid rate of mitochondrial evolution and evidence for paternal mtDNA inheritance

Mitochondrial genomes represent a valuable source of data for evolutionary research, but studies of their short-term evolution have typically been limited to invertebrates, humans and laboratory organisms. Here we present a detailed study of 12 mitochondrial genomes that span a total of 385 transmissions in a well-documented 50-generation pedigree in which two lineages of chickens were selected for low and high juvenile body weight. These data allowed us to test the hypothesis of time-dependent evolutionary rates and the assumption of strict maternal mitochondrial transmission, and to investigate the role of mitochondrial mutations in determining phenotype. The identification of a non-synonymous mutation in ND4L and a synonymous mutation in CYTB, both novel mutations in Gallus, allowed us to estimate a molecular rate of 3.13 × 10⁻⁷ mutations/site/year (95% confidence interval 3.75 × 10⁻⁸–1.12 × 10⁻⁶). This is substantially higher than avian rate estimates based upon fossil calibrations. Ascertaining which of the two novel mutations was present in an additional 49 individuals also revealed an instance of paternal inheritance of mtDNA. Lastly, an association analysis demonstrated that neither of the point mutations was strongly associated with the phenotypic differences between the two selection lines. Together, these observations reveal the highly dynamic nature of mitochondrial evolution over short time periods.     

Effects of metabolic rate on protein evolution

Since the modern evolutionary synthesis was first proposed early in the twentieth century, attention has focused on assessing the relative contribution of mutation versus natural selection on protein evolution. Here we test a model that yields general quantitative predictions on rates of protein evolution by combining principles of individual energetics with Kimura's neutral theory. The model successfully predicts much of the heterogeneity in rates of protein evolution for diverse eukaryotes (i.e. fishes, amphibians, reptiles, birds, mammals) from different thermal environments. Data also show that the ratio of non-synonymous to synonymous nucleotide substitution is independent of body size, and thus presumably of effective population size. These findings indicate that rates of protein evolution are largely controlled by mutation rates, which in turn are strongly influenced by individual metabolic rate.