The results show that codon bias and rates of protein evolution both have significant effects on levels of synonymous polymorphism (table 2) and these effects are working in opposite directions. This will allow for truly genome-wide population genetic analyses of the combined effects of natural selection and demography (see Macpherson et al. A synonymous mutation is when there is a change in the genetic sequence of DNA, but the change results in the same genetic output. The low GC content in noncoding regions in P. tremula suggests a mutation bias favoring GC to AT mutations, and because preferred codons in P. tremula end in G or C (Ingvarsson 2008a), this means a mutation bias favors unpreferred codons. One important goal of population genetics is to understand the relative importance of different evolutionary processes for shaping variation in natural populations. Previously such mutations were believed to be silent and have no impact, but scientists have more recently realized that synonymous mutations may be associated with diseases such as cancer. 2). 2007; Bachtrog 2008; Jensen et al. Recently, a study in scots pine (Pinus sylvestris), another long-lived tree species, has also documented patterns of polymorphism that are suggestive of the action of recurrent selective sweeps (Palmé et al. 2008). A mutation is defined as synonymous if it results in a codon that specifies the same amino acid as the original codon. To formally test these two alternative hypotheses, I fit a model including both shifts in γ and κ in equation (1) to the codon bias data from P. tremula and P. trichocarpa (dotted line in fig. Why selection on synonymous codon usage is stronger in P. tremula is at present an open question, but one plausible explanation is an increase in the effective population size of P. tremula compared with the ancestral population size of Populus and compared with many other extant species in the genus (Ingvarsson 2008a). It is conceivable that using this approach would also increase the likelihood of differentiating between selective and demographic effects, but more work is clearly needed to verify this. 2008). The data in this paper, with many, but rather short genome regions, are not well suited for separate estimation of these parameters, and there is thus still a great deal of uncertainty as to whether selection is generally rare and strong or common but weak in P. tremula. 2006.) As more data on nucleotide polymorphism accumulate in P. tremula, it should be possible to shed more light on this question. I used the ML method of Bierne and Eyre-Walker (2004), as implemented in the DoFE program (available at http://www.lifesci.sussex.ac.uk/home/Adam_Eyre-Walker/Software_files/DoFE-all_1.zip) to calculate the proportion of amino acid substitutions driven to fixation by positive selection in P. tremula, yielding an estimate of α of 0.159 with a 95% confidence interval of − 0.079 to 0.340. Posterior predictive simulations constitute an important “self-consistency check” of the model (Gelman et al. 2004). Diversity statistics were also calculated separately for noncoding, silent, and replacement sites. Posterior densities from the ABC analyses. If natural selection varies across at local populations, few mutations will spread throughout the species as they are not unconditionally favorable. The proportion of adaptive substitutions in P. tremula is slightly lower than estimates from Drosophila, where different studies have suggested that between 40% and 95% of all mutations are fixed by positive selection (Wright and Andolfatto 2008). From equation (1), it is clear that differences in codon bias between two closely related species can be driven by either changes in the strength of selection acting on codon usage (γ = 2Nes in eq. The recurrent hitchhiking model used in the ABC analyses assumes that the demography of the species conforms to the standard neutral model. The latter statistic requires the use of an outgroup sequence so that mutations can be polarized into ancestral or derived states (Fay and Wu 2000). Genes with high or low levels of codon bias will, however, show progressively greater change but in opposite directions (Akashi 1996). This study has been funded by grants from the Swedish Research Council to V.R. However, if positive selection is more common than postulated by the neutral or nearly neutral theories, many (or most) sites in the genome would have experienced the effects of selection, either directly or indirectly through selection acting on linked sites, through a process known as hitchhiking (Smith and Haigh 1974; Gillespie 2000; Sella et al. 1994) and adjusted manually using BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). We test this assumption by tracking synonymous mutations in longitudinal intrapatient data from the C2-V5 part of the env gene. On the other hand, if there is a systematic change in the strength of selection acting on synonymous codons, for instance, if the effective population size of a species is increased or reduced, genes with intermediate levels of codon bias will not show much change in codon bias. ‘Synonymous–Nonsynonymous Mutation Rates between Sequences Containing Ambiguous Nucleotides’ (Syn-SCAN), that calculates synonymous and non-synonymous substitution rates using a model that includes allelic mix-tures. Nevertheless, several studies have now show that recurrent hitchhiking appears to be common and/or strong enough in many species that most sites, even in high recombination regions of the genome, are affected by linked selection. The contour lines denote the quartiles of the bivariate posterior distribution. For instance, recent studies in Drosophila have documented reduced levels of synonymous polymorphism in rapidly evolving genes (Andolfatto 2007; Macpherson et al. The posterior predictive simulations were performed by generating a large number of data sets (105) from the posterior distributions of the parameters of the model. Genetics, Natural selection and the frequency distributions of "silent” DNA polymorphism in Drosophila, Hitchhiking effects of recurrent beneficial amino acid substitutions in the, Similar rates of protein adaptation in Drosophila miranda and D. melanogaster, two species with different current effective population sizes, Effect of misoriented sites on neutrality tests with outgroup, Species and recombination effects on DNA variability in the tomato genus, Approximate Bayesian computation in population genetics, Levels of naturally occurring DNA polymorphism correlate with recombination rates in, Linkage limits the power of natural selection in, The genomic rate of adaptive amino acid substitution in, The selection-mutation-drift theory of synonymous codon usage, Background selection and patterns of genetic diversity in Drosophila melanogaster, The effect of deleterious mutations on neutral molecular variation, The Mcdonald-Kreitman test and slightly deleterious mutations, Intragenic Hill-Robertson interference influences selection intensity on synonymous mutations in Drosophila, Ancestral inference on gene trees under selection, Selection intensity on preferred codons correlates with overall codon usage bias in, Selection at linked sites in the partial selfer, Base-calling of automated sequencer traces using phred. As a first step towards understanding the evolutionary effects of synonymous mutations, we estimated the distribution of fitness effects (DFE) for 39 synonymous, 65 nonsynonymous, and six nonsense substitutions at 34 sites along gtsB.Single nucleotide mutants were generated through site-directed mutagenesis and competed against the ancestor strain in glucose-limited medium. The effects of the rate of sweeps (2Neλ) and the strength of selection (s) are usually confounded because similar results can be obtained by either assuming strong and rare selection or common and selection. These data were used to calculate a number of summary statistics as suggested by Jensen et al. 2002; Foxe et al. The product of strength of selection acting on beneficial mutations and the rate by which these occur across the genome (2Neλs) equals 1.54×10 − 7, which is in line with estimates from Drosophila where recurrent hitchhiking has also been shown to have significant effects on standing levels of polymorphism. Strong selection, on the other hand, should leave very distinct signatures in the amount and patterns of nucleotide polymorphism in the genome region surrounding the site of an adaptive substitution (Kaplan et al. 2008; Sella et al. 2006), and Kelly's Zns (Kelly 1997). The vertical dotted line is the equilibrium value of Fop in the absence of selection on codon usage, that is, Fop calculated from the average GC content in noncoding regions in P. tremula. The posterior mode for 2Neλs equals 1.54×10 − 7, which is in line with estimates from Drosophila (reviewed in Jensen et al. Codon bias and protein evolution are influenced by different aspects of gene expression in P. tremula, with the level of gene expression being the main force shaping selection on codon usage, whereas protein evolution is largely determined by expression breadth, that is, how many tissue types a gene is expressed in (rather than maximum expression level per se; Ingvarsson 2007). Taking the amino acid Alanine as an example, this amino acid has 4 different codon formations: GCU, GCA, GCC, and GCG. 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These slightly deleterious mutations will to some extent mask the signature of adaptive evolution by introducing a negative bias into estimates of α. Although relatively simple demographic scenarios appear to explain patterns of intraspecific polymorphism in P. tremula (Ingvarsson 2008b), it is harder to envision a demographic explanation for the strong negative relationship observed between synonymous site diversity and rates of protein evolution. For instance, strong selection at linked sites reduces the efficiency of selection at sites under weak selection, such as selection on synonymous codons (Comeron and Guthrie 2005; McVean and Charlesworth 2000). A synonymous mutation is when there is a change in the genetic sequence of DNA, but the change results in the same genetic output. So a synonymous mutation is not essentially "silent" A non-synonymous mutation may not affect the phenotype (neutral mutation). However, when accounting for mutation rate variation between loci, using divergence at synonymous sites (dS) as a covariate, the association between the synonymous site diversity and the nonsynonymous substitution rate in fact becomes stronger, not weaker (partial correlation, ρC = − 0.324, P = 0.004; fig. Polymorphism data from a number of long-lived tree species have been shown to be consistent with a bottleneck sometime in their genealogical history (Heuertz et al. An alternative explanation is background selection where the removal of weakly deleterious mutations through purifying selection reduces local Ne of genome regions (Charlesworth et al. Nonsynonymous, obviously, changes the output enitrely, creating … In an effort to disentangle the direct and indirect effects of selection on synonymous codon usage and protein evolution on levels of polymorphism at synonymous sites in P. tremula, I included both variables in a multiple regression. 2004). At first hand, it may seem rather surprising that recurrent hitchhiking have such large effects on synonymous diversity, given that linkage disequilibrium does not extend more than a few hundred base pairs in P. tremula (Ingvarsson 2005; Ingvarsson 2008b). Multiple Regression of the Effects of Selection on Synonymous and Nonsynonymous Sites on Synonymous Site Diversity in Populus tremula. The results presented here show that two distinct selective processes are affecting levels of synonymous polymorphism in P. tremula as both selection on synonymous codon usage and protein evolution have significant effects on synonymous polymorphism.