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.

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

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

S. . . . . . . . . . . . . . . . noxia . . . . . . . . . . . . S. . . graminum . . . . . . . . . . . . . . . ECOR 60 . . . . . . . . . . . . . . . . suberi. . . . graminum . . . . . . . . .d AF038565b. . . . . . . S. Rhopalosiphum padi . . . . . . . . . . Uroleucon solidaginis. . . . . . . graminum . . . . . . . . . . . . . . . . . graminum . . . . . . . T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Uroleucon sonchi . . S. T. . . . . . . . . . . . . . S. . . . S. . . . . . . . . . . . . . chinensis .d AF058428e L46357b. . . . . D. . . . . . . . . . . . . . . . . T. . . . . . . . . graminum . . . . . typhimurium . . . . . . . . graminum . . . Macrosiphoniella ludovicianae . . .d X53376d Y11966b. . suberi. . . . . . . . . . graminum . . . . . . S. . . . graminum . .d J01810d U09185b. . . . . . . . . . . . . . S. . . graminum . . . . . . . . . Uroleucon obscurum . . . . . . .e AF058440d. . . R. . . . . . . . . . . . . . . . . . . . . . . . . . . graminum . . . .org/ by guest on August 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . pisum. .d Z21938b. .d X71612b. . .d AF041837b. . . . . . .e AF058436d. . . . . . . . . . . . . . . . . . . . . . .d M80817b L43549b 2827029b 2827030b 2827034b 2827033b U11066b AF041837b. . S. . . chinensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. . . . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . . noxia . . . . . . . graminum . . . . . . graminum . . . . . . . . typhimurium . . . S. . . . . . graminum . . . . . . . . . ECOR 29 . . . . . . . . . . pisum. . . . . . .d 2754807b. . . . . . . . S. . . . . . . . . . S. . . . . chinensis . . . . . . . . . . . . . . . . . .d L8124d Downloaded from http://mbe. . . . graminum . . . . . . . . . . . S. . . . . . . . . . . . . . . S. . . . . . . . . .d M31047d Y11966b. . . . . S. . . . . . . . . . . . . . . . . . . D. . S. . . T. . . . . . . graminum . . . . . . . .d AF041837b. . . . . . . . graminum . . . R. . . . . S. . . . . . . . . . . . . . . . . . . . . Tetraneura caerulescens. . . . . . graminum . . . . . . . . caligatum . graminum . . . . . . . typhimurium . . . . . graminum . . . . . . . . Schlechtendalia chinensis. ECOR 51 . . . . . . . . . . . . S. noxia . padi . . . . . . . . . . . . . . . . . . . . . . . . . . padi . . . . . . . .d Y11966b. . . . . . . . . . . A. . . . . . . . . . . . . U. . . . . . . . . . S. . . .g X71612b. . . . . .e AF058433d. . noxia . S. . . . . . . . . .e AF058435d. . S. . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . . . . . . . . . . . . padi . . . . . . . . . . maidis . . . . . . . . . . . . . . graminum . . . . . . ECOR 31 . . . . . . . . . . . . . . . . . S. . . . . . . S. . . . . Uroleucon helianthicola . . .d 2754808b. . . . . . . . . . . . . . . . . . . . . . .d Z19055b. . . . . . . . . . . S. . . . . . . graminum . . . . . Schizaphis graminum . Uroleucon astronomus . . .d L46356b. . . . S. . . . . . . . . . graminum . . . . . . . . . . . . . . . ECOR 71 . S. . S. graminum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . . . . . . . Thelaxes suberi .d X71612b. . . . .e U09185b Z19055b U09185b Z19055b L43555b. . . Uroleucon caligatum . . . . . . . . . . . . . S. . . . . . . ECOR 46 . . . . . . . . . . . . typhimurium . . . . . . . . . . . . . . .g AF041837b. . . . . . . . . . . . . . . . . . . . . . . . . . . noxia . .d L46358b. . . . . . . . . . . S. . . . . . . . . . . . . . . S. . . . . . .d AF041836b. . graminum . . . suberi. . . . . . S. . S. . . . . . . . . . . . . A. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . graminum . . . . . . . . . . graminum . . . chinensis . . Diuraphis noxia . S. . . Uroleucon erigeronense . . . . padi . .d D85628b. . . . . . . R. . . . . R. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . graminum . . . . . S. . . . . . . . . . . . . . . S. . . . . . . . suberi. .d U77380b. . . . . . . S. . . .e L81150d. . . .d 47968d AF041836b. . . . . . graminum . . . . . . . Melaphis rhois . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. graminum . . . .86 Wernegreen and Moran Table 1 Genetic Loci of Buchnera Strains Included in Study Gene Name Acyrthosiphon pisum. S. . . . . . pisum. . . . . .g Y11966b. . . . . . . . . . . . . . . . . . . . . . . . . . . . . suberi. . . . . S. . . . . . . padi . . Uroleucon rurale .d U23489e U25425e U23494e U23496e U23495e U23497e U25884e U23499e U23500e U25429e AF058431d. typhimurium . . . . . graminum . . . . . S. . . . . . . . . . . . . . . . . . . . . . . .e L81149d. . . . . . . .oxfordjournals. . . . . . . . . . . . .d L46769b. . . . . . . . . . . . . ECOR 17 . . Rhopalosiphum maidis . . . . . . . . . . . . . . graminum . . . . . . . padi . . . . Uroleucon rapunculoidis . graminum . . . . . . . . Uroleucon jaceicola . . . . . . . . . . . . . .d Y11966b. . . .d AF012886b 2827032b AF01288b X71612b 2827035b Taxona D. . . . . . . . . . . . . . S. . graminum . . . . . . 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. . . . . . .d. . . . . . . . . graminum . . . graminum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . padi . . S. . . . graminum . . . graminum . . . . . . . . . . . . . S. ECOR 72 . . . . . . . . . noxia . . . . . . . . . . . graminum . . . . . . . . . . . . . . .d X71612b. . . . . S. . graminum . . . . . . typhimurium . . . Uroleucon jaceae . . . . . . . . . . A. . . . . graminum . . . . . . . . . graminum . . . . . . . . A. . . . . . . . . . . . . . . . . . Sitobion avenae. .d AF041836b. . Uroleucon aeneum . . pisum. . . .e AF058437d. . . . . . . . . . padi . . . . . . . . . . . . . . . . . . . Myzus persicae . . . . . . . . . . . . . . . . . . . . . . S. . . . . Uroleucon rudbeckiae . . . . . . . . . . . . pisum. . . . . . . . . . . . . . . . . . . . . . . S. M. R. S. . . . . D. . . . . . . . . S. . . . . . . . . . D. . . . . . . A. . . . . . .d L43550b. .g Y11972b. . . S. . . . S. . . . . . . . S. . . . . . . . . . . . . . . . . . . . . . .d L43551b. . R. . . . D. . graminum . . . . . . . . . . . . . . . . . . ECOR 50 . graminum . .g Y11966b. . . . . . . . . . S. . . S. . . chinensis . . . . . . . S. . . . . . . . . . . . . . . . . 2015 Taxona GenBank Accession Number Table 1 Continued . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . persicae . graminum . . . . . . . graminum . . . . . . . . . . . . S. . . . . . . . graminum . . . . . . graminum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. S. . . . . . . . graminum . . . . . . . . . . . . R. S. . . . . . . . . . . . . . . . . .e AF058438d. padi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Salmonella typhimurium. . graminum . . . . . . .d X71612b. . . . . . . . . graminum . . R. . . . . . . .e AF058434d. . . . . . . . . . . . . . . . . . . . . . . .e AF058432d. . . . . . . . . . . . . . . . . . . . . ECOR 37 . . . . . . . .d U01039c U77379b. . graminum . . . . . . . . . . .d D85628b. . . . . S. . . . . . . . . S. . . . . . . graminum . . . . . . graminum . . . . . . . graminum . . . . . . . . . .g 2827037b M80817b L18927b M80817b Z11913b Z11913b M90644b L43549b M90644b U09185b U11066b L43549b 2827009b U09185b Z19055b L46355b. . Uroleucon ambrosiae . . . . . . . R. .g X71612b. . . . . . . . graminum . . . . . . . . . . . .d V01378d U09184b. graminum . . . . . . . . . . . . .d 47764d AF041836b. . chinensis . . . . . . . . . . . . . . . . S. . . . . . . . . . . . . . . . . . .e L81151d. . . . . . . . . . . . . . . . .d AF041837b. . S. . . . . . . .e 1137716b. . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . . S. . . . . . . . . . . . . . . . . . . . . . R. . . . . . . . . . . . . . . . . . . D. . . . . . . . . . . . . . . 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. . . . . . . . . . . . . . . . . . . . . . . . noxia . .e AF058439d. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

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

. . . graminum. .Ending Codons Across Several Buchnera Loci for Each of Eight Fourfold Degenerate Families Locus Schizaphis graminum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .or A.2069 2. Melaphis rhois . . . . . . . . . . . . . . . S. .915 3. . . . . . . . . . S. . . . graminum. S. . . T. . suberi . . . . . . . . . . S. pisum . . . . . . . .7299 2. . . graminum. graminum. S. .227 3. . . . . .2636 5. . . . . . . . . . .487 3. . . . graminum. . . . . . 2015 Taxona Amino Acid No. . . . . . . .706 (P . . . . . . graminum. . . . . . . . . . .754 2. .708 2. . graminum. .734 5. . . . . . . . . S. 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 6.817 3. . . . . .317 6. chinensis . . . . . . . . . . . . . . . S. . . . 0. . . . . . . . . . . . .734 2. . Critical values for these two-class x2 tests are 2. . . . . . . . . . . .8242 5. . . . suberi . . . . . .0635 3. . . .7963 3. . graminum. . . . . Downloaded from http://mbe. . .044 4. . . . . . . . . S. .81 3.2248 3. . . . .401 4. . . . .76 3. . . . . . . S. . .125 3. . . . . . . . . . . . . .841 (P .424 2. . . . .27 4. . . . avenae . . . . . . S. . . . . . . A. . . . . . . . . . . . . . . . . . . . .5667 4. . S. . . pisum . . . . . . S. . . . . . . S. . . . .613 3. . . . . . . . . . . . . . . . . . . . . . . . Sitobion avenae . . . . . . . . . . .777 3. . . . . . . . . . . . noxia . . . . . Rhopalosiphum maidis . . . . . . graminum.oxfordjournals. . . . . . . S. . . .784 3. . . . . . R. . . . . . . graminum. . . Diuraphis noxia .Evidence for Drift in Endosymbionts 89 Table 2 Significant Nonrandom Use of U. . . . . .05). . . . . .8263 7. . . . . . . . . chinensis . . graminum. . . . . . . S. . . A. . . S. . .0218 3. . . . . . . . . . graminum. . . . . . . . . . . . . . . . . . . . . . . . . S. S. . . . . . . Acyrthosiphon pisum . . . . . . . . . . . S.9462 3. . . .0547 6. . A. .532 3. .531 2. . . . . . . . . padi .8356 3. . . . . .9 2. . . . . . graminum. . . . . . Thelaxes suberi . S. . . . . . . . . . . . . . graminum. . S. . . . . . . . . . . . . chinensis . D. graminum. . graminum. . . . . graminum. . . . . . . . . . . . M. . rhois . . . . . . . graminum. . . . . . . .435 2. . . . . . . . . graminum. . . . . . .484 3. . . chinensis . T. . S. . .2 3 2. . . . . . . . A. pisum . .756 2. . . . . . . . . . . . . . . . . . . . . . . Schlechtendalia chinensis . . . S. . . . . . . . . . . . . . . . S. . . .2 3. . S. . . . . . . . . . . chinensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .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. . . . . . . .174 9. . T.624 3. graminum. . graminum. . .9269 6. graminum. . . . . . . . . . . . . . . . pisum . . . . . . . . . S. . . . . . . . . .1) and 3. . . S. . . . . . . .995 4 6. suberi . pisum . . .814 4. . . . . . . . . . . . S. . . . . S. . . . . . . S. . . . . graminum. of Amino Acid Residues in Protein . . . S. . A. . . . . . . . . . . . . . . A. . . . . . .7696 7. . . . . . . .803 2. . . . . . . . . . graminum. . . . . . . . . . . . S. . . . . . . . . graminum. . . . . . . . . . . . . . . . . . . . . . . . . . . . .452 3. . . . . . . . pisum . . . noxia . . . . . . . graminum. S. . avenae . . . . chinensis . S. . . .5219 5. . . . . . . . . D. . . . chinensis . .158 2. . . . . . . . . S. . . . . . . . . . . S.368 8. . . . . . .org/ by guest on August 5. . . . . .5715 9. . . . . . . . .265 3. . . .45 3. . . .5566 5. . . . graminum. . . . . . . . . . . . . . . . . . . graminum. . . . .417 3. S. . . . . . . . graminum. . . . . . . . . . . .858 3. . . . . .293 4. . . S. . S.875 2. . . . . .039 2. . chinensis . . . . . . . . . . . . graminum. . S. graminum. . . . . . . . . . . . .1565 3. . . S. . 0. . . . . . . . . . Pemphigus betae . . . . . . . . . . . . . . . . . graminum. . . . . . S. S. . . .184 3. . . . . . graminum. . . . . . . . . .

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

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

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... . 32 8 119 64 15 14 46 36 39 0.. ..... . distinct trpB clades were considered separately. . . . . . . . . however..) 0.01 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.. .. 4).77 0. . .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. ... coli strains (0.01 0. . . The ECOR and Buchnera trees each include nodes with relatively weak support (see bootstrap values. tuf . . . high ratios of nonsynonymous to synonymous divergence indicate that replacement substitutions accumulate at faster rates in a particular lineage (Brynnel et al. trpA . 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.12 0.083 0. . However. . . these relatively weak . G. . . . It should be noted. . . V.047 0. .6 24. .. leu genes ..8 43. . . . trpD . . . . .162 to 0.. .. . .. T .015 0.123 0. . . .. .. . .. however. .42 2. .. . 4). .oxfordjournals. .92 Wernegreen and Moran Table 3 Number of Buchnera Loci Showing a Slight Preference for U.018 0.. .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. . .154 0. ..64 P VALUE (.6 42.0328 across the E.. . .. coli or Buchnera isolates. 4). .09 5.. trpG . .13 8. S . .. . For the purposes of mapping nucleotide changes at each codon position. . 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. . . Therefore.. . . .. coli versus Buchnera Under the assumption that rates of synonymous substitutions reflect mutation rates. .. .org/ by guest on August 5. . nucleotide changes were summed across genealogies of trpB instead of calculated pairwise (fig.031 0.. c groEL had the greatest overrepresentation in the pool of significant tests compared to its frequency in the original sample.108 0.025 0. nodes do not affect the results obtained.092 A total of 772 x2 tests were performed.041 0. R.0 37. of x2 Tests Fraction of Total x2 Tests Performeda No. fig. coli trpB phylogeny. .. .015 0. .060 0. ... This assumption may be violated by differing levels of codon bias in Buchnera and E. ..... a b Downloaded from http://mbe. ... . . trpB . coli. .. ..019 0.... b The x2 value expresses the deviation of the observed from the expected number of loci with preference for A-ending codons. .8 31. divided by the length at third positions adjusted for the number of sites.32 6.9 NO. Significant x2 Tests Fraction of Significant x2 Testsb groELc . .0 46. . . Because of low levels of divergence within each group..3 45.. . relative to the E... .. .285 across subsets of the Buchnera trpB phylogeny and 0. Estimates of Synonymous Substitution in E.077 0.... Given the higher divergence among Uroleucon isolates. 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.. L . coli may depress rates of synonymous substitution and thus elevate Ka/Ks estimates. fig. coli strains and Buchnera associated with the aphid genus Uroleucon. . gave comparable values for the ratio of the tree length at first and second positions. groES .. Selection for translational efficiency in E. . Each clade.077 0.010 0. that the resolved trees presented are not intended to represent exact relationships among E. ... OF BUCHNERA LOCIa TOTAL LOCI x2 VALUEb 7. trpE .031 0.051 8 1 7 2 5 1 5 2 6 0.. .. ..07 0. .. . 1998). P . The total number of significant x2 tests was 65.. . This approximation of nonsynonymous divergence divided by synonymous divergence is considerably higher for the Uroleucon isolates...

relative-rate tests and comparisons of Ka/Ks ratios support previous conclusions that Buchnera lineages experience rapid sequence evolution at nonsynonymous sites. .21 0. 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. 1995. leuA . In Buchnera. Function .37 0. .6 4. . leuC.07 0. . . .66*** 1. . Buchnera provisions host insects with these nutrients.8 2. Baumann. . . 0. .5 2. . .29 0.58 0.10 0. influenzae Haemophilus ducreyi Vibrio cholerae Pseudomonas putida P. . since groEL is apparently single copy in at least one Buchnera species in which it is overexpressed.oxfordjournals.0 2.17 0. . .07 0.40 0. .31 0. coli) also suggest higher rates of fixation at replacement sites.14 0. . dnaJ. . both mutualistic (Aksoy 1995.3 2.01. in Buchnera.45 0. .16 0.14 zb K01/K02 7.6 1. .02 0. . leuB . which do not synthesize tryptophan and which depend on Buchnera to provide this essential nutrient (Baumann et al.9 1. However. aroA . . . . This increased copy number is considered an adaptation that benefits the aphid hosts. 2015 ilvC . . dnaK .71 1.20 0. comparisons of changes at first.06 0. . . .21 0.9 2. . influenzae H. . .0 3. .00*** 6. genes for anthranilate synthase (trpEG).36 0. . In addition.12 0.23* 3. 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. .6 a In each test. .24*** 1. . .14 0. . 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. influenzae Pseudomonas aeruginosa Azotobacter vinelandii A. . Baumann.06 0. secB. . 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.28 0. influenzae H. 1998a). 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.55 0.11 0. . and Baumann 1994. . . . influenzae H. .14 0.05. . influenzae H. Goloubinoff. .20 0. . tpiA . . . Hara et al. atpD .and second.vs.29 0. The mechanism for this overexpression is uncertain but likely involves changes in gene regulation rather than a gene duplication. . except for dnaJ and dnaK. In several Buchnera lineages. ** P . vinelandii Aeromonas salmonicida H. 1990). . . . relative to synonymous sites.56 0. b z scores were calculated as described by Muse and Weir (1992). .45 0. and translocation of other polypeptides (Bochdareva.18 0. .42 0.44 0.21 4. .22 0. influenzae 0.41 0.36 0. . and taxon 2 is always Escherichia coli. .65*** 5. ilvI . . . ilvD . .58 0.0001. . .05 0.27 0. .18 0. Baumann.3 6.09 0.3 1. 1996. . .25 0.59 0.09 0.04 0. . . . . c ‘‘(pt)’’ indicates that only a partial sequence is available in GenBank.37 0.org/ by guest on August 5. vertically inherited endosymbionts provide a good model system to test the effects of increased drift on sequence evolution in bacteria. 0.30 0. .24 0.07 0. . influenzae H. . and Lorimer 1989). . Rouhbakhsh et al.53 0.22 0.30 0.17 0. 1995.84*** 6.7 5. 1989. .4 4.33 0.7 2.25 0. Taxon 3 is a more distantly related reference taxon. . . . influenzae P.24 0. .43 0.13** 3. Likewise.25 0. . In this study. Gatenby. . which are Buchnera of Acyrthosiphon pisum. taxon 1 is Buchnera of Schizaphis graminum. observed duplications of leucine genes and their occurrence on plasmids may represent an adaptation for overexpression (Bracho et al. rpsA. . dnaN . . While its role Downloaded from http://mbe.16 0. In addition.48*** 7. .21 0. .20 2. . . . . 1997. Lissen.18*** 3.21 0. gidA.06 0. Conclusions Because of their small population sizes and limited opportunities for recombination.42 0. . . and Clark 1996) and pathogenic (Garduno et al.4 3. rpoB . Baumann et al. the first and limiting enzyme in the tryptophan biosynthetic pathway. . .38*** 4.77 0. . assembly. . .9 2. . The protein is known in E. third-codon positions across very shallow taxonomic levels (Buchnera isolates of Uroleucon and members of the ECOR collection of E. leuD. .36 0. . Douglas 1998). groEL comprises about 10% of the total protein produced (Ishikawa 1984. 1998). . . .32 0. . gapA . . 1997. . In addition.29 0. influenzae H. . coli.01*** 4. . . .36** 1.10 0. putida H.31 0. 1997.20 0. . . 0. A set of loci that might be suspected of experiencing unusual selection in Buchnera are those encoding enzymes for biosynthesis of essential amino acids. which are limiting in the plant phloem on which aphids feed. . 1998b). . coli to be a heat shock chaperonin involved in the folding. .38 0. putida H. Goloubinoff et al.12 0. .34 0. .05 0. cysE. van Ham et al. . Baumann et al. dnaG(pt)c . *** P . . . . .33 5.83*** 2.12 0. . are amplified in tandem repeats on multicopy plasmids (Lai.23 0.26*** 3.4 1. groEL is known to be constitutively overexpressed in several intracellular bacteria.22 0.6 3. . Probabilities for one-tailed t-test (H :K 0 01 # K02) are * P .21 0. . E. 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. .21*** 6. . the symbiont of Acyrthosiphon pisum (Ohtaka and Ishikawa 1993).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. compared to their free-living relative.37** 5.34** 3. .7 2. and Girshovich 1988. . .25 0.

12 have K01/K02 . (B) Ratios of nonsynonymous to synonymous substitutions. on the basis of pairwise divergence across Buchnera taxa [. 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. Of the first set. coli homologues. 2. All Ka/Ks ratios are based on Ks values less than one.94 Wernegreen and Moran in endosymbionts is less certain. however. selection is unlikely to be relaxed across all loci. as suggested by Moran (1996). even functionally important genes. 14 loci encode amino acid biosynthetic genes and 14 encode genes for other functions. typhimurium [x]. One alternative explanation for elevated evolutionary rates at nonsynonymous sites is positive selection for amino acid changes. and 2 have K01/K02 . Given their apparent significance in the symbiosis. argues for an increased effect of drift within endosymbiont populations.0 were excluded. 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. Of the second set.] taxa and across E. In this study. With the exception of groEL. 3—Comparison of levels of nonsynonymous substitutions (Ka) and synonymous substitutions (Ks) across several loci of the enteric bacteria and Buchnera. there is no evidence that the acceleration of nonsynonymous substitution rates is any higher across Buchnera biosynthetic genes than across other Buchnera loci. 2 and seven have K01/K02 . (A) Pairwise divergence at nonsynonymous and synonymous sites. and the trends we observed occur consistently across each locus examined. 2015 FIG. In addition. coli versus S. it may function to stabilize proteins that have accumulated amino acid substitutions. Perhaps the intracellular environment is more constant than that experienced by free-living bacteria. all Ka/ Ks estimates for Buchnera exceed the ratio calculated for the homologous gene of the enteric bacteria. amino acid biosynthetic loci tend to be less accelerated than other Buchnera genes. it is difficult to imagine relaxed selection on trp genes. table 2). . Ks values higher than 1. relative to E. such selection typically acts at specific loci rather than across the genome.oxfordjournals. sequence evolution of these functionally important Buchnera genes shows the same patterns as housekeeping genes that are not overexpressed.] and across E. First. coli versus Salmonella typhimurium [x]. The fact that the observed trends of depressed codon bias and accelerated evolutionary rates occur across all Buchnera loci included. In addition. While the results here cannot exclude the possibility of relaxed selection. two features of the data argue against it. 1. Combining loci from table 5 with those examined previously (Moran 1996. and Downloaded from http://mbe. calculated across Buchnera [. seven have K01/K02 . In particular.org/ by guest on August 5. Thus. 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. 2. leu genes.

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

96 Wernegreen and Moran aphid endosymbiont is homologous to an E. 1981. C. LAI. A. USA 91(38):19–23. and B. 1993. J. R. 36:96–99. E. Myzus persicae and Macrosiphum rosae. THAO. W. Mol. Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages. Bacteriol. 1995. How clonal are bacteria? Proc. ISHIKAWA. Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families. 41: 345–352. P. KAKEDA. J.. G. The guanine and cytosine content of genomic DNA and bacterial evolution. . Version 3. D. D. 35:18–21. Recombination in E. N. M. 1971. BULMER. and G. J. CLARK. VON DOHLEN. M. 1994. CLARK. Codon usage in bacteria: correlation with gene expressivity. 180:505–513.. 173:7257–7268. Testing for equality of evolutionary rates. BAUMANN. and L. KENGAKU. Y. 1984. Mol. Microbiol. C. Evol. M. A.. G. IKEMURA. A. HARA. Mol. 1998. A. INA. The control of the mycetocyte symbiotes of the aphids Brevicoryne brassicae. Evol. R. BAUMANN. physiology. MORAN. G. 1985. VOEGTLIN. Natl. 1987. Acad. MORAN. SPRATT. L. 1998b. and P. Y. Evol. MAYNARD SMITH. 36:121–126. Proc. 146:1–21. 1991. N. CHRISTELLER. Nature 246:96–98. S.. MUNSON. ISHIKAWA. P. New York. LORIMER. Nucleic Acids Res. 15:574–582. M. Mol.. and P. Mol. 1994. 1998. 1998. R. Evol. A. N. 2:13–34. C. coli transfer RNA’s and the occurrence of respective codons in its protein genes. OHTAKA. and evolutionary relationships of the genus Buchnera: intracellular symbionts of aphids. Rev. T. and B. Genetics 129:897–907. G. L. 1993. Discovery and molecular characterization of a plasmid localized in Buchnera sp. H. A. C. 25:283–290. 1998. Genetics 132:269–275. P. Proc. MORAN. 1965. BERG. 1998a. Nature 336: 254–257. P. L. ROUHBAKHSH. 173:6321–6324. GOLOUBINOFF. Sunderland. and rho. L. Sci. T. A. 40:190–226. H. MORAN. A. WEIR. P. 10: 7055–7074. MORTON. Models of nearly neutral mutations with particular implications for nonrandom usage of synonymous codons. Acad. 1992.. and P. coli and the definition of biological species. 46:449–459. TREVORS. CLARK. USA 84:166–169. Evol. M. Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria Buchnera. Sci. USA 95:4458–4462. and H. C. L. Acad. BAUMANN. Evol. Evol. Evol. VATS. Mol. Proc. V. Amplification of trpEG: adaptation of Buchnera aphidicola to an endosymbiotic association with aphids. OHTA. BOCHDAREVA. Mol. CLARK. N. W. Unbiased estimation of the rates of synonymous and nonsynonymous substitution.. BAUMANN. and H. Insect Physiol. B. and G. GOUY. and N. Genetics. S. Proc. D. 24:205–211. M. New methods for estimating the numbers of synonymous and nonsynonymous substitutions.. DYKHUIZEN. BAUMANN. Buchnera aphidicola: the endosymbiont of aphids. ISHIKAWA. OSAWA. J. A. Localized sex in bacteria. T. VOEGTLIN. OHTAKA. Bacteriol. D. Trends Ecol. Biol. D. 1997. Mol. A. N. Entomol. CAMPBELL.in press. bacterial endosymbiont of the aphid Rhopalosiphum padi. MORAN. N. 2015 BAUMANN. BRYNNEL. J. ASM News 64:203–209. MARTINEZ-TORRES. Kinetics of synonymous codon change for an amino acid of arbitrary degeneracy. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. ANDERSSON. GATENBY. 1995. N. and S. Biol. Microbiol. Nature 337:44–47. SPRATT. . and C. J.7-kb DNA segment from the genome of Buchnera aphidicola (endosymbiont of aphids) containing groEL. A. 1995. Ann. Natl. L. J.. J. Natl. P. Slightly deleterious mutant substitutions in evolution. E. LI. USA 93: 2873–2878. L. J. Evol. USA 90:4384–4388. M. LORIMER. P. and P. Sequence analysis of an aphid endosymbiont DNA fragment containing rpoB (B-subunit of RNA polymerase) and portions of rplL and rpoC. J. Bacteriol. C. Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria. MUSE. BAUMANN.. coli heat shock protein. 1982. J. Curr. and B. Characterization of the protein species synthesized in vivo and in vitro by an aphid endosymbiont. BAUMANN. BRACHO.. 1989... 9:15–20. J. and A. 1989. N. 1993. Evol. SANDSTROM. E. M. Accumulation of adenine and thymine in a groE-homologous operon of an intracellular symbiont. O’ROURKE. J. 41:67–73. and M.0. Faster evolutionary rates in endosymbiotic bacteria than in cospeciating insect hosts. Genetic characterization of plasmids containing genes encoding enzymes of leucine biosynthesis in endosymbionts (Buchnera) of aphids. BAUMANN. Symbiosis 8:271–283. BAUMANN. and M. MUTO. CLARK. and M. G. Correlation between the abundance of E. Wiley and Sons. 1973. Nature 342:884– 889. and A. Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP. E. and D. Nature 349:29–31. MADDISON. LISSEN. MORAN. BAUMANN. GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli. GATENBY. BAUMANN. LAI.. 49:55–94. BAUMANN. J. LAMBERT. P. 1991.. T. The predominant protein in an . Endosymbionts (Buchnera) of the aphid Uroleucon sonchi contain plasmids with trpEG and remnants of trpE pseudogenes. 17:1791–1800. O.. Insect Biochem.oxfordjournals. Evol. KURLAND. A. M. Downloaded from http://mbe. GARDUNO. 1998. D. A. Codon usage and tRNA content in unicellular and multicellular organisms. A. Immunolocalization of Hsp60 in Legionella pneumonphila. Mol. MORAN. Sci. MOYA. A. A. P. dnaA. J.. gidA. L.. BAUMANN. J. Phylogenetics of cytoplasmically inherited microorganisms or arthropods. S. MADDISON. J.. 36:158–163. 1990. and B. Sci.. MORAN. GOLOUBINOFF. and S. U. 1998. HOFFMAN. ROUHBAKHSH. 43:17–37. Mass. A. LATORRE. Nucleotide composition and kinetic complexity of the genomic DNA of an intracellular symbiont in the pea aphid Acyrthosiphon pisum. E. J. M.. S. Mol. C. DOWSON. 1993. 1991. 1996. J. Natl. DOUGLAS. GAUTIER. M. MacClade: analysis of phylogeny and character evolution. Annu. 24:337–345. Evol. H. Mol. HINDE.. K.. and D. S. Acad. and P. THAO. A. Mol. 1987. Curr. Sequence analysis of a 34. Natl. 1987. GREEN. .. B. GIRSHOVICH. A. . 1992. R. N. Microbiol. 1995. 1988. L. Evolutionary rates for tuf genes in endosymbionts of aphids. C. J. J.org/ by guest on August 5. A. BAUMANN. FUKATSU. 1992. FAULKNER. 14:417–425. The selection-mutation-drift theory of synonymous codon usage. Endosymbiosis of animals with plant microorganisms. MAYNARD SMITH. N. Accelerated evolution and Muller’s rachet in endosymbiotic bacteria. Sci. Biol. MORAN. Acad. 1995. Rev. J. BUCHNER. D. N. Evol. BAUMANN. Microbiol. H.. 41:727–731. Curr. Mol. A. the atp operon. SMITH. Sinauer Associates.. M.

1992. P. and F. ROUHBAKHSH. R. GEOFFREY I. 5:704–716. reviewing editor Accepted September 24. TUOHY. BAUMANN. and J.org/ by guest on August 5. melanogaster and H. cerevisiae. SWOFFORD. Mol. Genetic structure and variation in natural populations of Escherichia coli. R. D. J. Champaign. CAUGANT. A. 1988. M.. H. SHARP. Y. 179:4768–4777. F. Acad. Nucleic Acids Res. P. HIGGINS.oxfordjournals. and P. B. P. NEIDHARDT. H. C. coli. D. ‘‘Silent’’ sites in Drosophila genes are not neutral: evidence of selection among synonymous codons. SHARP. Codon usage patterns in E. MOSURSKI. sapiens: a review of the considerable within-species diversity. T. coli groE mutants. Proc. 174:1869–74. 1997. BAUMANN. BAUMANN. M. and W. SELANDER. A. MORAN. 1993. 97 . Mol. Evolution in Mendelian populations. L. VAN HAM. ROHLF. and T. Sci. LATORRE. 1987. WRIGHT. VOEGTLIN.. Freeman and Co. 1986. A.. pombe. and P. and F. 1981. C. Correlation between base composition of DNA and amino acid composition of protein. C. Nucleic Acids Res.. 31: 71–80. and K. Gene 113:55–65. CLARK. S. 1989. and F. 15: 1281–1295. C. Evolution of the tryptophan biosynthetic pathway in Buchnera (aphid endosymbionts): studies of plasmid-associated trpEG within the genus Uroleucon. WOLFE. R. C.. N. Evol.. R. L. Biometry. D. Nucleic Acids Res. Version 3. D. A. American Society of Microbiology. D. D. DEVINE. D. CLARK. J.. J. New York. BIBB. SHARP. and D. D.. Natl. 16:8207–8211. D. S. D. K. 1987. D. L. P. Genetics 16:97–159. P. G. Mol. WRIGHT. 1988. Phylogenet. 1998 Downloaded from http://mbe. MORAN. 8:167–176. Washington. Evol. D. C. Putative evolutionary origin of plasmids carrying the genes involved in leucine biosynthesis in Buchnera aphidicola. Nucleic Acids Res. Codon usage and gene expression level in Dictyostelium discoideum: highly expressed genes do ‘prefer’ optimal codons. SOKAL. K. S.H. M. D. and K. The codon adaptation index—a measure of directional synonymous codon usage bias. MCFADDEN. 42:414–421. Evol. 1997. PAUP: phylogenetic analysis using parsimony. N. 17:5029–39. 1992. Biol. and M. H. VON DOHLEN. SHARP. LAI. Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes. Bacteriol.1. M. J. 14:5125– 5143.C. Evol. N. SHIELDS. SHIELDS. Illinois Natural History Survey. WRIGHT. subtilis. C.. MOYA. S. ISHIKAWA. ed. G. 16:8023–8040.1. SUOEKA. HIGGINS. SHARP. The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae. A. 1996. R. P. Escherichia coli and Salmonella typhimurium: cellular and molecular biology. Structures of chaperonins from an intracellular symbiont and their functional expression in E. Synonymous codon usage in Bacillus subtilis reflects both translational selection and mutational biases. F. SHIELDS. 1625–1648 in F. E. 1931.. M. M.. SHIELDS. 2015 OHTAKA. J. J. J. 1990. and A.Evidence for Drift in Endosymbionts SHARP. A.. Nucleic Acids Res. COWE. USA 47:1141–1149. WHITTAM. W. Switches in species-specific codon preferences: the influence of mutation biases. ROUHBAKHSH. NAKAMURA. WRIGHT. 1961. M. 1987. BAUMANN.. LI. Pp.. M. and its potential applications. Mol. Bacteriol. Codon usage in the G1Crich Streptomyces genome.

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