The nearly neutral theory of molecular evolution predicts that slightly deleterious mutations subject to purifying selection are widespread in natural populations, particularly those of large effective population size. To test this hypothesis, the standardized difference between pairwise nucleotide difference and number of segregation sites (corrected for number of sequences) was estimated for 149 population data sets from 84 species of bacteria. This quantity (Tajima's D-statistic) was estimated separately for synonymous (D_syn) and nonsynonymous (D_non) polymorphisms. D_syn was positive in 70% of data sets, and the overall median D_syn (0.873) was positive. By contrast D_non was negative in 68% of data sets, and the overall median D_non (–0.656) was negative. The preponderance of negative values of D_non is evidence that there are widespread rare nonsynonymous polymorphisms in the process of being eliminated by purifying selection, as predicted to occur in populations with large effective size by the nearly neutral theory. The major exceptions to this trend were seen among surface proteins, particularly those of bacteria parasitic on vertebrates, which included a number of cases of polymorphisms apparently maintained by balancing selection.

---

THE concept of purifying or negative natural selection—i.e., natural selection acting to decrease the frequency of deleterious alleles—is one of the key ideas of modern evolutionary biology (KIMURA and OHTA 1974; KIMURA 1983). As first pointed out by KIMURA (1977), strong evidence for the widespread occurrence of purifying selection is provided by the fact that, in most comparisons between homologous genes, the number of synonymous nucleotide substitutions per synonymous site (d_S) generally exceeds the number of nonsynonymous nucleotide substitutions per nonsynonymous site (d_N; LI et al. 1985). However, this evidence provides no information regarding the overall strength of purifying selection, which is an important factor in the extent to which nonsynonymous polymorphisms are observed in populations.

If most nonsynonymous mutations are strongly deleterious, they will be eliminated from populations very quickly. On the other hand, the fate of slightly deleterious nonsynonymous mutations depends on effective population size (OHTA 1976). Purifying selection against slightly deleterious mutations is not efficient in populations with small effective size but is much more efficient as effective population size increases. Ohta's "nearly neutral" theory of molecular evolution emphasizes the importance of such slightly deleterious mutations in the evolutionary process (OHTA 1973, 1976, 2002). Some recent evidence in support of this theory is based on the gene diversity at single-nucleotide polymorphism (SNP) loci in the human population. Nonsynonymous SNPs—particularly those having radical effects on protein structure—tend to have lower average gene diversities than synonymous and noncoding SNPs in the same genes (FREUDENBERG-HUA et al. 2003; HUGHES et al. 2003; SUNYAEV et al. 2003; ZHAO et al. 2003). This pattern suggests that slightly deleterious alleles, which had drifted to relatively high allelic frequencies when the effective size of the human population was low (HARPENDING et al. 1998), have decreased in frequency as a result of purifying selection after population expansion (HUGHES et al. 2003).

TAJIMA (1989) pointed out that important inferences regarding population processes can be obtained from a sample of allelic DNA sequences by comparing the average number of pairwise nucleotide differences () with the number of segregating sites, corrected for the number of sequences compared. Specifically, if S is the number of segregating (or polymorphic) sites, we define S* = S/a₁, where

(1)

Both and S* are estimators of the population parameter M = 4Nu, where N is the effective population size and u is the mutation rate per generation per sequence under investigation. However, when rare variants are present in a population, S* will tend to be larger than . The difference between these two quantities, divided by its standard error, is known as Tajima's D-statistic; D represents an index of the action of natural selection on a sample of allelic sequences (TAJIMA 1989). When D is strongly positive, there are few rare variants, a situation found under balancing selection. When D is strongly negative, rare variants are abundant, a situation indicating purifying selection (TAJIMA 1989).

Several authors have extended TAJIMA's (1989) logic to examine synonymous and nonsynonymous polymorphisms separately (e.g., RAND and KANN 1996; WISE et al. 1998; NAVARRO-SABATé et al. 2003). Here I applied this method to 149 nucleotide sequence data sets from 84 species of bacteria. To avoid ambiguity, the analysis was restricted to codons at which only a single polymorphic site was observed. Computing the D-statistic separately for synonymous (D_syn) and nonsynonymous (D_non) polymorphisms makes it possible to test the hypothesis that rare variants are particularly common at nonsynonymous sites, indicating purifying selection against slightly deleterious alleles.

On the nearly neutral theory, purifying selection against slightly deleterious alleles is expected in bacteria, because most bacterial species are believed to have very large effective population sizes (LYNCH and CONERY 2003). FEIL et al. (2003) recently reported a high level of nonsynonymous nucleotide polymorphisms in Staphylococcus aureus and suggested that many of these may be deleterious mutations in the process of elimination by purifying selection. On the other hand, the bacterial species analyzed include several that are parasitic on vertebrates; certain proteins, particularly surface proteins exposed to the host immune system in these species, are known to be subject to positive selection (REID et al. 1999). Thus, these data make it possible to compare the effects on sequence polymorphism of both positive and purifying selection.

Data sets of protein-coding genes from bacteria (eubacteria) were chosen from the NCBI Popset database. No sets that represented laboratory isolates of a single strain were included, but only sets that had been collected as patient or environmental isolates or reference strains (e.g., American Type Culture Collection). I included only data sets for which functional information about the encoded protein was available, data sets containing at least four allelic sequences, and data sets including both synonymous and nonsynonymous polymorphisms at codons with only a single polymorphic site. Using these criteria, it was possible to find usable data sets for 7 of 21 bacterial phyla. The mean number of sequences per data set was 13.11 (±0.86 SE); the median was 11.00; the range was 4–64.

The sequences were aligned at the amino acid level using the CLUSTALW program (THOMPSON et al. 1994), and the resulting alignment was imposed on the DNA. Portions of any alignment that appeared unreliable on account of large numbers of gaps or poor sequence conservation and codons including undetermined nucleotides were removed. The numbers of synonymous substitutions per synonymous site (d_S) and of nonsynonymous substitutions per nonsynonymous site (d_N) were estimated by NEI and GOJOBORI's (1986) method. In preliminary analyses, more complex methods of estimating d_S and d_N (LI 1993; ZHANG et al. 1998; YANG and NEILSEN 2000) yielded essentially identical results, as is expected in a case like the present one where numbers of substitutions per site are low (NEI and KUMAR 2000). d_S and d_N were estimated for each pairwise comparison between homologous sequences in two ways: (1) for the entire sequence and (2) for the sequence excluding codons with two or more polymorphic sites. From these values, _S (the mean of all pairwise d_S values) and _N (the mean of all pairwise d_N values) were estimated for each data set.

After excluding codons with two or more polymorphic sites, the following quantities were computed: the average number of pairwise synonymous differences (_S) and the average number of nonsynonymous pairwise differences (_N), the number of synonymous segregating sites (S_S), and the number of nonsynonymous segregating sites (S_N). Then, I computed and , where a₁ is as in Equation 1 above. D_syn was defined as k_S – S^*_S, divided by the standard error of that difference (computed as described by TAJIMA 1989). Likewise, D_non was defined as k_N – S^*_N, divided by the standard error of that difference (computed as described by TAJIMA 1989).

Bacterial species were characterized as primarily parasitic on vertebrates if a vertebrate host constitutes a major portion of the species niche. Thus species that can be vertebrate pathogens but whose primary niche is not parasitism on vertebrates (e.g., Bacillus anthracis and Vibrio cholerae) were not included in the group of vertebrate parasites. Because surface proteins may be particularly important in interactions with the immune system of vertebrates, all proteins were categorized on the basis of surface or nonsurface expression.

Because the distributions of _S, _N, D_syn, and D_non across the 149 data sets deviated significantly from normality (Kolmogorov-Smironov test), nonparametric methods were used for testing hypotheses regarding differences among data sets.

The occurrence of polymorphic sites per codon was compared with that expected, assuming a Poisson distribution with µ equal to the observed frequency of polymorphic sites per codon (Figure 1). The hypothesis that the median differerence between observed and expected proportions of codons having a given number of polymorphic sites equaled zero was tested by Wilcoxon signed-rank tests (Figure 1). There were significantly fewer codons than expected with zero or two substitutions, but significantly more codons than expected with one substitution (Figure 1). Thus, the distribution of polymorphic sites in these data showed overall a more dispersed pattern than did a Poisson distribution.

View larger version (10K): In this window In a new window Download PPT slide   FIGURE 1.— Median pairwise difference between observed and expected (assuming a Poisson distribution) proportions of codons with zero to three polymorphic nucleotides in the 149 data sets. Tests of the equality of the median proportion observed and the median proportion expected (Wilcoxon signed-rank test) are **P < 0.01 and ***P < 0.001.

 
For each data set, similar results were obtained when _S and _N were estimated for the entire sequence and for the sequence excluding codons with two or three polymorphic sites. In both cases, median _S (0.0836 and 0.0835, respectively) was significantly greater than median _N (0.0070 and 0.0044, respectively). In seven data sets _N exceeded _S by both methods of computation, but in none of these was there a significant difference between _N and _S (z-test).

D_syn was positive in 104 (69.8%) of the 149 data sets, and median D_syn (0.8726) was positive (Figure 2). On the other hand, D_non was negative in 102 (69.4%) of data sets, and median D_non (–0.6555) was negative (Figure 2). Median D_syn exceeded D_non in 123 (82.6%) of 149 data sets, and the two medians differed significantly from each other (Wilcoxon signed-rank test; P < 0.001; Figure 2). Also both median D_syn and median D_non were significantly different from zero (Wilcoxon signed-rank test; P < 0.001 in each case).

View larger version (10K): In this window In a new window Download PPT slide   FIGURE 2.— Plot of Dnon vs. Dsyn for the 149 data sets. The line is a 45° line. Median Dsyn was significantly greater than median Dnon (Wilcoxon signed-rank test, P < 0.001).

 
Overall, D_syn and D_non were positively correlated (Spearman's rank correlation coefficient, r_S = 0.236; P = 0.004; Figure 2). An examination of the data showed six data sets with high positive values of both D_non and D_syn. These unusual data sets were the following (with D_syn and D_non values, respectively, in parentheses): Borrelia burgdoerferi flagellin (8.8576, 2.4235), Clostridium difficile adhesin (4.2776, 2.9993) and S-layer protein (3.5102, 1.9122), Streptococcus pneumoniae cspB (4.9233, 1.6862), Vibrio cholerae toxin coregulated pilus subunit (10.1966, 2.0389), and Wolbachia pipientis surface protein wsp (4.2608, 3.1798). When these six data sets were excluded, there was no longer a significant correlation between D_syn and D_non (r_S = 0.147; NS).

Bacteria were categorized in terms of parasitism on vertebrates and in terms of surface expression of the proteins, and median _S and _N were computed for each category (Figure 3). Figure 3 illustrates median _S and _N computed excluding codons with two or three polymorphic sites; similar results were seen when the latter codons were included (data not shown). There was no significant difference with respect to median _S among categories (Figure 3). However, there was a highly significant difference among categories with respect to median _N (Kruskal-Wallis test; P < 0.001). Median _N was much higher for surface proteins from bacteria parasitic on vertebrates than for the other three categories (Figure 3B).

View larger version (15K): In this window In a new window Download PPT slide   FIGURE 3.— Median values of (A) S and (B) N for data sets categorized by parasitism on vertebrates and surface expression of the protein. There was no significant difference among categories with respect to median S (Kruskal-Wallis test), but median N differed significantly among categories (Kruskal-Wallis test, P < 0.001).

 
Similar patterns were seen when median D_syn and D_non were compared across categories (Figure 4). No significant differences among categories were observed with respect to median D_syn, but median D_non differed significantly among categories (Kruskal-Wallis test; P = 0.035). Median D_non was higher for surface proteins from bacteria parasitic on vertebrates than for the other three categories (Figure 4B). Four of the six data sets identified as having unusually high values of both D_syn and D_non were surface proteins of bacteria primarily parasitic on vertebrates, namely B. burgdoerferi flagellin, C. difficile adhesin and S-layer protein, and S. pneumoniae cspB. Yet even when these six data sets were excluded from the analysis, there was still a significant difference among categories with respect to median D_non (Kruskal-Wallis test; P = 0.04), and the highest median D_non (–0.1683) was still seen in surface proteins of bacteria primarily parasitic on vertebrates.

View larger version (12K): In this window In a new window Download PPT slide   FIGURE 4.— Median values of (A) Dsyn and (B) Dnon for data sets categorized by parasitism on vertebrates and surface expression of the protein. There was no significant difference among categories with respect to median Dsyn (Kruskal-Wallis test), but median Dnon differed significantly among categories (Kruskal-Wallis test, P < 0.001).

 
It is well known that bacterial species have characteristic patterns of codon usage, and it has been proposed that bacterial codon usage is selectively maintained (GRANTHAM et al. 1980). To test whether the trends in _N and D_non (Figures 3B and 4B) were correlated with codon usage, the median percentage of G + C at third-codon positions (GC3) was compared across data sets (Figure 5). The data in Figure 5 were computed excluding codons with two or three polymorphic sites; similar results were seen when the latter codons were included (data not shown). Although there were significant differences among categories (Figure 5), there was no obvious relationship between the pattern of GC3 variation and that of _N and D_non.

View larger version (11K): In this window In a new window Download PPT slide   FIGURE 5.— Median percentage of G + C at third-codon positions (GC3) for data sets categorized by parasitism on vertebrates and surface expression of the protein. There was a significant difference among categories with respect to median GC3 (Kruskal-Wallis test, P = 0.004).

 
TAJIMA (1989) provides a formula for estimating the total number of deleterious mutants per DNA sequence when the D-statistic is negative. For codons with a single polymorphic site in the present data set, this method yielded the estimates that 25 of 5248 synonymous polymorphisms (0.48%) are deleterious and 58 of 1840 nonsynonymous polymorphisms (3.15%) are deleterious. Thus the ratio of the proportions of deleterious mutations and nonsynonymous sites to that at synonymous sites is 6.6:1. This ratio is over twice the ratio of nonsynonymous to synonymous sites in the data set (79,806:24,621 or 3.24:1).

An examination of DNA sequence of polymorphism in 149 data sets from 84 bacterial species showed a significant preponderance of negative D_non values but not of D_syn values. This pattern indicates the widespread occurrence of relatively rare nonsynonymous polymorphisms but not of synonymous polymorphisms. The abundance of such rare polymorphisms at nonsynonymous but not synonymous sites in the same genes is in turn evidence that bacterial populations harbor abundant slightly deleterious nonsynonymous allelic substitutions, which are subject to ongoing purifying selection.

It is believed that, in many bacterial species, synonymous codon usage is subject to selection relating to tRNA abundance or other factors (GRANTHAM et al. 1980). Thus, purifying selection at synonymous as well as nonsynonymous sites might be expected to occur in bacteria. The present results suggested that such selection on synonymous sites is a minor factor in comparison to that on nonsynonymous sites. Deleterious mutations were estimated to be present at only 0.48% of synonymous sites, in comparison to 3.15% of nonsynonymous sites. These results are consistent with earlier estimates that the selection coefficients at synonymous sites in bacteria are quite low (BULMER 1991).

Deleterious mutations present in the bacterial populations examined here presumably largely represent nearly neutral mutations, i.e., those whose selection coefficients are close to the reciprocal of the effective population size (OHTA and GILLESPIE 1996). Deleterious synonymous mutations are expected to have very low selection coefficients (BULMER 1991). By contrast, at least some nonsynonymous mutations are expected to be strongly deleterious because they damage protein structure and thereby render the cell nonviable. These mutations are likely to be eliminated very quickly and thus not appear as polymorphisms within populations. The low d_N/d_S ratios reported for bacteria largely reflect the elimination of such strongly deleterious mutations. For example, in a comparison of >14,000 phylogenetically independent pairs of genes between closely related pairs of bacterial species, FRIEDMAN et al. (2004) found a mean d_N/d_S ratio of 0.14; and JORDAN et al. (2002) reported similar values. On the other hand, the present results indicate that there are abundant nearly neutral nonsynonymous mutations present in bacterial populations, where they are subject to ongoing purifying selection.

The eventual fate of nearly neutral mutations is predicted by the nearly neutral theory of molecular evolution to be determined by a combination of selection and drift (OHTA 1973, 1976, 2002). The present results support the prediction of this theory that selection against slightly deleterious mutations is likely to be efficient in species with large effective population sizes, since most bacterial species have very large effective population sizes (LYNCH and CONERY 2003). Thus, the present results are consistent with JORDAN et al.'s (2002) observation of a lower d_N/d_S ratio in Escherichia coli than in three other bacterial species. Since E. coli was believed to have a larger effective population size than the other species examined, JORDAN et al. (2002) interpreted their results as support for the prediction of the nearly neutral theory that purifying selection will be most effective in species with large effective population sizes.

In addition, the fact that evidence for purifying selection was found in the case of nonsynonymous but not synonymous sites supports the hypothesis that slightly deleterious mutations are more likely to be nonsynonymous than synonymous because of the effect of the former on protein structure. In the present data, deleterious nonsynonymous mutations were estimated to exceed deleterious synonymous mutations by a ratio over twice the ratio of nonsynonymous to synonymous sites.

The fact that a majority of data sets analyzed had positive D_syn is most likely to be due to a certain degree of population subdivision in the bacterial species studied (KREITMAN 2000). In the case of bacteria, limits on the extent of recombination are expected to impose limits on gene flow among clonal lineages and thus to create population subdivision. However, it is important to recognize that the present results are inconsistent with an entirely clonal population structure in the bacterial species examined. The evidence of purifying selection reducing the frequency of slightly nonsynonymous variants implies recombination, since without recombination such variants cannot be purged from a given genetic background. The present results are thus consistent with numerous other studies showing evidence of both small-scale and large-scale recombination events among bacterial genomes (e.g., NELSON et al. 1997; MCGRAW et al. 1999; HUGHES and FRIEDMAN 2004).

A small number of the data sets analyzed here showed high positive values of both D_non and D_syn, suggestive of balancing selection. Of the six data sets with the highest D_non and D_syn, all encoded surface proteins, and four encoded surface proteins of bacterial species classified as parasitic on vertebrates. Because vertebrates possess an immune system capable of specific recognition of foreign proteins, it is expected that the parasitic organisms exposed to vertebrate immune mechanisms will be under selective pressure to evade this recognition (FRANK 2002). As a result, organisms parasitic on vertebrates are expected to be subject to balancing selection favoring amino acid sequence diversity of immunogenic proteins, including cell-surface proteins (e.g., HUGHES and HUGHES 1995).

The data sets with high positive values of both D_non and D_syn that were not classified among surface proteins of vertebrate parasites were the toxin coregulated pilus subunit of V. cholerae and the W. pipientis surface protein wps. As mentioned previously (see MATERIALS AND METHODS), V. cholerae was not classified as a vertebrate parasite for the purpose of analyses because the vast majority of known V. cholerae strains belong to the natural flora of the aquatic environment (FARUQUE and MEKALANOS 2003). However, strains pathogenic on humans have acquired by horizontal gene transfer plasmids, phages, and chromosomal gene clusters ("pathogenicity islands"). The toxin coregulated pilus subunit gene forms part of such a pathogenicity island and the protein it encodes is thus exposed to vertebrate immune recognition (KOTETISHVILI et al. 2003).

On the other hand W. pipientis is a maternally transmitted obligate parasite of insects that influences its host's life history (TSUTSUI et al. 2003). The present results provide evidence for balancing selection on a surface protein (wsp) of this species. This in turn suggests that polymorphism at the locus encoding this protein may play a role in the evolutionary dynamics of the host-parasite relationship.

This research was supported by grant GM43940 from the National Institutes of Health.

BULMER, M., 1991 The selection-mutation-drift theory of synonymous codon usage. Genetics 129: 897–907.[Abstract]

FARUQUE, S. M., and J. J. MEKALANOS, 2003 Pathogenicity islands and phages in Vibrio cholerae evolution. Trends Microbiol. 11: 505–510.[CrossRef][Medline]

FEIL, E. J., J. E. COOPER, H. GRUNDMANN, D. A. ROBINSON, M. C. ENRIGHT et al., 2003 How clonal is Staphylococcus aureus? J. Bacteriol. 185: 3307–3316.[Abstract/Free Full Text]

FRANK, S. A., 2002 Immunology and Evolution of Infectious Disease. Princeton University Press, Princeton, NJ.

FREUDENBERG-HUA, Y., J. FREUDENBERG, N. KLUCK, S. CICHON, P. PROPPING et al., 2003 Single nucleotide variation analysis in 65 candidate genes for CNS disorders in a representative sample of the European population. Genome Res. 13: 2271–2276.[Abstract/Free Full Text]

FRIEDMAN, R., J. DRAKE and A. L. HUGHES, 2004 Genome-wide patterns of nucleotide substitution reveal stringent functional constraints on the protein sequences of thermophiles. Genetics 167: 1507–1512.[Abstract/Free Full Text]

GRANTHAM, R., C. GAUTIER, M. GOUY, R. MERCIER and A. PAVE, 1980 Codon catalog usage and the genome hypothesis. Nucleic Acids Res. 8: r49–r62.

HARPENDING, H. C., M. A. BATZER, M. GURVEN, L. B. JORDE, A. R. ROGERS et al., 1998 Genetic traces of ancient demography. Proc. Natl. Acad. Sci. USA 95: 1961–1967.[Abstract/Free Full Text]

HUGHES, A. L., and R. FRIEDMAN, 2004 Patterns of sequence divergence in 5' intergenic spacers and linked coding regions in 10 species of pathogenic bacteria reveal distinct recombinational histories. Genetics 168: 1795–1803.[Abstract/Free Full Text]

HUGHES, A. L., B. PACKER, R. WELCH, A. W. BERGEN, S. J. CHANOCK et al., 2003 Widespread purifying selection at polymorphic sites in human protein-coding loci. Proc. Natl. Acad. Sci. USA 100: 15754–15757.[Abstract/Free Full Text]

HUGHES, M. K., and A. L. HUGHES, 1995 Natural selection on Plasmodium surface proteins. Mol. Biochem. Parasitol. 71: 99–113.[CrossRef][Medline]

JORDAN, I. K., I. B. ROGOZIN, Y. I. WOLF and E. V. KOONIN, 2002 Microevolutionary genomics of bacteria. Theor. Popul. Biol. 61: 435–447.[CrossRef][Medline]

KIMURA, M., 1977 Preponderance of synonymous changes as evidence for the neutral theory of molecular evolution. Nature 267: 275–276.[CrossRef][Medline]

KIMURA, M., 1983 The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge, UK.

KIMURA, M., and T. OHTA, 1974 On some principles governing molecular evolution. Proc. Natl. Acad. Sci. USA 71: 2848–2852.[Abstract/Free Full Text]

KOTETISHVILI, M., O. C. STINE, Y. CHEN, A. KREGER, A. SULAKVELIDZE et al., 2003 Multilocus sequence typing has better discriminatory ability for typing Vibrio cholerae than does pulsed-field gel electrophoresis and provides a measure of phylogenetic relatedness. J. Clin. Microbiol. 41: 2191–2196.[Abstract/Free Full Text]

KREITMAN, M., 2000 Methods to detect selection in populations with applications to the human. Annu. Rev. Genomics Hum. Genet. 1: 539–559.[CrossRef][Medline]

LI, W.-H., 1993 Unbiased estimates of the rates of synonymous and nonsynonymous substitution. J. Mol. Evol. 36: 96–99.[CrossRef][Medline]

LI, W.-H., C.-C. LUO and C.-I WU, 1985 Evolution of DNA sequences, pp. 1–94 in Molecular Evolutionary Genetics, edited by R. J. MACINTYRE. Plenum Press, New York.

LYNCH, M., and J. S. CONERY, 2003 The origins of genome complexity. Science 302: 1401–1404.[Abstract/Free Full Text]

MCGRAW, E. A., J. LI, R. K. SELANDER and T. S. WHITTAM, 1999 Molecular evolution and mosaic structure of , ß, and intimins of pathogenic Escherichia coli. Mol. Biol. Evol. 16: 12–22.[Abstract]

NAVARRO-SABATé, À., M. AGUADé and C. SEGARRA, 2003 Excess of nonsynonymous polymorphism at Acph-1 in different gene arrangements of Drosophila subobscura. Mol. Biol. Evol. 20: 1833–1843.[Abstract/Free Full Text]

NEI, M., and T. GOJOBORI, 1986 Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3: 418–426.[Abstract]

NEI, M., and S. KUMAR, 2000 Molecular Evolution and Phylogenetics. Oxford University Press, New York.

NELSON, K., F.-S. WANG, E. F. BOYD and R. K. SELANDER, 1997 Size and sequence polymorphism in the isocitrate dehydrogenase kinase/phosphatase gene (aceK) and flanking regions in Salmonella enterica and Escherichia coli. Genetics 147: 1509–1520.[Abstract]

OHTA, T., 1973 Slightly deleterious mutations in evolution. Nature 246: 96–98.[CrossRef][Medline]

OHTA, T., 1976 Role of very slightly deleterious mutations in molecular evolution and polymorphism. Theor. Popul. Biol. 10: 254–275.[CrossRef][Medline]

OHTA, T., 2002 Near-neutrality in evolution of genes and gene regulation. Proc. Natl. Acad. Sci. USA 99: 16134–16137.[Abstract/Free Full Text]

OHTA, T., and J. H. GILLESPIE, 1996 Development of neutral and nearly neutral theories. Theor. Popul. Biol. 49: 128–142.[CrossRef][Medline]

RAND, D. M., and L. M. KANN, 1996 Excess amino acid polymorphism in mitochondrial DNA: contrasts among Drosophila, mice, and humans. Mol. Biol. Evol. 13: 735–748.[Abstract]

REID, S. D., R. K. SELANDER and T. S. WHITTAM, 1999 Sequence diversity of flagellin (fliC) alleles in pathogenic Escherichia coli. J. Bacteriol. 181: 153–160.[Abstract/Free Full Text]

SUNYAEV, S., F. A. KONDRASHOV, P. BORK and V. RAMENSKY, 2003 Impact of selection, mutation rate and genetic drift on human genetic variation. Hum. Mol. Genet. 15: 3325–3330.

TAJIMA, F., 1989 Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123: 585–595.[Abstract/Free Full Text]

THOMPSON, J. D., D. G. HIGGINS and T. GIBSON, 1994 CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680.[Abstract/Free Full Text]

TSUTSUI, N. D., S. N. KAUPPINEN, A. F. OFAYUSO and R. K. GROSBERG, 2003 The distribution and evolutionary history of Wolbachia infection in native and introduced populations of the invasive argentine ant (Linepithema humile). Mol. Ecol. 12: 3057–3068.[CrossRef][Medline]

WISE, C. A., M. SRAML and S. EASTEAL, 1998 Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans, but not in chimpanzees. Genetics 148: 409–421.[Abstract/Free Full Text]

YANG, Z., and R. NIELSEN, 2000 Estimating synonymous and nonsynonymous substitution rates under realistic evolutionary models. Mol. Biol. Evol. 17: 32–43.[Abstract/Free Full Text]

ZHANG, J., H. F. ROSENBERG and M. NEI, 1998 Positive Darwinian selection after gene duplication in primate ribonuclease genes. Proc. Natl. Acad. Sci. USA 98: 3708–3713.

ZHAO, Z., Y.-X. FU, D. HEWETT-EMMETT and E. BOERWINKLE, 2003 Investigating single nucleotide polymorphism (SNP) density in the human genome and its implications for molecular evolution. Gene 312: 207–213.[CrossRef][Medline]

This article has been cited by other articles:

				J. Seger, W. A. Smith, J. J. Perry, J. Hunn, Z. A. Kaliszewska, L. L. Sala, L. Pozzi, V. J. Rowntree, and F. R. Adler Gene Genealogies Strongly Distorted by Weakly Interfering Mutations in Constant Environments Genetics, February 1, 2010; 184(2): 529 - 545. [Abstract] [Full Text] [PDF]

				A. L. Hughes, R. Friedman, P. Rivailler, and J. O. French Synonymous and Nonsynonymous Polymorphisms versus Divergences in Bacterial Genomes Mol. Biol. Evol., October 1, 2008; 25(10): 2199 - 2209. [Abstract] [Full Text] [PDF]

				S. J. Irausquin and A. L. Hughes Distinctive pattern of sequence polymorphism in the NS3 protein of hepatitis C virus type 1b reflects conflicting evolutionary pressures J. Gen. Virol., August 1, 2008; 89(8): 1921 - 1929. [Abstract] [Full Text] [PDF]

				J. Charlesworth and A. Eyre-Walker The McDonald-Kreitman Test and Slightly Deleterious Mutations Mol. Biol. Evol., June 1, 2008; 25(6): 1007 - 1015. [Abstract] [Full Text] [PDF]

				J. Charlesworth and A. Eyre-Walker The other side of the nearly neutral theory, evidence of slightly advantageous back-mutations PNAS, October 23, 2007; 104(43): 16992 - 16997. [Abstract] [Full Text] [PDF]

				O. G. Pybus, A. Rambaut, R. Belshaw, R. P. Freckleton, A. J. Drummond, and E. C. Holmes Phylogenetic Evidence for Deleterious Mutation Load in RNA Viruses and Its Contribution to Viral Evolution Mol. Biol. Evol., March 1, 2007; 24(3): 845 - 852. [Abstract] [Full Text] [PDF]

				M. S. Snoke, T. U. Berendonk, D. Barth, and M. Lynch Large Global Effective Population Sizes in Paramecium Mol. Biol. Evol., December 1, 2006; 23(12): 2474 - 2479. [Abstract] [Full Text] [PDF]

				J. Charlesworth and A. Eyre-Walker The Rate of Adaptive Evolution in Enteric Bacteria Mol. Biol. Evol., July 1, 2006; 23(7): 1348 - 1356. [Abstract] [Full Text] [PDF]

				J. H. McDonald Apparent Trends of Amino Acid Gain and Loss in Protein Evolution Due to Nearly Neutral Variation Mol. Biol. Evol., February 1, 2006; 23(2): 240 - 244. [Abstract] [Full Text] [PDF]

				M. Nei Selectionism and Neutralism in Molecular Evolution Mol. Biol. Evol., December 1, 2005; 22(12): 2318 - 2342. [Abstract] [Full Text] [PDF]

				B. Tummler and P. Cornelis Pyoverdine Receptor: a Case of Positive Darwinian Selection in Pseudomonas aeruginosa J. Bacteriol., May 15, 2005; 187(10): 3289 - 3292. [Full Text] [PDF]

International Access Link

Online ISSN: 1943-2361  Print ISSN: 0016-6731
Copyright 2009 by the Genetics Society of America
phone: 412-268-1812   fax: 412-268-1813   email: genetics-gsa{at}andrew.cmu.edu