Studying Patterns of Recent Evolution at Synonymous Sites and Intronic Sites in Drosophila melanogaster

Most previous studies of the evolution of codon usage bias (CUB) and intronic GC content (iGC) in Drosophila melanogaster were based on between-species comparisons, reflecting long-term evolutionary events. However, a complete picture of the evolution of CUB and iGC cannot be drawn without knowledge of their more recent evolutionary history. Here, we used a polymorphism dataset collected from Zimbabwe to study patterns of the recent evolution of CUB and iGC. Analyzing coding and intronic data jointly with a model which can simultaneously estimate selection, mutational, and demographic parameters, we have found that: (1) natural selection is probably acting on synonymous codons; (2) a constant population size model seems to be sufficient to explain most of the observed synonymous polymorphism patterns; (3) GC is favored over AT in introns. In agreement with the long-term evolutionary patterns, ongoing selection acting on X-linked synonymous codons is stronger than that acting on autosomal codons. The selective differences between preferred and unpreferred codons tend to be greater than the differences between GC and AT in introns, suggesting that natural selection, not just biased gene conversion, may have influenced the evolution of CUB. Interestingly, evidence for non-equilibrium evolution comes exclusively from the intronic data. However, three different models, an equilibrium model with two classes of selected sites and two non-equilibrium models with changes in either population size or mutational parameters, fit the intronic data equally well. These results show that using inadequate selection (or demographic) models can result in incorrect estimates of demographic (or selection) parameters.     

Evidence of Selection upon Genomic GC-Content in Bacteria

The genomic GC-content of bacteria varies dramatically, from less than 20% to more than 70%. This variation is generally ascribed to differences in the pattern of mutation between bacteria. Here we test this hypothesis by examining patterns of synonymous polymorphism using datasets from 149 bacterial species. We find a large excess of synonymous GC→AT mutations over AT→GC mutations segregating in all but the most AT-rich bacteria, across a broad range of phylogenetically diverse species. We show that the excess of GC→AT mutations is inconsistent with mutation bias, since it would imply that most GC-rich bacteria are declining in GC-content; such a pattern would be unsustainable. We also show that the patterns are probably not due to translational selection or biased gene conversion, because optimal codons tend to be AT-rich, and the excess of GC→AT SNPs is observed in datasets with no evidence of recombination. We therefore conclude that there is selection to increase synonymous GC-content in many species. Since synonymous GC-content is highly correlated to genomic GC-content, we further conclude that there is selection on genomic base composition in many bacteria.     

Synonymous and nonsynonymous polymorphisms versus divergences in bacterial genomes

Comparison of the ratio of nonsynonymous to synonymous polymorphisms within species with the ratio of nonsynonymous to synonymous substitutions between species has been widely used as a supposed indicator of positive Darwinian selection, with the ratio of these 2 ratios being designated as a neutrality index (NI). Comparison of genome-wide polymorphism within 12 species of bacteria with divergence from an outgroup species showed substantial differences in NI among taxa. A low level of nonsynonymous polymorphism at a locus was the best predictor of NI < 1, rather than a high level of nonsynonymous substitution between species. Moreover, genes with NI < 1 showed a strong tendency toward the occurrence of rare nonsynonymous polymorphisms, as expected under the action of ongoing purifying selection. Thus, our results are more consistent with the hypothesis that a high relative rate of between-species nonsynonymous substitution reflects mainly the action of purifying selection within species to eliminate slightly deleterious mutations rather than positive selection between species. This conclusion is consistent with previous results highlighting an important role of slightly deleterious variants in bacterial evolution and suggests caution in the use of the McDonald-Kreitman test and related statistics as tests of positive selection.     

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.     

Recurrent positive selection at bgcn, a key determinant of germ line differentiation, does not appear to be driven by simple coevolution with its partner protein bam

Surveys of nucleotide sequence polymorphism in Drosophila melanogaster and Drosophila simulans were performed at 2 interacting loci crucial for gametogenesis: bag-of-marbles (bam) and benign gonial cell neoplasm (bgcn). At the polymorphism level, both loci appear to be evolving under the expectations of the neutral theory. However, ratios of polymorphism and divergence for synonymous and nonsynonymous mutations depart significantly from neutral expectations for both loci consistent with a previous observation of positive selection at bam. The deviations suggest either an excess of synonymous polymorphisms or an excess of nonsynonymous fixations at both loci. Synonymous evolution appears to conform to neutrality at bam. At bgcn, there is evidence of positive selection affecting preferred synonymous mutations along the D. simulans lineage. However, there is also a significantly higher rate of nonsynonymous fixations at bgcn within D. simulans. Thus, the deviation from neutrality detected by the McDonald-Kreitman test at these 2 loci is likely due to the selective acceleration of nonsynonymous fixations. Differences in the pattern of amino acid fixations between these 2 interacting proteins suggest that the detected positive selection is not due to a simple model of coevolution.     

Selection intensity on preferred codons correlates with overall codon usage bias in Caenorhabditis remanei

Adaptive codon usage provides evidence of natural selection in one of its most subtle forms: a fitness benefit of one synonymous codon relative to another. Codon usage bias is evident in the coding sequences of a broad array of taxa, reflecting selection for translational efficiency and/or accuracy as well as mutational biases. Here, we quantify the magnitude of selection acting on alternative codons in genes of the nematode Caenorhabditis remanei, an outcrossing relative of the model organism C. elegans, by fitting the expected mutation-selection-drift equilibrium frequency distribution of preferred and unpreferred codon variants to the empirical distribution. This method estimates the intensity of selection on synonymous codons in genes with high codon bias as N(e)s = 0.17, a value significantly greater than zero. In addition, we demonstrate for the first time that estimates of ongoing selection on codon usage among genes, inferred from nucleotide polymorphism data, correlate strongly with long-term patterns of codon usage bias, as measured by the frequency of optimal codons in a gene. From the pattern of polymorphisms in introns, we also infer that these findings do not result from the operation of biased gene conversion toward G or C nucleotides. We therefore conclude that coincident patterns of current and ancient selection are responsible for shaping biased codon usage in the C. remanei genome.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Genome-wide selection on codon usage at the population level in the fungal model organism Neurospora crassa

Many organisms exhibit biased codon usage in their genome, including the fungal model organism Neurospora crassa. The preferential use of subset of synonymous codons (optimal codons) at the macroevolutionary level is believed to result from a history of selection to promote translational efficiency. At present, few data are available about selection on optimal codons at the microevolutionary scale, that is, at the population level. Herein, we conducted a large-scale assessment of codon mutations at biallelic sites, spanning more than 5,100 genes, in 2 distinct populations of N. crassa: the Caribbean and Louisiana populations. Based on analysis of the frequency spectra of synonymous codon mutations at biallelic sites, we found that derived (nonancestral) optimal codon mutations segregate at a higher frequency than derived nonoptimal codon mutations in each population; this is consistent with natural selection favoring optimal codons. We also report that optimal codon variants were less frequent in longer genes and that the fixation of optimal codons was reduced in rapidly evolving long genes/proteins, trends suggestive of genetic hitchhiking (Hill-Robertson) altering codon usage variation. Notably, nonsynonymous codon mutations segregated at a lower frequency than synonymous nonoptimal codon mutations (which impair translational efficiency) in each N. crassa population, suggesting that changes in protein composition are more detrimental to fitness than mutations altering translation. Overall, the present data demonstrate that selection, and partly genetic interference, shapes codon variation across the genome in N. crassa populations.     

Molecular evolution of cadherin-related neuronal receptor/protocadherin(alpha) (CNR/Pcdh(alpha)) gene cluster in Mus musculus subspecies

The mouse cadherin-related neuronal receptor/protocadherin (CNR/Pcdh) gene clusters are located on chromosome 18. We sequenced single-nucleotide polymorphisms (SNPs) of the CNR/Pcdh(alpha)-coding region among 12 wild-derived and four laboratory strains; these included the four major subspecies groups of Mus musculus: domesticus, musculus, castaneus, and bactrianus. We detected 883 coding SNPs (cSNPs) in the CNR/Pcdh(alpha) variable exons and three in the constant exons. Among all the cSNPs, 586 synonymous (silent) and 297 nonsynonymous (amino acid exchanged) substitutions were found; therefore, the K(a)/K(s) ratio (nonsynonymous substitutions per synonymous substitution) was 0.51. The synonymous cSNPs were relatively concentrated in the first and fifth extracellular cadherin domain-encoding regions (ECs) of CNR/Pcdh(alpha). These regions have high nucleotide homology among the CNR/Pcdh(alpha) paralogs, suggesting that gene conversion events in synonymous and homologous regions of the CNR/Pcdh(alpha) cluster are related to the generation of cSNPs. A phylogenetic analysis revealed gene conversion events in the EC1 and EC5 regions. Assuming that the common sequences between rat and mouse are ancestral, the GC content of the third codon position has increased in the EC1 and EC5 regions, although biased substitutions from GC to AT were detected in all the codon positions. In addition, nonsynonymous substitutions were extremely high (11 of 13, K(a)/K(s) ratio 5.5) in the laboratory mouse strains. The artificial environment of laboratory mice may allow positive selection for nonsynonymous amino acid variations in CNR/Pcdh(alpha) during inbreeding. In this study, we analyzed the direction of cSNP generation, and concluded that subspecies-specific nucleotide substitutions and region-restricted gene conversion events may have contributed to the generation of genetic variations in the CNR/Pcdh genes within and between species.     

Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans,but not in chimpanzees.

To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA''s D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies.     

Determinants of synonymous and nonsynonymous variability in three species of Drosophila

We estimated the intensity of selection on preferred codons in Drosophila pseudoobscura and D. miranda at X-linked and autosomal loci, using a published data set on sequence variability at 67 loci, by means of an improved method that takes account of demographic effects. We found evidence for stronger selection at X-linked loci, consistent with their higher levels of codon usage bias. The estimates of the strength of selection and mutational bias in favor of unpreferred codons were similar to those found in other species, after taking into account the fact that D. pseudoobscura showed evidence for a recent expansion in population size. We examined correlates of synonymous and nonsynonymous diversity in these species and found no evidence for effects of recurrent selective sweeps on nonsynonymous mutations, which is probably because this set of genes have much higher than average levels of selective constraints. There was evidence for correlated effects of levels of selective constraints on protein sequences and on codon usage, as expected under models of selection for translational accuracy. Our analysis of a published data set on D. melanogaster provided evidence for the effects of selective sweeps of nonsynonymous mutations on linked synonymous diversity, but only in the subset of loci that experienced the highest rates of nonsynonymous substitutions (about one-quarter of the total) and not at more slowly evolving loci. Our correlational analysis of this data set suggested that both selective constraints on protein sequences and recurrent selective sweeps affect the overall level of codon usage.     

Schematic for efficient computation of GC, GC3, and AT3 bias spectra of genome

Selection of synonymous codons for an amino acid is biased in protein translation process. This biased selection causes repetition of synonymous codons in structural parts of genome that stands for high N/3 peaks in DNA spectrum. Period-3 spectral property is utilized here to produce a 3-phase network model based on polyphase filterbank concepts for derivation of codon bias spectra (CBS). Modification of parameters in this model can produce GC, GC3, and AT3 bias spectra. Complete schematic in LabVIEW platform is presented here for efficient and parallel computation of GC, GC3, and AT3 bias spectra of genomes alongwith results of CBS patterns. We have performed the correlation coefficient analysis of GC, GC3, and AT3 bias spectra with codon bias patterns of CBS for biological and statistical significance of this model.     

GC-biased segregation of noncoding polymorphisms in Drosophila

The study of base composition evolution in Drosophila has been achieved mostly through the analysis of coding sequences. Third codon position GC content, however, is influenced by both neutral forces (e.g., mutation bias) and natural selection for codon usage optimization. In this article, large data sets of noncoding DNA sequence polymorphism in D. melanogaster and D. simulans were gathered from public databases to try to disentangle these two factors-noncoding sequences are not affected by selection for codon usage. Allele frequency analyses revealed an asymmetric pattern of AT vs. GC noncoding polymorphisms: AT --> GC mutations are less numerous, and tend to segregate at a higher frequency, than GC --> AT ones, especially at GC-rich loci. This is indicative of nonstationary evolution of base composition and/or of GC-biased allele transmission. Fitting population genetics models to the allele frequency spectra confirmed this result and favored the hypothesis of a biased transmission. These results, together with previous reports, suggest that GC-biased gene conversion has influenced base composition evolution in Drosophila and explain the correlation between intron and exon GC content.     

Neutral Evolution of Synonymous Base Composition in the Brassicaceae

The GC content of synonymous sites is elevated in genes from both Brassica oleraceae and Arabidopsis lyrata compared with Arabidopsis thaliana. However, this shift in base composition is independent of gene expression level, and there is no evidence for a similar difference in the frequency of codons preferred by translational selection. The results suggest that composition evolution is caused by a change in mutation bias or biased gene conversion, rather than by a reduction in the efficacy of natural selection in selfing Arabidopsis. [Reviewing Editor: Dr. Magrus Nordborg]     

Vanishing GC-rich isochores in mammalian genomes

To understand the origin and evolution of isochores-the peculiar spatial distribution of GC content within mammalian genomes-we analyzed the synonymous substitution pattern in coding sequences from closely related species in different mammalian orders. In primate and cetartiodactyls, GC-rich genes are undergoing a large excess of GC --> AT substitutions over AT --> GC substitutions: GC-rich isochores are slowly disappearing from the genome of these two mammalian orders. In rodents, our analyses suggest both a decrease in GC content of GC-rich isochores and an increase in GC-poor isochores, but more data will be necessary to assess the significance of this pattern. These observations question the conclusions of previous works that assumed that base composition was at equilibrium. Analysis of allele frequency in human polymorphism data, however, confirmed that in the GC-rich parts of the genome, GC alleles have a higher probability of fixation than AT alleles. This fixation bias appears not strong enough to overcome the large excess of GC --> AT mutations. Thus, whatever the evolutionary force (neutral or selective) at the origin of GC-rich isochores, this force is no longer effective in mammals. We propose a model based on the biased gene conversion hypothesis that accounts for the origin of GC-rich isochores in the ancestral amniote genome and for their decline in present-day mammals.     

A dissection of volatility in yeast

It has been suggested that volatility, the proportion of mutations which change an amino acid, can be used to infer the level of natural selection acting upon a gene. This conjecture is supported by a correlation between volatility and the rate of nonsynonymous substitution (dN), or the ratio of nonsynonymous and synonymous substitution rates, in a variety of organisms. These organisms include yeast, in which the correlations are quite strong. Here we show that these correlations are a by-product of a correlation between synonymous codon bias toward translationally optimal codons and dN. Although this analysis suggests that volatility is not a good measure of the selection, we suggest that it might be possible to infer something about the level of natural selection, from a single genome sequence, using translational codon bias.     

Patterns of mutation and selection at synonymous sites in Drosophila

That natural selection affects molecular evolution at synonymous sites in protein-coding sequences is well established and is thought to predominantly reflect selection for translational efficiency/accuracy mediated through codon bias. However, a recently developed maximum likelihood framework, when applied to 18 coding sequences in 3 species of Drosophila, confirmed an earlier report that the Notch gene in Drosophila melanogaster was evolving under selection in favor of those codons defined as unpreferred in this species. This finding opened the possibility that synonymous sites may be subject to a variety of selective pressures beyond weak selection for increased frequencies of the codons currently defined as "preferred" in D. melanogaster. To further explore patterns of synonymous site evolution in Drosophila in a lineage-specific manner, we expanded the application of the maximum likelihood framework to 8,452 protein coding sequences with well-defined orthology in D. melanogaster, Drosophila sechellia, and Drosophila yakuba. Our analyses reveal intragenomic and interspecific variation in mutational patterns as well as in patterns and intensity of selection on synonymous sites. In D. melanogaster, our results provide little statistical evidence for recent selection on synonymous sites, and Notch remains an outlier. In contrast, in D. sechellia our findings provide evidence in support of selection predominantly in favor of preferred codons. However, there is a small subset of genes in this species that appear to be evolving under selection in favor of unpreferred codons, which indicates that selection on synonymous sites is not limited to the preferential fixation of mutations that enhance the speed or accuracy of translation in this species.     

Positive and negative selection on noncoding DNA in Drosophila simulans

There is now a wealth of evidence that some of the most important regions of the genome are found outside those that encode proteins, and noncoding regions of the genome have been shown to be subject to substantial levels of selective constraint, particularly in Drosophila. Recent work has suggested that these regions may also have been subject to the action of positive selection, with large fractions of noncoding divergence having been driven to fixation by adaptive evolution. However, this work has focused on Drosophila melanogaster, which is thought to have experienced a reduction in effective population size (N(e)), and thus a reduction in the efficacy of selection, compared with its closest relative Drosophila simulans. Here, we examine patterns of evolution at several classes of noncoding DNA in D. simulans and find that all noncoding DNA is subject to the action of negative selection, indicated by reduced levels of polymorphism and divergence and a skew in the frequency spectrum toward rare variants. We find that the signature of negative selection on noncoding DNA and nonsynonymous sites is obscured to some extent by purifying selection acting on preferred to unpreferred synonymous codon mutations. We investigate the extent to which divergence in noncoding DNA is inferred to be the product of positive selection and to what extent these inferences depend on selection on synonymous sites and demography. Based on patterns of polymorphism and divergence for different classes of synonymous substitution, we find the divergence excess inferred in noncoding DNA and nonsynonymous sites in the D. simulans lineage difficult to reconcile with demographic explanations.     

On the varied pattern of evolution of 2 fungal genomes: a critique of Hughes and Friedman

A number of statistical tests have been proposed to detect positive Darwinian selection affecting a few amino acid sites in a protein, exemplified by an excess of nonsynonymous nucleotide substitutions. These tests are often more powerful than pairwise sequence comparison, which averages synonymous (d(S)) and nonsynonymous (d(N)) rates over the whole gene. In a recent study, however, Hughes AL and Friedman R (2005. Variation in the pattern of synonymous and nonsynonymous difference between two fungal genomes. Mol Bio Evol. 22: 1320-1324) argue that d(S) and d(N) are expected to fluctuate along the sequence by chance and that an excess of nonsynonymous differences in individual codons is no evidence for positive selection. The authors compared codons in protein-coding genes from the genomes of 2 yeast species, Saccharomyces cerevisiae and Saccharomyces paradoxus. They calculated the proportions of synonymous and nonsynonymous differences per site (p(S) and p(N)) in every codon and discovered that p(N) is often greater than p(S) and that among some codons p(S) and p(N) are negatively correlated. The authors argued that these results invalidate previous tests of codons under positive selection. Here I discuss several errors of statistics in the analysis of Hughes and Friedman, including confusion of statistics with parameters, arbitrary data filtering, and derivation of hypotheses from data. I also apply likelihood ratio tests of positive selection to the yeast data and illustrate empirically that Hughes and Friedman's criticisms on such tests are not valid.     

Unconstrained evolution in short introns? – An analysis of genome‐wide polymorphism and divergence data from Drosophila

An unconstrained reference sequence facilitates the detection of selection. In Drosophila, sequence variation in short introns seems to be least influenced by selection and dominated by mutation and drift. Here, we test this with genome‐wide sequences using an African population (Malawi) of D. melanogaster and data from the related outgroup species D. simulans, D. sechellia, D. erecta and D. yakuba. The distribution of mutations deviates from equilibrium, and the content of A and T (AT) nucleotides shows an excess of variance among introns. We explain this by a complex mutational pattern: a shift in mutational bias towards AT, leading to a slight nonequilibrium in base composition and context‐dependent mutation rates, with G or C (GC) sites mutating most frequently in AT‐rich introns. By comparing the corresponding allele frequency spectra of AT‐rich vs. GC‐rich introns, we can rule out the influence of directional selection or biased gene conversion on the mutational pattern. Compared with neutral equilibrium expectations, polymorphism spectra show an excess of low frequency and a paucity of intermediate frequency variants, irrespective of the direction of mutation. Combining the information from different outgroups with the polymorphism data and using a generalized linear model, we find evidence for shared ancestral polymorphism between D. melanogaster and D. simulans, D. sechellia, arguing against a bottleneck in D. melanogaster. Generally, we find that short introns can be used as a neutral reference on a genome‐wide level, if the spatially and temporally varying mutational pattern is accounted for.