Evidence for Genetic Drift in Endosymbionts (Buchnera): Analyses of

Protein-Coding Genes
J. J. Wernegreen and N. A. Moran
Department of Ecology and Evolutionary Biology, University of Arizona

Introduction
The rate of fixation of mutations with fitness consequences depends not only on the strength of selection
for or against them but also on the effectiveness of such
selection as influenced by effective population size. In
populations with low rates of recombination and small
effective sizes, slightly deleterious mutations may experience increased rates of fixation through drift (Ohta
1973). This predicted relationship between population
structure and rate of fixation of slightly deleterious mutations can be tested among prokaryotes. Free-living
bacteria are thought to have large effective population
sizes (Selander, Caugant, and Whittam 1987), and even
clonal groups experience recombination that is important in their evolutionary dynamics (Maynard Smith,
Dowson, and Spratt 1991; Dykhuizen and Green 1993;
Maynard Smith et al. 1993).
In contrast, endosymbiotic bacteria associated with
several insect groups have relatively small effective population sizes and have restricted opportunities for interstrain recombination because of their mode of transmission. Bacteria associated with specialized insect cells
(i.e., mycetocytes) are maternally transmitted by the infection of ovaries or of internally developing embryos
(reviewed in Buchner 1965; Moran and Baumann 1994;
Baumann et al. 1995). The effective population size of
the bacteria is reduced by the bottleneck at each inoculation of progeny, where relatively few bacteria are
Abbreviations: CAI5 Codon Adaptation Index; Nc5 effective number of codons; GC35 percent G1C content at third-codon positions.
Key words: Buchnera, endosymbionts, codon bias, drift, population
size.
Address for correspondence and reprints: Jennifer Wernegreen, Department of Ecology and Evolutionary Biology, University of Arizona,
Biological Sciences West, Room 310, Tucson, Arizona 85721. E-mail:
werjen@u.arizona.edu.
Mol. Biol. Evol. 16(1):83–97. 1999
q 1999 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038

transmitted (Hinde 1971; A. Mira, personal communication). In addition, modeling indicates that insect host
population sizes may be the primary determinant of the
effective population size of intracellular genomes (C.
Rispe, personal communication). Insect population sizes, while relatively large among animals, are much
smaller than those of free-living bacteria (reviewed in
Lambert and Moran 1998). Finally, any lateral gene
transfer among endosymbionts would be confined to the
bacterial genotypes present in the same host individual,
and the tight bottleneck at transmission implies that
these would be similar or identical. Buchnera, the endosymbionts of aphids, are particularly well characterized, and the perfect congruence between symbiont and
host phylogenies supports anatomical evidence for their
stable, vertical inheritance (Munson et al. 1991; Moran
and Baumann 1994). The goals of this study are to explore the effects of this strict asexuality and small population size on sequence evolution in Buchnera and to
test the hypothesis that Buchnera lineages experience
increased rates of substitution of slightly deleterious mutations.
Codon Bias
The use of alternative codons may be shaped by
biases in mutation rates among the four bases (Suoeka
1961; Muto and Osawa 1987), by selection for the use
of optimal codons to maximize rates and efficiency of
translation (Ikemura 1981, 1985), or by a combination
of these processes. Studies of codon usage often attempt
to distinguish the relative importance of genome nucleotide composition and selection for translational efficiency by testing alternative predictions of these models.
In cases where patterns of codon usage largely reflect
mutational pressure and drift rather than translational selection, codon bias is expected to correspond with local
base compositional biases. This pattern characterizes genomes of vertebrates and some bacterial genomes with
83

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Buchnera, the bacterial endosymbionts of aphids, undergo severe population bottlenecks during maternal transmission through their hosts. Previous studies suggest an increased effect of drift within these strictly asexual, small
populations, resulting in an increased fixation of slightly deleterious mutations. This study further explores sequence
evolution in Buchnera using three approaches. First, patterns of codon usage were compared across several homologous Escherichia coli and Buchnera loci, in order to test the prediction that selection for the use of optimal
codons is less effective in small populations. A x2-based measure of codon bias was developed to adjust for the
overall A1T richness of silent positions in the endosymbionts. In contrast to E. coli homologues, adaptive codon
bias across Buchnera loci is markedly low, and patterns of codon usage lack a strong relationship with gene
expression level. These data suggest that codon usage in Buchnera has been shaped largely by mutational pressure
and drift rather than by selection for translational efficiency. One exception to the overall lack of bias is groEL,
which is known to be constitutively overexpressed in Buchnera and other endosymbionts. Second, relative-rate tests
show elevated rates of sequence evolution of numerous protein-coding loci across Buchnera, compared to E. coli.
Finally, consistently higher ratios of nonsynonymous to synonymous substitutions in Buchnera loci relative to the
enteric bacteria strongly suggest the accumulation of nonsynonymous substitutions in endosymbiont lineages. Combined, these results suggest a decreased effectiveness of purifying selection in purging endosymbiont populations
of slightly deleterious mutations, particularly those affecting codon usage and amino acid identity.

For example. Wright and Bibb 1992. Nc plots. relatively weak selection may produce strong codon biases. and the A1T richness of the Buchnera genome (Ishikawa 1987) is considered in testing for codon bias. In the A1T–rich Rickettsia genome. Ohtaka and Ishikawa 1993). 1998.oxfordjournals. for example. Therefore. asexual populations are expected to exhibit lower overall levels of codon bias because of weakness of translational selection. These studies suggest that codon usage in Buchnera largely reflects strong A1T mutational bias and fixation of nonoptimal codons through drift. and CAI estimates may fail to detect slight preferences among synonyms. a correlation between the degree of codon bias and the gene expression level (Gouy and Gautier 1982. Ohtaka. coli homologues are included to represent a wide range of gene expression levels. In particular. In the analysis presented in this study. similar levels of codon bias across Rickettsia genes with very different expression levels indicate that mutational bias has a stronger effect than translational selection. coli (Clark. In contrast. Among bacterial genomes with base compositional biases. and Ishikawa 1992. drift may also allow rare transitions in coadapted states (Wright 1931). previous estimates of codon bias in Buchnera do not account for local base composition. ‘‘Nc-plots’’ show an agreement between observed Nc values and those expected. Bulmer 1991). Where they do exhibit bias. patterns of codon usage often reflect a combination of mutational pressure and translational selection (Shields and Sharp 1987. In other taxa. In large populations. Ohtaka and Ishikawa 1993). In Escherichia coli and in yeast. 1988. This combination of mutational bias and translational selection is also apparent for other genomes with mutational biases. However. to an expected distribution of CAIs based on genome-wide nucleotide composition (Morton 1998). the effective number of codons. relative to chloroplasts of flowering plants.org/ by guest on August 5. which may allow the maintenance of nonoptimal codons. is reduced by preferences for particular codons or biased base composition. such as the links between codon frequencies and tRNA abundances (Shields 1990). Brynnel et al. given the strong A1T bias at synonymous sites. preferred codons in tuf are also preferred by another G1C–rich bacterium. and the fact that. small. Nakamura. previous analyses of protein-coding genes in Buchnera show a general A1T richness at synonymous sites and a lack of strong preferences for the optimal codons of the closely related species E.84 Wernegreen and Moran sample of Buchnera and E. Baumann. Ohtaka. Population genetic models indicate that effective population size influences the balance between the effects of any mutational bias and selection for optimal codons (Li 1987. such as Micrococcus luteus (Ohtaka. weak translational selection is countered by a strong effect of drift. and Baumann 1992. 2015 strong A1T or G1C mutational biases (Sharp et al. an effect of translational selection is evidenced by a positive relationship between the extent of codon bias and level of gene expression. However. In addition. or bias toward a pool of preferred codons (here. CAI estimates are possible only when the optimal codons for a particular genome are known. indicating that codon usage reflects local base composition and may therefore be attributed largely to mutational bias (Andersson and Sharp 1996). In this study. and Ishikawa 1992. translational selection will be effective in a haploid population only when the selective coefficient (s) for a particular codon is greater than 2/Ne (Li 1987). Sharp et al. In addition. expected values of Nc may be adjusted to account for local base composition. the combined effects of mutational bias and translational selection are apparent. Ohtaka and Ishikawa 1993. apparently. Nakamura. since these methods derive a single estimate across all amino acids in a locus. The relative importance of selection and genome composition in shaping codon usage of several A1T–biased chloroplast genomes was recently tested by comparing an observed CAI (Sharp and Li 1987). Clark. correspondence analysis. Ohtaka and Ishikawa 1993. the bias may favor synonyms other than the optimal codons of related lineages because of switches in codon preferences. Relatively few empirical studies explicitly test the effect of population size on the balance between these two processes. Baumann. estimates of codon bias across Buchnera loci are also Downloaded from http://mbe. The analyses above test the null hypothesis that codon usage may be explained solely by local base composition. Wright and Bibb 1992. However. A slight effect of translational selection on the highly expressed Streptomyces tuf gene is supported by the relatively low Nc for this locus. a more extensive . and Bacillus subtilis (Shields and Sharp 1987). The high levels of codon bias in chloroplasts of most algal lineages. on the basis of a highly expressed chloroplast gene). a strong effect of mutational bias is suggested by the correspondence of GC3 and Nc and by a correlation between the GC3 of a locus and the locus’ position along the major axis in correspondence analysis of codon usage (Wright and Bibb 1992). Previous studies of codon usage in A1T– and G1C–rich genomes highlight methods for assessing codon usage in genomes with strong mutational biases (Shields and Sharp 1987. Nc. its clear distinction from other loci in correspondence analysis. Likewise. so it is difficult to identify any preferences for particular codons. in small populations. 1988) is thought to reflect selection for the rapid translation of highly expressed genes through the use of optimal codons and a lack of such selection on lowly expressed genes and their retention of nonoptimal codons (Shields 1990). Across several Streptomyces loci. One such study compares levels of codon bias of chloroplast genes across several algal and angiosperm lineages (Morton 1998). In order to test the null hypothesis that codons are used randomly except for the influence of local mutational bias. Organelle genomes may also show strong nucleotide biases. given the GC3. Micrococcus luteus (Wright and Bibb 1992). Tuohy. and Ishikawa 1992. Andersson and Sharp 1996). Andersson and Sharp 1996). Dictyostelium discoideum (Sharp and Devine 1989). and Mosurski 1986. Likewise. Nakamura. and Baumann 1998). Sharp. supports the hypothesis that selection for translational efficiency is more effective in large (algal) populations than in relatively small (angiosperm) populations.

the x2 values in this study reflect the deviation of relative frequencies of U. Regions of loci with ambiguous amino acid alignments were excluded from the analysis. For example. Codon Bias Estimation of Codon Bias at Fourfold Degenerate Sites For each Buchnera locus. Expected relative frequencies of U. Madison. 1988). codon preferences in Buchnera. 1998). coli. personal communication). Similar to the scaled x2 (Shields et al. we compared observed codon frequencies to those expected if codon usage reflects local base composition at synonymous sites. The x2 analysis involved several steps: first. as modified by Akashi and Shaeffer (1997) to adjust for A1T content at silent positions.and A-ending codons for each fourfold codon family were based on the relative frequencies of A’s and T’s at fourfold degenerate sites (calculated by MEA). leucine. and observed changes in endosymbiont 16S rRNA genes destabilize the secondary structure of the molecule (Lambert and Moran 1998). (Arginine. and serine were treated as fourfold degenerate by considering only the fourfold degenerate synonyms. Since purifying selection against nonoptimal codons is likely to be weaker than selection against replacement substitutions. and patterns of nonsynonymous and synonymous substitutions.and A-ending codons from frequencies expected if codon usage reflects local base composition. adaptive codon bias is expected to be much lower than that observed in E. coli homologues (Moran 1996. Wis.org/ by guest on August 5. In this study. Genetics Computer Group. observed and expected relative values of U.).) Cand G-ending codons were excluded from the analysis because of their small sample sizes and low expected values in this A1T-rich genome (see below). 2015 Rates of Sequence Divergence The decreased effectiveness of selection in small. coli are compared across an extensive sample of available protein-coding loci. and nucleotide alignments were adjusted to conform to amino acid alignments.5–2 times faster (Moran. Alignments Inferred protein sequences of homologous loci were aligned using Pileup of GCG (Wisconsin Sequence Analysis Program. x2 values were averaged across the fourfold codon families after excluding those amino acids with fewer than five representatives. Moriyama. previous evidence for the accumulation of nonsynonymous substitutions through drift strongly suggests that nonoptimal codons will accumulate in Buchnera lineages.and A-ending codons within fourfold degenerate codon families was assessed using a x2 analysis. compare levels of bias with homologues in E. This approach may be generally applicable to other organisms in which codon preferences may be absent or subtle. von Dohlen. several protein-coding genes in Buchnera have been shown to evolve more rapidly than their E.200.Evidence for Drift in Endosymbionts adjusted for local base composition. rates of sequence evolution. the x2-based method developed here tests for nonrandom-use codons for single amino acids and does not require prior knowledge of preferred codons. In addition. Downloaded from http://mbe. Second. such as in taxa with small effective population sizes and/or strong mutational biases. Values near saturation are known to have high standard errors. nucleotide composition at fourfold degenerate sites was determined for each Buchnera locus (by the computer package Molecular Evolutionary Analysis [MEA]. However. possibly different. in contrast to previous estimates. as suggested by Sokal and Rohlf (1981) for sample sizes . were limited by high levels of synonymous divergence (Moran 1996). and Baumann 1995). and explore the possibility of subtle. rates of sequence evolution of Buchnera and E.and A-ending codons were compared by a twoclass x2 test adjusted for a small sample size. which may be exacerbated by the strong A1T bias of Buchnera genomes (Berg 1995). Brynnel et al. Amino acids with less than five residues in a given locus were omitted. the 16S rRNA gene of several endosymbiotic lineages has been shown to evolve 1. Here we assess patterns of codon usage across several Buchnera loci. E. Previous estimates of the ratio of nonsynonymous to synonymous divergence in Buchnera 85 . This study combines several types of analyses to further explore whether Buchnera loci experience increased rates of fixation of deleterious mutations. In order to estimate overall bias at a given locus. so that codon usage in Buchnera is shaped by A1T mutational bias and by the fixation of nonoptimal codons through drift. asexual populations. the consideration of shallower taxonomic levels allows for more reliable estimates of ratios of nonsynonymous to synonymous substitutions. However. the magnitude of the x2 value reflects the deviation of random use of synonymous codons. in order to test whether previously observed rate elevation is a general phenomenon across the Buchnera genome. compared to freeliving relatives in the enterics. By applying this method to several homologous loci in Buchnera and in their free-living relative. coli. (1988). nonrandom use of U. 1998). asexual populations is also expected to accelerate the fixation of replacement substitutions. In addition. Previous studies provide strong evidence for differences in rates and patterns of sequence evolution of endosymbiotic and freeliving bacterial lineages. Relatively high ratios of nonsynonymous divergence (Ka) to synonymous divergence (Ks) imply that these substitutions are concentrated at sites that affect amino acid sequences (Moran 1996. as would be expected in small. Similar to the scaled x2 of Shields et al. we test the hypothesis that translational selection is relatively ineffective in the endosymbionts. Methods Loci Sampled Several loci from Buchnera taxa and the enteric bacteria were included (table 1) in estimates of codon bias. coli. E. Therefore.oxfordjournals. Brynnel et al.

. . . suberi. . . . . . . . . . . S. . .org/ by guest on August 5. . . . . . . . . . . S. . . . . . . . . . . . . . . . . . graminum . . . . . . . . . . . . . caligatum . . . . . . . suberi. . . . A. . . . . Myzus persicae . . R. . . . . . ECOR 51 . . . . . . . . . graminum . . . . . . . . . noxia . . . . . . . . . . . . . . . . . . . . . . . . . . graminum . . . . . . Uroleucon jaceae . . . . . . . . . . . . . . . ECOR 72 . . . . . . 2015 Taxona GenBank Accession Number Table 1 Continued . . . . . .e AF058432d. . . . . . . . . . . . graminum . . . . . . . . . . . . . . . . . . . . . . . . . . . .d X71612b. . . . graminum . . . . . . . . . . . . . . . . R. . . . . . Uroleucon obscurum . . . . . . . R. . . . . . . . . . . S. ECOR 17 . . . . . . . . . . . . . .e U09185b Z19055b U09185b Z19055b L43555b. . . S. . . . . . . . . .86 Wernegreen and Moran Table 1 Genetic Loci of Buchnera Strains Included in Study Gene Name Acyrthosiphon pisum. . . . . . . .d L43551b. . . . . Tetraneura caerulescens. . . S. chinensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . . . . . graminum . . . . . . . . . . . . . . . . . S. . . . . . . .d AF041836b. . . . . . . . . .g X71612b. . .d 47764d AF041836b. . . . . . graminum . . . . . . . . . . . . . . . . . . . . ECOR 71 . .e AF058435d. . . . . . . . . . S. . . . . . . . Macrosiphoniella ludovicianae . . . . . . . . . .d Z21938b. . . . . . . . . . . . . . . . . . S. . . . S. . . .e L81151d. . . . . Diuraphis noxia . padi . . . . . . . . . .g Y11972b. . . . . . . . . . . S. . S. . . . . . . . S. graminum . U. . . . . . . . S. . . . . . . . S. . graminum . . . . . . graminum . . . . . . . . . . . . . . . S. . . . . . . . . ECOR 50 . . . . . . . . . S. . . . . . . S. . . . . . . . . . . . . . . . . . . . . . . . . . .d X71612b. . . . . . . Uroleucon caligatum . . . . . . . . . . . . . . S. . . . . . . S. . . . . graminum . . . . . . . . . . . . . . . . graminum . . . . . . . . . . . . . . . . . . . . . typhimurium . . . . . . . . . . . . . . . . . Uroleucon solidaginis. graminum . . . . . . . . . suberi. . . . . . . . . . S. S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . graminum . . . . . noxia . . . . . . . . . . . . pisum.e L81150d. . . . . . . graminum . . . . . . .d M31047d Y11966b. . . . . ECOR 29 . . . . . . . . . . . . .d U01039c U77379b. graminum . S. . . . . . . . . . . .g X71612b. . . . . . . . . . . . . . . . . . . . . . . . . . . . R. . . . . . . . . . . . . . . . . . . . . graminum . . . . . . . . . chinensis . . . . . . S. . . . . . typhimurium . . . . . . . . . . . . . graminum . . . . . T. . . chinensis . . noxia . . R.e AF058440d. . . . . . . . . . . . . . . . . . S. A. . . . . . graminum . . . . . . . . . . . . . R. . . . . . . . . . . S. . Uroleucon aeneum . . S. . . . . . . . . . . . S. graminum . . . S. . . . . . . . . . . . . . . . . . . . . . . . . . suberi. . . .g AF041837b. . . S. . . . . . . . A. . . . . . . .g Y11966b. . . . . padi . . . . . . . . . . . . . . S. . . . . . . . . graminum . . . . . S. . . . . . . . . . . . . . . . graminum . . . . . padi . . . . . . . . . . . . . .d L46769b. . . . . . . . . . . . . . . . S. S. .d AF041836b. argS argS aroA aroE aroH atpA atpB atpC atpD atpE atpF atpG atpH cysE cysS ddlB dnaA dnaG(pt)c dnaJ dnaK dnaN dnaQ fdx ftsA ftsZ gapA gidA groEL groEL groEL groEL groEL groEL groES groES gyrB himD hscA hscB ilvC ilvD infC leuA leuA leuA leuA leuA leuB leuB leuB leuB leuB leuC leuC leuC leuC leuC leuD leuD leuD leuD leuD murC nifS pfs rep rep L18933b L18932b L43549b U09230b U11066b 2827020b 2827024b 2827018b 2827017b 2827023b 2827022b 2827019b 2827021b M90644b U09230b 2738587b M80817b M90644b D88673b D88673b M80817b L18927b 2827028b 2738589b 2738588b U11045b 2827025b X61150b. . . . S. . . . . . . . . . . . . . . . graminum . . . . . .d J01810d U09185b. . . . . S. . persicae . . . . . . . . . . ECOR 31 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. Melaphis rhois . . . . . . . . . . . . S. pisum. . . . graminum . . . . . . . . . . . . . . . . . . . . Schlechtendalia chinensis. . . . . S. . . . ECOR 37 . S. . . . S.e AF058434d. . . Sitobion avenae. .e AF058439d. . . . . . . . . . . .g Y11966b. . . graminum . . . . noxia .d X53376d Y11966b. . graminum . . . . . . . . . . . . . S. . . . . . . . . . . . . . . . S. graminum . . . . . . . . . . . . . graminum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .d Y11966b. . . . . . . . . . . . . . . . . . . . S. . . . . . .e AF058437d. . . . . . . . . . . . . . . . . . . . . . .e AF058438d. . . . S. . . . graminum . . . . . . . . . . . . graminum . . . . .e L81149d.e 1137716b. . . . . . . . . . . . . . . . . . . . . . . . padi . . . . . . . . . . . . . . . . . . . . . . .d V01378d U09184b. . . . . . . . . . . . S. . . . pisum. . . . . . . . . . . . . . . . . . . . .e AF058436d. . . . . . . . . . S. . . . . . . . . . . . . . . . . . . . . .d Y11966b. . . . . . . . . . . . . . . . . . . . . . . . . D. Uroleucon helianthicola . . . . . . D. . . . . . . . . . . . . . graminum . . . . . . graminum . . . . . . . . . chinensis . . . chinensis . . . . . . . . . . . . . . . . . . . . . . A. . . . . . . . . S. . .d 47968d AF041836b. . . . . . . . . . . . . . . . . Uroleucon rudbeckiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .oxfordjournals. . . .d U23489e U25425e U23494e U23496e U23495e U23497e U25884e U23499e U23500e U25429e AF058431d. . . . . S. . . Uroleucon erigeronense . . . . padi . . . . . . . . . . . . . . . . .d U77380b. . . graminum . . . . . . . . ECOR 46 . . . . . . . . . . . Rhopalosiphum padi . . . . . . . . . . . . . . . . . . . . . . . . Uroleucon ambrosiae . . . . . . padi . . . . . . . . . . . Schizaphis graminum . . . . . . . . . Salmonella typhimurium. . . noxia . . . . . . . . . . . . . . T. . . . graminum . . . . . .d AF041837b. . . . S. . . . . . . . . . . . . . . . graminum . .d M80817b L43549b 2827029b 2827030b 2827034b 2827033b U11066b AF041837b. . . T. . . . . . S. . . . . . . . . . . . . . . . . A. . . . Uroleucon astronomus . . noxia . . . . . . S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .d 2754808b. .d D85628b. . . . . . . . . . . . Uroleucon jaceicola . . S. . . . . . . . . . . . . S. . . . . . . . . . . . .d AF012886b 2827032b AF01288b X71612b 2827035b Taxona D. . . . . D. . . . .d AF058428e L46357b. . . . . . . . . . . . . . . S. . suberi. . . . . . . . . . Uroleucon sonchi . S. . . . . Gene Name repA1 repA1 repA1 repA1 repA2 repA2 repA2 rho rmph rnh rnpA rpoB rpoC rpoD rpsA secB sohB thrS tpiA trmE trpA trpA trpB trpB trpB trpB trpB trpB trpB trpB trpB trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpB(pt) trpC trpC trpD trpD trpE trpE trpE trpE trpE trpE trpE trpE GenBank Accession Number AF041837b. R. . . typhimurium . . . . . . . . . . . . . typhimurium . . . . . S. . . . . . . S. pisum. . . T. typhimurium . . . . . . . . . . padi . .e AF058433d. . . . . . ECOR 60 . . . . . . . graminum . . T. . . . graminum . . . .d AF041837b. . . . . . . . . . . . . graminum . . S. . . . . . . .d L46356b. .d X71612b. . . . . . . . . . . . graminum . . . . graminum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . graminum . . . . . . .d Z19055b. . . . . D. . . . . . . . . . . . . . . . . . . .d L8124d Downloaded from http://mbe. . . . . . . . . . . .d L46358b. . . . .g 2827037b M80817b L18927b M80817b Z11913b Z11913b M90644b L43549b M90644b U09185b U11066b L43549b 2827009b U09185b Z19055b L46355b. . . . . . . . . . . . . . padi . . . . . . . . . . . . . . . . graminum . . . . . . . . . S. . . . . . . . . . . . . . . graminum . . . . . . . . . . . .d AF041837b. . . . maidis . . . . . . . . . . . . . . graminum . . . . . . . . . . . . .d AF038565b. . . . . . . . . . . . . . . . . . . . . . . . . graminum . . . . graminum . . . . . . . . . . . . . . . . chinensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. . . . D. . . . S. . . . . . . . S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rhopalosiphum maidis . . . Uroleucon rurale . Thelaxes suberi . . . . . . . . . . graminum . . . . . . . . S. . . . . . . . . . . . . . . . . . . . . graminum . . . . . . . . . . S. . . . . . . .d 2754807b. . . . . .d X71612b. . . . . R. . . . . S. . . pisum. . . . . . . . . . . . . . . . . . . M. . . . . . . . . R. . . . graminum . . . . .d D85628b. . . . . . graminum . . . . . . . . . . graminum . . . . . . . . . . . . . noxia . . . . . . . . . . . . . . . . . typhimurium . . . . . . . . . . . padi . . . . . . . . . . Uroleucon rapunculoidis . . . . . . . . . . . R. graminum . . . . . . . S. . . . . .d. . . . . . . . . . . . graminum . . . . graminum . . . graminum .d L43550b. . . . . . . . . . . D. . . S. . . . . . . . . . . . . . graminum . .

. . . . In order to avoid inaccurate estimates due to saturation. chinensis . . . . Codon usage in Buchnera was also explored by testing for the overrepresentation of particular loci among significant x2 test results and by identifying consistent preferences for U. coli and Buchnera (see above). respectively). pisum. . . . typhimurium .d L05002f U85774f U25996f Y14237f X14791f U29655f Y11280f Y11280f X15840f a Buchnera strains are labeled by the aphid species from which they were isolated. . . .Evidence for Drift in Endosymbionts Table 1 Continued Taxona Gene Name trpE trpE trpE trpG trpG trpG trpG trpG trpG trxA tufA tufA tufA tufA tufA tufA aroA dnaG dnaJ dnaK dnaN leuB leuC leuD rpoB L81123d L8112d 1137712d L43555b. . . . . . . . . . . the average x2 values of homologous loci. . . . maidis . . . . . 1. . . sonchi . . .d X55116d L43549b. . . . . . A. . . . . . . . . . c ‘‘(pt)’’ indicates that only a partial sequence is available in GenBank. . . . . . g No homologue in E. . . . . since the two tests have different numbers of classes and therefore different degrees of freedom. . . Tests were performed as described previously (Moran 1996). coli isolates in the ECOR collection. . . . . . Individual loci of these two species are not listed. Only amino acids with five or more representatives in a given locus were considered. . M. the latter two analyses allow a visual comparison of levels and patterns of codon bias. . . . . . . . . graminum . . putida . . . .g 2827036b 2369691b. . . Phylogenies of trpB partial sequences were estimated by parsimony analysis (by PAUP. . . . . . . . . . . . . . . . . aeruginosa . . . A. Mapping of Nucleotide Changes Across Genealogies A portion of trpB is available for Buchnera of several Uroleucon species and for several E. . . . . . . . . . . . . 2015 U. Analyses of Codon Usage Based on x2 Test Results and Values E. Although the actual x2 values are not directly com- parable between E. . . P. The ratios of these estimates were compared across the ECOR phylogeny and subsets of the Buchnera phylogeny.0 were included in the calculation of Ka/Ks ratios. . . . . . . . . .oxfordjournals. graminum . U. The magnitudes of x2 values are not directly comparable between E. in addition to the trpB sequence of E. . . .d 2369695b. . . . . Aeromonas salmonecida. Instead. . .d 2369697b. . . . Pemphigus betae . . erigeronense. . . . . . All sequences from Escherichia coli and Haemophilus influenzae are accessible from the full genome sequences of these two species (GenBank accession numbers U00096 and L42023. . . . For each data set. . . . . . . . . . . . . . . . . . chinensis . . . . . . . Rates of Nucleotide Divergence Relative-Rate Tests Relative-rate tests were used to compare rates of nonsynonymous divergence at homologous loci of Buchnera and E. . . . . . . . . . . . . . . . . . . putida . . . . . . . . . . . . . coli loci was estimated by the deviation from random use of all four synonyms (not just U. . . .g L43550b. and a single mostparsimonious tree was selected. . . . D. . . . . . . . Pseudomonas putida. . .org/ by guest on August 5. . . . .or A-ending codons for particular amino acids. two most-parsimonious trees were found. . . . . . divided by the length at third positions. . . . . .and second-codon positions was used to approximate nonsynonymous divergence. . Li’s (1993) method has been shown to be reliable for loci with biased base composition (Ina 1995). Within each of these two groups. . . . . rurale . . Expected values were based on the nucleotide composition at fourfold degenerate sites for a particular locus (calculated using MEA). . . . . and the x2 values for individual amino acids across homologous loci. . . S. . . . . coli Loci Codon bias in E. . . . S. . nucleotide changes at each codon position were mapped across the selected tree (by MacClade. . . GenBank Accession Number 87 . . . . . . . . . R. . . coli. . . . . . . . Estimates of Codon Bias in Homologous E. .g L46769b. . . . Changes at each codon position were mapped with parsimony across genealogies of the partial trpB sequence and summed across each phylogeny. . S. . . . . . . . . . . . . . . .g U09184b.and A-ending codons) at fourfold degenerate sites. . . . . . . . . . . . . . . . . S. . . . . . . . . . GCG). . . .g L43551b. . . . Compared to other commonly used estimates of nucleotide divergence. . U. . . . . . . . . . . . Downloaded from http://mbe. . . . . . Swofford 1993). . . For the ECOR and Buchnera trpB data sets. f Taxon 3 in relative-rate tests. padi .and second-codon positions (452 nt) to approximate percent change at nonsynonymous sites. . Azotobacter vinelandii . S. noxia . . . coli compared in codon usage analysis. . . . . . . graminum . . . . . . . . . . . . . . . . . changes at third positions were summed and divided by the total number of third-codon positions (227 nt) to approximate percent change at synonymous sites. . S. . Azotobacter vinelandii . roughly approximates the ratio of divergence at replacement versus silent positions. . only pairwise comparisons with Ks . Haemophilus ducreyi . .and second-codon positions. . . coli and the enteric species listed) used in comparison of Ka/Ks. . Estimation of Ka/Ks Estimates of nonsynonymous and synonymous pairwise divergence and standard deviations were calculated using Li’s (1993) method (DIVERGE. . R. The tree length at first. . . . the percent change at first.d 2369693b. Maddison and Maddison 1992). . . P. b Sequences of Buchnera taxa (listed) and E. . . . . . d Sequences of Buchnera and enteric bacteria (including E. . . . . . . . coli and Buchnera loci were compared in terms of the proportion of total x2 tests with significant results. . . . Likewise. .and second-codon positions were summed across each tree and divided by the total number of first. . . and deviation from expected was tested using a four-class x2 test. . . . . . . adjusted for the number of sites. . . . . . P. . locus excluded from comparison of CAI values. coli with sufficient similarity. . .g Z21938b. . coli and Buchnera. . . . . . . pisum. . . . . . and percent change at third positions was used to approximate synonymous divergence. . . . . . . . . . rhois . . . . . . . . . Ka values were too low to estimate reliable Ka/ Ks ratios for pairwise divergences. . coli K12. . . . . . Vibrio cholerae . . e Sequences used in mapping of nucleotide changes across phylogenies. Changes at first. . .

org/ by guest on August 5. This is consistent with previous observations of A1T richness across the Buchnera genome (Ishikawa 1987. Only 23 tests were significant at the 5% level. and alanine tends to be encoded by the A-ending codon. Since expected values of these tests are based on gene-specific base composition. codon preferences for alanine in E.oxfordjournals. which is thought to represent the optimal codon for this amino acid. x2 values are relatively high for individual amino acids across most E. Of a total of 772 x2 tests performed across Buchnera loci. In contrast. Baumann. which is known to be highly correlated with gene expression level (Sharp and Li 1987). fewer tests were significant than expected by chance alone. such as trp genes (Sharp. mann. 1). as highly expressed loci are biased toward GCU. Comparison of x2 Averaged Across Amino Acids The depression of codon bias reflected in Buchnera is also apparent in the low x2 values. coli. table 3). Clark. Preference for the U-ending codon of the fourfold degenerate families of serine and arginine agrees with the codon preferences of highly expressed genes of E. coli homologues and provides further evidence against effective translational selection in Buchnera. 1988). and 35 more were significant at the 10% level (data not shown). Baumann. 2015 Comparison of E. Likewise. codon usage cannot be attributed solely to mutational bias.or A-ending codons for individual amino acids. However.88 Wernegreen and Moran Results and Discussion Evidence for Strong A1T Mutational Pressure Across Buchnera Loci All Buchnera loci sampled were extremely A1T rich at fourfold degenerate sites (the average across loci was 88. which is known to be highly expressed in Buchnera (Baumann.1% in original Downloaded from http://mbe. the majority of E. possibly because of an expansion of aphid population sizes. coli genes.841 for df 5 1). for loci showing slight (nonsignificant) codon preferences. Although the magnitude of x2 values is not directly comparable for E. In addition. and Baumann 1998). These apparent codon preferences across several Buchnera loci and taxa suggest that purifying selection may effectively reduce the frequency of A-ending codons for serine and arginine and reduce the U-ending codon for alanine. averaged across amino acids for each locus (fig. the preferred codon within the fourfold degenerate family for serine tends to be UCU. For these amino acids. whereas no Buchnera loci have an average x2 value that exceeds the critical value significance at 5% (3. In contrast.4%). Bau- Evidence for Preferential Use of U-ending and Aending Codons in Buchnera Despite the severe depression of codon bias in Buchnera. even for groEL. 2). coli loci showed significant nonrandom use of alternative codons for most amino acids. Ohtaka and Ishikawa 1993. coli loci have average x2 values greater than the critical value for significance at the 5% level (7. relative to its frequency in the original sample (4. coli loci indicates that codon bias in Buchnera does not correspond with levels of gene expression of E. a strong relationship exists between the average x2 values for loci of E. but lowly expressed loci are biased toward GCA. Tuohy. Buchnera populations may occasionally go through periods where selection is more effective. Buchnera loci rarely show significantly nonrandom use of U. This gene had the greatest overrepresentation in the pool of significant x2 tests. Of a total of 528 x2 tests performed across E.815 for df 5 3). Clark. . and alanine tends to be encoded by GCA (P .01 in each case. these significant results cannot be attributed to local variation in A1T content. Strong mutational bias and drift may largely shape patterns of codon use. These preferences agree with the general trends found in a previous analysis of codon usage across several loci of the endosymbiont of Schizaphis graminum. 274 were significant at the 5% level. many of which are included in the genes sampled here (Clark. but these occasional periods of effective purifying selection may reduce the frequency of nonoptimal codons. coli is also evident in the narrow range of x2 values for each fourfold degenerate family across Buchnera loci (fig. coli and Buchnera Number of Significant x2 Tests The null hypothesis that codon usage in Buchnera reflects local base composition was tested by performing a x2 analysis of observed and expected numbers of Uor A-ending codons for eight fourfold degenerate families across several loci. Baumann. In contrast. and Baumann 1998). coli and Buchnera (see Methods). coli and the CAI on the basis of preferred codons for this species. coli depend on gene expression levels. serine and arginine are generally encoded by U-ending codons. the lack of correspondence between average x2 estimates of Buchnera genes and the CAI of homologous E. The preference for GCA across Buchnera loci may reflect a change in the optimal codon. coli (Sharp et al. coli loci. This paucity of significant x2 tests indicates that codon usage generally reflected local base composition. Evidence for Bias in Buchnera groEL Genes A second line of evidence that codon use may be under effective (although weak) selection in Buchnera is the slightly higher levels of bias observed at the overexpressed gene. and Mosurski 1986). 0. including those considered low expression in E. In particular. possibly precipitated by drift in small populations. Comparison of x2 Values for Individual Amino Acids The contrast between levels of bias in Buchnera and in E. a large proportion of E. It has been suggested that such switches in codon preference among the enteric bacteria may relate to differences among lineages in effective population sizes (Shields 1990). and 42 more were significant at the 10% level (table 2). significant x2 tests across loci almost always reflect a higher frequency of one particular (U. and Baumann 1992.or A-ending) codon (table 2). and Clark 1996). groEL.

. S. graminum. . . . . . . . . . . . . Critical values for these two-class x2 tests are 2. . . . . .417 3. . . . . . . S. . . . graminum.706 (P . . .oxfordjournals. . graminum. . . . . .7299 2. . . . . . . . . . . . . . . . graminum. . . . . . . . 0. . . . . T. .708 2. . . . S. . . . . .2 3. . . . . S. . . . .81 3. . . . . . . . . .756 2. . . . . . . . . . . . . . .05). . . .915 3. . . . S. . . . . . .044 4. . . . A. avenae . . . .org/ by guest on August 5. . suberi .435 2. . . . . . . S. . S. . graminum. . . Sitobion avenae . . . . .734 5. graminum. .0218 3. .7963 3. . . . . . .814 4. . . . . . . . . .875 2. . . . . . . . . . . . . . . .1565 3.613 3. T. . . . . . . . . . . . . . . .158 2. . . . . . . .265 3. . . . . . . S. . .452 3. . . graminum. S. . . . . . . . . . . graminum. . . . . . . . .2 3 2. . . . . Rhopalosiphum maidis . .2636 5. . . . . Schlechtendalia chinensis . M. . chinensis . graminum. . . . . . . . . S. . graminum.039 2. . . . . 0. . . . . . S. . . . . . .424 2. . . chinensis .841 (P . . . . . . . .5566 5. . . . . . . . . . .777 3.487 3. . . . . . . . . . . . . . . . . . . . . . . . . . S. . . . . .484 3. . . . .858 3. . . .817 3. . . . . . . . . S. . . .Evidence for Drift in Endosymbionts 89 Table 2 Significant Nonrandom Use of U. . . . . . . . . S. . D. . S. . . . . . . . . . . . . . . S. . . .2248 3. . . . . . . . . . . . . . . . . . graminum. . . chinensis . . . S.7696 7. . . . . . . . . . . . . . . . S. . . . S. . . . . . . .754 2. . . . . . . . graminum. . . . . . .8242 5.227 3. . . . . . . . . . graminum. . . . . . .734 2. . . . . . . . . . . . pisum . . . . . Diuraphis noxia . .624 3. .8263 7. . . . . . . . . . . . graminum. . . . . . . . . T. . . graminum. chinensis . . . . . . . . . . . . . .or A. . . . . . . . . S. . . .5715 9. . . . . . . . . . . . . . . . . . . . . . tpiA groES trpG leuA tufA groEL trpD trpA groEL rpoD atpA trpE trpA tuf rpoB rep leuB trpE leuD dnaN trpA ftsA hscA trpE trpB groEL dnaA aroH groEL leuA leuC dnaG murC ilvD groEL trpB rpoB dnaA infC dnaK groEL dnaJ tuf ddlB trpE groEL repAl ftsZ rpoB trpG trpE groEL rhn leuA ilvD argS tufA rpoB ilvC trpA tuAf gidA tufA leuD trpA A A A A A A A A A A A G G G G G G G G L L L L L P P P P P P P P P R R R R R R R R R S S S S S S S S S S T T T T T T T V V V V V V a b x2 Value for Nonrandom Useb Codon Ending 14 8 7 35 22 54 12 11 51 27 35 18 17 30 90 17 19 16 8 9 5 9 15 18 11 15 10 10 15 7 12 8 11 14 17 6 35 17 11 9 18 10 10 16 20 29 11 17 64 7 33 30 10 18 26 5 22 58 18 7 30 26 31 9 11 6. . S. . . . . . . S. . . . S. S. avenae . . . . . . Pemphigus betae . . . . . . . . . . . . . . . . . . . .45 3. . . . . . . . noxia . . . . . . .0635 3. . . . . . . A. . .401 4. A. . . . . . . . . . . 2015 Taxona Amino Acid No. . . . . . chinensis . . . . .174 9. . graminum. . . . . . . . . . . .5219 5. . . . graminum. suberi . . . S. . .803 2. . . . . pisum . . . . . . . . . . Thelaxes suberi . . . . . . . . . Melaphis rhois . . . . . . . . . . . . . . . . . . . . .Ending Codons Across Several Buchnera Loci for Each of Eight Fourfold Degenerate Families Locus Schizaphis graminum . . . . . . . . graminum. S. . . . graminum. graminum. . . . . . . . graminum. . . . . . . S. . . . pisum . . . . . . . . . . . . .967 A A A A A A A A A A U A A A A A A U U A U U U U A A A A A U U U U A U U U U U U U U U U U U U U U U U U A A A A U U U A A A A A U Buchnera taxa are labeled by the aphid species from which they were isolated. . . . noxia . . graminum. . . . . . . . . . . . . . pisum . . .76 3. . Acyrthosiphon pisum . . S. . . . . . . . . . . . . . . S. . . . . graminum. . . S. . . . . . . . A. . A. . . . . Downloaded from http://mbe. . . . . . . . . S. . graminum. . .1) and 3. . . S. . . . . . . . . . . . . graminum. . . . . . . . .995 4 6. .317 6. . . . . . . . . . . . . . . D. . . S. . . . . . . . . . . . . . . . . . . .184 3. . . . . chinensis .5667 4. . . . . . . . graminum.532 3. . . . . . pisum . S. . . graminum. . . . . .9269 6. . S. . . . . . . . . . . . . . . . .9 2. . . . . . . . . . . . graminum. . . . . . R. . . . . . graminum. A. . .531 2. graminum. . . S. . . . . . . . . . . . . S. . . . . . . . . chinensis . . . . . . . . . . . .125 3. . . . . . . . . . . . . .784 3. . . . . . . . S. . . . . . . . . . . . . . . . . pisum . . . . . . . . . . . . . . . . .8356 3. . . . . . . S. graminum. graminum.0547 6. . . . . . . . . .293 4. . . . S. . chinensis . . suberi . of Amino Acid Residues in Protein . . .9462 3. . . . S. . . . . . . S. . rhois . . .27 4. . . . . . . . . . . . . padi . . . . . . . . . . . .2069 2. . . . . . . . . . . . .368 8. . . graminum. . S. . . . . . . . . . . . . . . . . . . .

coli versus Salmonella typhimurium.3% in the pool of significant x2 tests. coli (fig. E. Points represent a single locus and are positioned on the y-axis by the x2 value averaged across each fourfold degenerate amino acid.90 Wernegreen and Moran sample versus 12. 2015 FIG. coli or Buchnera loci. Across each locus sampled. 1. These pairwise divergences were used to estimate ratios of nonsynonymous to synonymous divergence (Ka/Ks) for Buchnera and for the enterics. 3). Relative-Rate Tests In agreement with previous studies. groEL of Buchnera from Acyrthosiphon pisum has the highest average x2 (see fig. In contrast to Buchnera genes. table 4). The maximum value for Ka/Ks for Buch- Downloaded from http://mbe. coli genes (table 5). Rates of sequence evolution in Buchnera genes were 1. Compared to other Buchnera loci. coli loci have consistently higher average x2 values and a strong relationship with CAI. for symbionts of Acyrthosiphon pisum. pairwise divergences at nonsynonymous and synonymous sites were calculated across several Buchnera lineages and across E. coli homologues. 1—Relationship between the x2-based estimate of codon bias and the Codon Adaptation Index (CAI) for several E. .oxfordjournals. Slight codon bias at groEL is also suggested by the relatively high x2 values at this locus (figs. coli) or the homologous E. coli gene (Buchnera). relative-rate tests demonstrated higher rates of nonsynonymous substitution in Buchnera loci relative to E. Average x2 includes only x2 values for amino acids with five or more residues in a given locus. Points are positioned on the x-axis by the CAI of either the same gene (E. four of the eight amino acids considered showed significant x2 values (table 2). Comparisons of Ka/Ks For several protein-coding loci.3 to 6. 2).org/ by guest on August 5. In particular.9 times faster than in E. 2). values of Ka/Ks were generally higher in Buchnera than in E.

2—Comparison of x2 values for individual amino acids. For illustrative purposes only. Pb 5 Pemphigus beta. Sc 5 Schlechtendalia chinensis. Mp 5 Myzus persicae. Except for groEL. The locus showing the strongest evidence for bias in Buchnera is groEL. coli Relative Synonymous Codon Usage table (Sharp et al. When several loci were available for a given pair of Buchnera taxa. The relatively low Ka/Ks ratios for this gene may be attributed to decreased nonsynonymous divergence that is more extreme than the observed depression at synonymous sites.oxfordjournals.org/ by guest on August 5. 1988). 3b). levels of bias in E. 3a). Rm 5 R. Ts 5 Thelaxes suberi). coli are much higher than those in Buchnera. nera always exceeded that of the enterics. . Sa 5 Sitobion avenae. x2 values of E. 2015 FIG. Compared to other genes in Buchnera. Dn 5 Diuraphis noxia. Mr 5 Melaphis rhois. x2 values of Buchnera are graphed as negative values if the A-ending codon is preferred. across several homologous E. Ap 5 Acyrthosiphon pisum. coli are graphed as negative values if the preferred codon is a nonoptimal codon as defined by the E. particularly that of Acyrthosiphon pisum. Usn 5 Uroleucon sonchi. the minimum Ka/Ks value also exceeded the estimate for the enterics (fig.Evidence for Drift in Endosymbionts 91 Downloaded from http://mbe. For each locus. maidis.] loci. groEL generally proved to have the lowest Ka and the lowest Ks (fig. purifying selection is apparently more effective against replacement substitutions at this highly expressed locus. coli [x] and Buchnera [. Rp 5 Rhopalosiphum padi. Buchnera loci are labeled by the taxon from which a gene was sampled (Sg 5 Schizaphis graminum.

R.8 43. c groEL had the greatest overrepresentation in the pool of significant tests compared to its frequency in the original sample. .. . . . .. .. gave comparable values for the ratio of the tree length at first and second positions..07 0.025 0. . . ... divided by the length at third positions adjusted for the number of sites.. L ... . trpA .. The ECOR and Buchnera trees each include nodes with relatively weak support (see bootstrap values. . S . .. ..01 0. .. For the purposes of mapping nucleotide changes at each codon position.6 24. . coli may depress rates of synonymous substitution and thus elevate Ka/Ks estimates. coli versus Buchnera Under the assumption that rates of synonymous substitutions reflect mutation rates. distinct trpB clades were considered separately. relative to the E.77 0. however. .. .015 0. However.or A-Ending Codons for Each of Eight Fourfold Degenerate Families AMINO ACID xxU xxA CONSIDERED EXPECTED NUMBER OF LOCI SHOWING A PREFERENCE FOR AENDING CODON A. . .. . . . that the resolved trees presented are not intended to represent exact relationships among E.. .010 0.. trpG . . . . coli trpB phylogeny. . .. . coli strains and Buchnera associated with the aphid genus Uroleucon. coli.. of x2 Tests Fraction of Total x2 Tests Performeda No.019 0.42 2. tuf ... . . . coli strains (0.8 31.077 0. . .123 0. . these relatively weak . 4). . . b The x2 value expresses the deviation of the observed from the expected number of loci with preference for A-ending codons.. .031 0.92 Wernegreen and Moran Table 3 Number of Buchnera Loci Showing a Slight Preference for U.. Because of low levels of divergence within each group. . Given the higher divergence among Uroleucon isolates. ... groES . .. It should be noted.64 P VALUE (.162 to 0.. . Estimates of Synonymous Substitution in E.. .. ..108 0. . .12 0.09 5. .031 0. . . 4). V. fig.. high ratios of nonsynonymous to synonymous divergence indicate that replacement substitutions accumulate at faster rates in a particular lineage (Brynnel et al. however. trpD .0 46. 32 8 119 64 15 14 46 36 39 0. . ... .. .154 0. 1998). . a b Downloaded from http://mbe. . The total number of significant x2 tests was 65. .9 NO.015 0.. this discrepancy in codon bias would only dispose the Ka/ Ks comparison away from the previously observed trend: higher Ka/Ks ratios in Buchnera..... Selection for translational efficiency in E. OF BUCHNERA LOCIa TOTAL LOCI x2 VALUEb 7. ..047 0. coli or Buchnera isolates.. .. .6 42.. nucleotide changes were summed across genealogies of trpB instead of calculated pairwise (fig.01 0.0328 across the E.. leu genes . . trpB . . nodes do not affect the results obtained..3 45.051 8 1 7 2 5 1 5 2 6 0. low levels of adaptive codon bias in Buchnera only strengthen the interpretation that relatively high Ka/Ks ratios across Buchnera loci reflect an elevation of nonsynonymous substitutions. . . Significant x2 Tests Fraction of Significant x2 Testsb groELc . . . 4). .0 37.... . Therefore.092 A total of 772 x2 tests were performed. .. ..32 6. . ..018 0.13 8.. . . .077 0.041 0.. .org/ by guest on August 5. This approximation of nonsynonymous divergence divided by synonymous divergence is considerably higher for the Uroleucon isolates. P ...083 0. 2015 Mapping of Base Changes Across Genealogies Sequence evolution at a portion of trpB was compared across two very shallow taxonomic groups: the ECOR collection of E. Table 4 Frequencies of Buchnera Loci in the Original Sample Compared to Their Representation in the Pool of Loci for Which There Is Evidence of Significant Codon Bias Locus No. . . .. This assumption may be violated by differing levels of codon bias in Buchnera and E. . .oxfordjournals. Each clade. trpE . .. . fig. . T . . ..01 a Number of Buchnera loci showing slight preference for the A-ending codon of that amino acid and number showing a preference for the U-ending codon.... . 37 52 33 37 32 72 41 55 53 47 37 48 23 31 54 51 90 99 70 85 55 103 95 106 39. . .060 0...285 across subsets of the Buchnera trpB phylogeny and 0. G. . .. . . .) 0. .

c ‘‘(pt)’’ indicates that only a partial sequence is available in GenBank. . .29 0. .02 0. influenzae Pseudomonas aeruginosa Azotobacter vinelandii A. . . .07 0. .34** 3.32 0.25 0.6 1. . .40 0. cysE.4 4.org/ by guest on August 5.43 0.38 0.16 0. influenzae H.33 0.09 0.21 4. the first and limiting enzyme in the tryptophan biosynthetic pathway. . 1998b). influenzae H.25 0. . . .14 zb K01/K02 7. .14 0. 0. .34 0. These results suggest that selection is ineffective in eliminating two types of weakly deleterious mutations from Buchnera populations: those resulting in nonoptimal codons and those resulting in amino acid replacements. relative-rate tests and comparisons of Ka/Ks ratios support previous conclusions that Buchnera lineages experience rapid sequence evolution at nonsynonymous sites. influenzae H.12 0.10 0.21 0.18*** 3. groEL is known to be constitutively overexpressed in several intracellular bacteria. and taxon 2 is always Escherichia coli. . . . dnaG(pt)c . . 1996. . However.12 0. .9 2. .3 6. . . . compared to their free-living relative. . .48*** 7. . rpoB .71 1. except for dnaJ and dnaK. 2015 ilvC .17 0. and Clark 1996) and pathogenic (Garduno et al. . A set of loci that might be suspected of experiencing unusual selection in Buchnera are those encoding enzymes for biosynthesis of essential amino acids. . are amplified in tandem repeats on multicopy plasmids (Lai. . . . .20 0. influenzae H.21*** 6.36** 1. 1990).7 5. Gatenby.00*** 6. .22 0. E. and Lorimer 1989). leuD.42 0. leuA . 0.3 2.66*** 1. Probabilities for one-tailed t-test (H :K 0 01 # K02) are * P . In several Buchnera lineages. . 1995. . .53 0. . . 1998). . Codons Isoleucine valine biosynthesis 492 Isoleucine valine biosynthesis 617 Isoleucine valine biosynthesis 568 Leucine biosynthesis 507 Leucine biosynthesis 365 Leucine biosynthesis 465 Leucine biosynthesis 208 Aromatic amino acid biosynthesis 226 Serine/glycine family amino acid biosynthesis 251 Heat-shock protein 380 Heat-shock protein 638 Chromosome replication 368 Replication 321 Protein export. .21 0. . . Baumann et al. which are Buchnera of Acyrthosiphon pisum. . coli) also suggest higher rates of fixation at replacement sites. . *** P . .21 0. . .18 0.20 0. .20 2. comparisons of changes at first. This increased copy number is considered an adaptation that benefits the aphid hosts. . . . since groEL is apparently single copy in at least one Buchnera species in which it is overexpressed. leuB . 1997. . dnaN . .3 1. . the calculation of Ka/Ks ratios across shallow taxonomic levels avoided the problem of saturation at synonymous sites and the large standard errors that accompany high divergence estimates. . In this study. .9 2. .04 0. Conclusions Because of their small population sizes and limited opportunities for recombination. leuC. In addition.45 0. vinelandii Aeromonas salmonicida H. . . .01. .8 2. and Baumann 1994. .37 0.25 0. In addition.10 0. . assembly.41 0. .83*** 2. . Hara et al. . Function . . .14 0. .30 0.22 0. dnaJ.Evidence for Drift in Endosymbionts 93 Table 5 Relative-Rates Test for Substitutions at Nondegenerate Sites in Loci of Buchnera Versus Escherichia coli Gene No. .30 0. .05 0. . .36 0.5 2. groEL comprises about 10% of the total protein produced (Ishikawa 1984. .24 0.12 0. ilvI . .28 0.16 0. Taxon 3 is a more distantly related reference taxon. More problematic is the possibility that Ks in Buchnera is underestimated because of the strong A1T bias across loci and more rapid saturation at silent sites. b z scores were calculated as described by Muse and Weir (1992). and translocation of other polypeptides (Bochdareva.18 0. . influenzae H. which are limiting in the plant phloem on which aphids feed.09 0. .31 0.65*** 5. . . . In Buchnera. .20 0. atpD . 0.26*** 3.58 0.7 2. .37** 5.06 0. influenzae P. coli to be a heat shock chaperonin involved in the folding. . . .31 0. tpiA . . .and second. both mutualistic (Aksoy 1995. . in Buchnera. molecular chaperonin 155 Transcription (RNA polymerase) 1360 Translation (ribosomal protein) 557 Energy metabolism (ATP synthase) 465 Electron transport 253 Electron transport 336 Chromosome replication 629 Taxon 3a K12 K13 K23 K13–K23 Haemophilus influenzae H. dnaK . .44 0. Baumann et al. Douglas 1998). . genes for anthranilate synthase (trpEG). 1995. taxon 1 is Buchnera of Schizaphis graminum.56 0. . Buchnera provisions host insects with these nutrients. observed duplications of leucine genes and their occurrence on plasmids may represent an adaptation for overexpression (Bracho et al. . .29 0. Likewise. . . . .14 0. Baumann.42 0. influenzae Haemophilus ducreyi Vibrio cholerae Pseudomonas putida P.9 1. ilvD .vs.05 0. Goloubinoff. .77 0.11 0. secB.22 0. . and Girshovich 1988. Goloubinoff et al.07 0.38*** 4.59 0.07 0. van Ham et al. .21 0. which do not synthesize tryptophan and which depend on Buchnera to provide this essential nutrient (Baumann et al.06 0.33 5. influenzae H. 1997.36 0. . coli. The protein is known in E.23 0. rpsA. .6 3. . . .24*** 1. . . .05. The mechanism for this overexpression is uncertain but likely involves changes in gene regulation rather than a gene duplication. . .4 3. Rouhbakhsh et al. In addition. influenzae 0. . relative to synonymous sites.45 0.06 0. .13** 3. .0001. vertically inherited endosymbionts provide a good model system to test the effects of increased drift on sequence evolution in bacteria. . ** P . gapA . .55 0. 1998a). putida H. . Baumann.0 3. . . 1997. gidA.oxfordjournals.37 0.4 1. . Lissen. . .27 0.58 0. putida H.21 0.23* 3.7 2. . While its role Downloaded from http://mbe.36 0. Baumann.0 2.17 0. the symbiont of Acyrthosiphon pisum (Ohtaka and Ishikawa 1993). . third-codon positions across very shallow taxonomic levels (Buchnera isolates of Uroleucon and members of the ECOR collection of E.01*** 4.25 0.24 0. 1989.6 4. .84*** 6. aroA . . . the lack of adaptive codon bias across several Buchnera loci suggests that codon usage is shaped primarily by A1T mutational bias rather than by translational selection.29 0.6 a In each test. . .

] taxa and across E. it is difficult to imagine relaxed selection on trp genes. One alternative explanation for elevated evolutionary rates at nonsynonymous sites is positive selection for amino acid changes. relative to E. In particular. Thus. Ks values higher than 1. all Ka/ Ks estimates for Buchnera exceed the ratio calculated for the homologous gene of the enteric bacteria. (B) Ratios of nonsynonymous to synonymous substitutions. as suggested by Moran (1996). Combining loci from table 5 with those examined previously (Moran 1996. seven have K01/K02 .org/ by guest on August 5. such selection typically acts at specific loci rather than across the genome. however. leu genes. calculated across Buchnera [. Perhaps the intracellular environment is more constant than that experienced by free-living bacteria. there is no evidence that the acceleration of nonsynonymous substitution rates is any higher across Buchnera biosynthetic genes than across other Buchnera loci. The fact that the observed trends of depressed codon bias and accelerated evolutionary rates occur across all Buchnera loci included. Of the second set.oxfordjournals. and the trends we observed occur consistently across each locus examined. amino acid biosynthetic loci tend to be less accelerated than other Buchnera genes. In this study. 1. 2. While the results here cannot exclude the possibility of relaxed selection. coli homologues. typhimurium [x]. it may function to stabilize proteins that have accumulated amino acid substitutions. A second alternative hypothesis for lack of codon bias and accelerated evolutionary rates in Buchnera is a relaxation of purifying selection against nonoptimal codons and replacement substitutions. 12 have K01/K02 . and 2 have K01/K02 . 2015 FIG. 14 loci encode amino acid biosynthetic genes and 14 encode genes for other functions. coli versus Salmonella typhimurium [x]. argues for an increased effect of drift within endosymbiont populations.94 Wernegreen and Moran in endosymbionts is less certain.] and across E. First. even functionally important genes. Of the first set. genes in the tryptophan (trpABC(F)DE) and leucine (leuABCD) biosynthetic pathways and groEL each show low levels of codon bias and rapid rates of nonsynonymous substitution. (A) Pairwise divergence at nonsynonymous and synonymous sites. sequence evolution of these functionally important Buchnera genes shows the same patterns as housekeeping genes that are not overexpressed. All Ka/Ks ratios are based on Ks values less than one. In addition. table 2). 2 and seven have K01/K02 . With the exception of groEL. . two features of the data argue against it. 3—Comparison of levels of nonsynonymous substitutions (Ka) and synonymous substitutions (Ks) across several loci of the enteric bacteria and Buchnera. In addition. selection is not likely to be relaxed at loci that are functionally important in the symbiosis and that are known to be overexpressed in Buchnera. Given their apparent significance in the symbiosis. coli versus S. selection is unlikely to be relaxed across all loci. and Downloaded from http://mbe. on the basis of pairwise divergence across Buchnera taxa [. 2.0 were excluded.

rurale.. Levels of Buchnera aphidicola chaperonin groEL during growth of the aphid Schizaphis graminum. Codon usage and base composition in Rickettsia prowazekii. Acknowledgments We thank Joana Silva for her comments on an earlier version of this paper. Curr. Downloaded from http://mbe. CLARK. The number of changes at first. sonchi. Nucleotide changes were summed across the entire ECOR tree. Uo 5 U. Usn 5 U. ambrosiae. albeit at much lower levels than for the E.000 replicates). Urp 5 U. rudbeckiae. 1996. S. erigeronense). In contrast to trp and leu genes. SHARP. SCHAEFFER. BAUMANN. roughly approximates Ka/Ks. obscurum. 4—Mapping of nucleotide changes across genealogies of trpB for (A) strains in the ECOR collection of E. (DEB9527635). ANDERSSON. helianthicola. Usl 5 U. AKSOY. LITERATURE CITED AKASHI. jaceicola. H. M. Insect Mol. 1997. Ujl 5 U. and by an NSF grant to N.J. W.. and its constitutive overexpression may depend on rapid. While the addition of more loci would be desirable. Biol.M. and P. Genetics 146:295–307. For both data sets.Evidence for Drift in Endosymbionts 95 groEL.W. 42:525–536. Natural selection and the frequency distributions of ‘‘silent’’ DNA polymorphism in Drosophilia. is followed by the number of changes at third-codon positions (in b). Molecular analysis of the endosymbionts of tsetse flies: 16S rDNA locus and over-expression of a chaperonin. and we thank two anonymous reviewers for their helpful suggestions. solidaginis. 1995.. Uja 5 U. astronomus. Evol. caligatum. coli isolates and (B) several Buchnera isolates from the aphid genus Uroleucon (Uae 5 Uroleucon aeneum. groEL does not appear to be duplicated in Buchnera (Ohtaka and Ishikawa 1993). 32:279– 285. Microbiol. Uas 5 U. P. rapunculoidis. coli homologue. jaceae. This work was supported by a Center for Insect Science postdoctoral fellowship to J.oxfordjournals. The percent change at first and second positions. Ue 5 U. Uam 5 U. the consistency of the results across a variety of loci best supports the view that the effects of mutational bias and drift in Buchnera are sufficiently strong to override the effect of purifying selection against the nonoptimal codons and amino acid replacements. therefore. and across subsets of taxa in the Buchnera tree (see figure insert). that slight codon bias was detected for Buchnera groEL. Urd 5 U. It is not surprising. The number of unambiguous nucleotide changes along branches is given in parentheses. BAUMANN. S. Mol. J. and S. Urr 5 U. 4:23–29. Confidence in nodes was assessed using bootstrapping (1. trees presented are one of two most-parsimonius trees. and M. L. E. . Uh 5 U. divided by the percent change at third positions. G. efficient translation of a limited number of mRNA molecules. Uc 5 U. 2015 FIG. 1996.and second-codon positions. TrpB phylogenies were estimated using parsimony analysis of all sites (679 nucleotides).org/ by guest on August 5.

P. 15:574–582. 36:121–126. S. Evolutionary rates for tuf genes in endosymbionts of aphids. 1987. USA 93: 2873–2878. BAUMANN. 1998a. Acad. A. T. Evol. Kinetics of synonymous codon change for an amino acid of arbitrary degeneracy. 1993. S. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. New York. L. 1993. USA 91(38):19–23. Natl. 1995. P. G. and B. and P. VATS. J. A.org/ by guest on August 5. MORTON. 1973. 14:417–425. MOYA. Correlation between the abundance of E. Nature 246:96–98. A. MUTO. A. coli heat shock protein. Version 3. G. OHTA. Y. LISSEN. J. Evol. Genetics 129:897–907. J. BUCHNER. and S. Curr. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. J. A. BRACHO. J. DOWSON. Characterization of the protein species synthesized in vivo and in vitro by an aphid endosymbiont. GOLOUBINOFF. 25:283–290. A. 1995. Proc. A. 41: 345–352. GOUY. Evol. Curr. Mol. C. A. MARTINEZ-TORRES. Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria. 1992. P. . 1995. BOCHDAREVA. M. 1995. L. 24:205–211. BAUMANN. and P. Mol. THAO. Sci. Evol. USA 90:4384–4388. 36:96–99. and A. Mol. VOEGTLIN. Evol. L. Bacteriol.. LORIMER. and N. M. GATENBY. Microbiol. Endosymbiosis of animals with plant microorganisms.. SPRATT. M. Acad. N. and L. Mol. Evol. Genetics. WEIR. Evol. 1998. J. 1998. 41:727–731.0. 1993. LAMBERT. FAULKNER. Biol. Insect Biochem. gidA. Microbiol. physiology. Evol. 1991. ISHIKAWA. KAKEDA. M. J. Proc. LORIMER. Accelerated evolution and Muller’s rachet in endosymbiotic bacteria. J. A. New methods for estimating the numbers of synonymous and nonsynonymous substitutions. BRYNNEL. D. IKEMURA. A. and M. and M. Rev. MORAN. L. GIRSHOVICH.. FUKATSU. 1984. Amplification of trpEG: adaptation of Buchnera aphidicola to an endosymbiotic association with aphids. 1985. MORAN.. BAUMANN. Annu. BAUMANN. G. 9:15–20. A. D. MADDISON. Endosymbionts (Buchnera) of the aphid Uroleucon sonchi contain plasmids with trpEG and remnants of trpE pseudogenes. Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families. MORAN. dnaA.. Natl. R. Sequence analysis of a 34. Entomol. Acad. 1994. N. How clonal are bacteria? Proc. and P. coli and the definition of biological species. 10: 7055–7074. C. BAUMANN. The guanine and cytosine content of genomic DNA and bacterial evolution.. and D. 43:17–37. CLARK. TREVORS. M. C.oxfordjournals. 1993. J. Proc. 17:1791–1800. Symbiosis 8:271–283. Proc. N. G. W. and M. MORAN. L. Mol. Evol. and B. C. R... 1996. ISHIKAWA. Acad.. Nature 337:44–47. J. Mol. Rev.. N. C. H. Recombination in E. VON DOHLEN. Buchnera aphidicola: the endosymbiont of aphids. C. J. the atp operon. MORAN. 1992. 1992. J. 1990. BULMER. Microbiol. HINDE. CLARK.. . D. L. L. V. HOFFMAN. CLARK. MUSE. BAUMANN. Testing for equality of evolutionary rates. BAUMANN. BAUMANN. Sinauer Associates. BAUMANN. 1994. A. Faster evolutionary rates in endosymbiotic bacteria than in cospeciating insect hosts. 1998.. E. P. 173:6321–6324. D. P. SMITH. INA. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP. DOUGLAS. J. Evol.. A. and P. 173:7257–7268. G. Biol. J. Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria Buchnera. THAO. P. Natl. U. H. S. Mass. Trends Ecol. Evol. Sci. 35:18–21. Biol. Mol. C. N. MORAN. Immunolocalization of Hsp60 in Legionella pneumonphila. 1981. M. ROUHBAKHSH. A. Bacteriol. M... Wiley and Sons. Genetics 132:269–275. Natl. GREEN. L. E.. A. BERG. ANDERSSON. Models of nearly neutral mutations with particular implications for nonrandom usage of synonymous codons. 1998b. J.. ISHIKAWA. Y. T. Mol. CHRISTELLER. M. Ann. Sci. Phylogenetics of cytoplasmically inherited microorganisms or arthropods. BAUMANN. 1982. A. Nucleotide composition and kinetic complexity of the genomic DNA of an intracellular symbiont in the pea aphid Acyrthosiphon pisum. H. BAUMANN. H. S. BAUMANN. Microbiol. GAUTIER. O’ROURKE. The selection-mutation-drift theory of synonymous codon usage. Mol. M. D. and H. SPRATT. P. Nucleic Acids Res. N. coli transfer RNA’s and the occurrence of respective codons in its protein genes. D. R. CLARK. Insect Physiol. OSAWA. B. D. 1998. . MORAN. USA 95:4458–4462. 1971. J. 1995. N.... T. Mol. Codon usage and tRNA content in unicellular and multicellular organisms. OHTAKA. Nature 336: 254–257. GATENBY. W. N. 2:13–34. LATORRE. BAUMANN.in press. L. 2015 BAUMANN. Genetic characterization of plasmids containing genes encoding enzymes of leucine biosynthesis in endosymbionts (Buchnera) of aphids. Sci. MUNSON. S. 146:1–21.. and D. Accumulation of adenine and thymine in a groE-homologous operon of an intracellular symbiont. MORAN. J. Localized sex in bacteria. . Codon usage in bacteria: correlation with gene expressivity. The predominant protein in an . VOEGTLIN. Sunderland. M. 1998. 40:190–226. MacClade: analysis of phylogeny and character evolution. and B. 1965. and C. and A.96 Wernegreen and Moran aphid endosymbiont is homologous to an E. and rho. BAUMANN. 1998.7-kb DNA segment from the genome of Buchnera aphidicola (endosymbiont of aphids) containing groEL. KENGAKU. 1991. CLARK. HARA. LI. and G. 1989. Evol.. Mol. A. Mol. R. The control of the mycetocyte symbiotes of the aphids Brevicoryne brassicae. Curr. ROUHBAKHSH. Mol. 1989. BAUMANN. M. and G. N. GOLOUBINOFF. 1997. SANDSTROM. Downloaded from http://mbe. Unbiased estimation of the rates of synonymous and nonsynonymous substitution. N. 24:337–345. 1987. O. J. J. USA 84:166–169.. P. MADDISON. Slightly deleterious mutant substitutions in evolution. M. and S. Acad. 1991. OHTAKA. and B. Natl. ASM News 64:203–209. Sci.. DYKHUIZEN. J. Bacteriol. and evolutionary relationships of the genus Buchnera: intracellular symbionts of aphids. 46:449–459. 180:505–513. J. MAYNARD SMITH. E. Discovery and molecular characterization of a plasmid localized in Buchnera sp. Nature 349:29–31. and P. 1987. 1988. Nature 342:884– 889. MORAN. 49:55–94. Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages. B. C. bacterial endosymbiont of the aphid Rhopalosiphum padi.. K. N. LAI. 36:158–163. LAI. A. T. CAMPBELL. E. Myzus persicae and Macrosiphum rosae. 41:67–73. MAYNARD SMITH. Evol.. A. E. GARDUNO. and H. A. P. A. Sequence analysis of an aphid endosymbiont DNA fragment containing rpoB (B-subunit of RNA polymerase) and portions of rplL and rpoC. J. KURLAND.

1986. W.. P. J. Proc. Escherichia coli and Salmonella typhimurium: cellular and molecular biology. and K. D. Version 3. C. ROUHBAKHSH. S. The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae. pombe. H. Phylogenet. NAKAMURA. P. D. M. 16:8207–8211. D. Genetics 16:97–159. CLARK. S. TUOHY.C. A. 1931. SHIELDS. GEOFFREY I. Biometry. Mol. R. D. J.1. 1992. Nucleic Acids Res. Nucleic Acids Res. N. LAI. Evol. N.. Putative evolutionary origin of plasmids carrying the genes involved in leucine biosynthesis in Buchnera aphidicola. 17:5029–39.Evidence for Drift in Endosymbionts SHARP. and W. and F. SOKAL. Y. WRIGHT. Illinois Natural History Survey. Correlation between base composition of DNA and amino acid composition of protein. COWE. Nucleic Acids Res. Gene 113:55–65. sapiens: a review of the considerable within-species diversity. ed. Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases. C.. Evolution of the tryptophan biosynthetic pathway in Buchnera (aphid endosymbionts): studies of plasmid-associated trpEG within the genus Uroleucon. cerevisiae. H.. K. Evolution in Mendelian populations. SWOFFORD. B.. CLARK. T. C. MORAN. 179:4768–4777. Bacteriol. PAUP: phylogenetic analysis using parsimony. J. and its potential applications. SELANDER. BAUMANN. 1993. Pp. and J. M. 16:8023–8040. Evol. 97 . R. D. Natl. D. 31: 71–80. J. MOSURSKI. 1625–1648 in F. WOLFE. 1996. and P. 2015 OHTAKA. D. H. 1989. Switches in species-specific codon preferences: the influence of mutation biases. Genetic structure and variation in natural populations of Escherichia coli. and K.. ‘‘Silent’’ sites in Drosophila genes are not neutral: evidence of selection among synonymous codons.. 1987. 8:167–176.1. L. Nucleic Acids Res. New York.. HIGGINS. BAUMANN. J. MORAN. ROHLF. P. ISHIKAWA. S. R. J. The codon adaptation index—a measure of directional synonymous codon usage bias. WRIGHT. DEVINE. Mol. R. Structures of chaperonins from an intracellular symbiont and their functional expression in E. and F. G. reviewing editor Accepted September 24. R. M. J. ROUHBAKHSH. melanogaster and H. NEIDHARDT. SUOEKA. 174:1869–74. 14:5125– 5143. SHARP. and A. and T. D. BAUMANN. Bacteriol. 5:704–716. A. D. Evol. A. Freeman and Co. Acad. M. MCFADDEN. BIBB. F. G. L. and D. SHIELDS.oxfordjournals. 1997. subtilis. A.. coli groE mutants. BAUMANN. SHARP. F. Champaign. coli. and F. SHARP. SHARP. 1988. 1997. 1981. Sci. Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. MOYA. M. 1988. LATORRE. Nucleic Acids Res. American Society of Microbiology. C. 1998 Downloaded from http://mbe. P. Evol. VOEGTLIN. D. Washington. K. D. 1992. Codon usage patterns in E. Biol.H. SHARP. Codon usage in the G1Crich Streptomyces genome. 1961. VAN HAM. E. USA 47:1141–1149. S. N.. and M. 42:414–421. C. M.org/ by guest on August 5. 1987. Mol. C. CAUGANT. M.. WRIGHT. VON DOHLEN. C. P. 15: 1281–1295. Mol.. SHIELDS. 1987. D. HIGGINS.. 1990. L. D. M. P.. SHIELDS. WRIGHT. LI. A. Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do ‘prefer’ optimal codons. and P. A. WHITTAM.