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.

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

nonrandom use of U. we test the hypothesis that translational selection is relatively ineffective in the endosymbionts. Amino acids with less than five residues in a given locus were omitted. Brynnel et al.and A-ending codons were compared by a twoclass x2 test adjusted for a small sample size. Expected relative frequencies of U. By applying this method to several homologous loci in Buchnera and in their free-living relative. compare levels of bias with homologues in E. coli. 1998). and nucleotide alignments were adjusted to conform to amino acid alignments. previous evidence for the accumulation of nonsynonymous substitutions through drift strongly suggests that nonoptimal codons will accumulate in Buchnera lineages. as modified by Akashi and Shaeffer (1997) to adjust for A1T content at silent positions. 1998). coli are compared across an extensive sample of available protein-coding loci. and explore the possibility of subtle. von Dohlen.) 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). Therefore. Genetics Computer Group. personal communication). However.200. adaptive codon bias is expected to be much lower than that observed in E. Values near saturation are known to have high standard errors.). However. For example. nucleotide composition at fourfold degenerate sites was determined for each Buchnera locus (by the computer package Molecular Evolutionary Analysis [MEA]. coli. Second.and A-ending codons from frequencies expected if codon usage reflects local base composition. rates of sequence evolution of Buchnera and E. E. were limited by high levels of synonymous divergence (Moran 1996). Codon Bias Estimation of Codon Bias at Fourfold Degenerate Sites For each Buchnera locus. Previous estimates of the ratio of nonsynonymous to synonymous divergence in Buchnera 85 . Alignments Inferred protein sequences of homologous loci were aligned using Pileup of GCG (Wisconsin Sequence Analysis Program. (Arginine. Previous studies provide strong evidence for differences in rates and patterns of sequence evolution of endosymbiotic and freeliving bacterial lineages. as suggested by Sokal and Rohlf (1981) for sample sizes . as would be expected in small. leucine. the magnitude of the x2 value reflects the deviation of random use of synonymous codons. the x2 values in this study reflect the deviation of relative frequencies of U.and A-ending codons within fourfold degenerate codon families was assessed using a x2 analysis.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). The x2 analysis involved several steps: first. which may be exacerbated by the strong A1T bias of Buchnera genomes (Berg 1995). asexual populations is also expected to accelerate the fixation of replacement substitutions. Here we assess patterns of codon usage across several Buchnera loci. in order to test whether previously observed rate elevation is a general phenomenon across the Buchnera genome. rates of sequence evolution. Downloaded from http://mbe. 2015 Rates of Sequence Divergence The decreased effectiveness of selection in small. Methods Loci Sampled Several loci from Buchnera taxa and the enteric bacteria were included (table 1) in estimates of codon bias. Wis. and serine were treated as fourfold degenerate by considering only the fourfold degenerate synonyms. the 16S rRNA gene of several endosymbiotic lineages has been shown to evolve 1. asexual populations. the x2-based method developed here tests for nonrandom-use codons for single amino acids and does not require prior knowledge of preferred codons.org/ by guest on August 5.5–2 times faster (Moran.oxfordjournals. 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. (1988). x2 values were averaged across the fourfold codon families after excluding those amino acids with fewer than five representatives. Similar to the scaled x2 of Shields et al. This approach may be generally applicable to other organisms in which codon preferences may be absent or subtle. This study combines several types of analyses to further explore whether Buchnera loci experience increased rates of fixation of deleterious mutations. such as in taxa with small effective population sizes and/or strong mutational biases. coli homologues (Moran 1996. observed and expected relative values of U. 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. we compared observed codon frequencies to those expected if codon usage reflects local base composition at synonymous sites. In addition. and observed changes in endosymbiont 16S rRNA genes destabilize the secondary structure of the molecule (Lambert and Moran 1998). In this study. in contrast to previous estimates. and patterns of nonsynonymous and synonymous substitutions. and Baumann 1995). coli. several protein-coding genes in Buchnera have been shown to evolve more rapidly than their E. compared to freeliving relatives in the enterics. Madison. codon preferences in Buchnera. Brynnel et al. Moriyama. the consideration of shallower taxonomic levels allows for more reliable estimates of ratios of nonsynonymous to synonymous substitutions. E. possibly different. Since purifying selection against nonoptimal codons is likely to be weaker than selection against replacement substitutions. In addition. 1988). Similar to the scaled x2 (Shields et al. Regions of loci with ambiguous amino acid alignments were excluded from the analysis.Evidence for Drift in Endosymbionts adjusted for local base composition.

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

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

Clark. but lowly expressed loci are biased toward GCA. coli is also evident in the narrow range of x2 values for each fourfold degenerate family across Buchnera loci (fig.815 for df 5 3).or A-ending codons for individual amino acids. the lack of correspondence between average x2 estimates of Buchnera genes and the CAI of homologous E. coli. In particular. coli (Sharp et al. This paucity of significant x2 tests indicates that codon usage generally reflected local base composition. groEL. a strong relationship exists between the average x2 values for loci of E. and Baumann 1998). relative to its frequency in the original sample (4.1% in original Downloaded from http://mbe. Bau- Evidence for Preferential Use of U-ending and Aending Codons in Buchnera Despite the severe depression of codon bias in Buchnera. coli depend on gene expression levels.4%). This gene had the greatest overrepresentation in the pool of significant x2 tests. coli genes. serine and arginine are generally encoded by U-ending codons. which is known to be highly expressed in Buchnera (Baumann. Baumann. the majority of E.org/ by guest on August 5. In addition. which is known to be highly correlated with gene expression level (Sharp and Li 1987). Clark.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. Comparison of x2 Values for Individual Amino Acids The contrast between levels of bias in Buchnera and in E.or A-ending) codon (table 2). In contrast. Buchnera loci rarely show significantly nonrandom use of U. Strong mutational bias and drift may largely shape patterns of codon use. 1988). even for groEL. and alanine tends to be encoded by the A-ending codon. these significant results cannot be attributed to local variation in A1T content. 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. . possibly precipitated by drift in small populations. table 3). coli loci indicates that codon bias in Buchnera does not correspond with levels of gene expression of E. fewer tests were significant than expected by chance alone. averaged across amino acids for each locus (fig. This is consistent with previous observations of A1T richness across the Buchnera genome (Ishikawa 1987. 0. In contrast. and 42 more were significant at the 10% level (table 2). coli loci have average x2 values greater than the critical value for significance at the 5% level (7. significant x2 tests across loci almost always reflect a higher frequency of one particular (U. 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. Tuohy. 1). and Clark 1996). coli loci showed significant nonrandom use of alternative codons for most amino acids. and Mosurski 1986). The preference for GCA across Buchnera loci may reflect a change in the optimal codon. and Baumann 1998). 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. coli loci. Buchnera populations may occasionally go through periods where selection is more effective. and Baumann 1992. the preferred codon within the fourfold degenerate family for serine tends to be UCU. 2015 Comparison of E. codon usage cannot be attributed solely to mutational bias. whereas no Buchnera loci have an average x2 value that exceeds the critical value significance at 5% (3.oxfordjournals. x2 values are relatively high for individual amino acids across most E. codon preferences for alanine in E. Likewise. In contrast. However.841 for df 5 1). and alanine tends to be encoded by GCA (P . possibly because of an expansion of aphid population sizes. 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. 274 were significant at the 5% level. coli and Buchnera (see Methods). Of a total of 528 x2 tests performed across E. such as trp genes (Sharp. but these occasional periods of effective purifying selection may reduce the frequency of nonoptimal codons. including those considered low expression in E.01 in each case. 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). as highly expressed loci are biased toward GCU. Since expected values of these tests are based on gene-specific base composition. coli and the CAI on the basis of preferred codons for this species. Although the magnitude of x2 values is not directly comparable for E. Baumann. For these amino acids. mann. coli homologues and provides further evidence against effective translational selection in Buchnera. and 35 more were significant at the 10% level (data not shown). Of a total of 772 x2 tests performed across Buchnera loci. 2). These preferences agree with the general trends found in a previous analysis of codon usage across several loci of the endosymbiont of Schizaphis graminum. which is thought to represent the optimal codon for this amino acid. for loci showing slight (nonsignificant) codon preferences. Baumann. Only 23 tests were significant at the 5% level. Comparison of x2 Averaged Across Amino Acids The depression of codon bias reflected in Buchnera is also apparent in the low x2 values. Ohtaka and Ishikawa 1993. many of which are included in the genes sampled here (Clark.

. . . .1565 3. . . graminum. . . . . . .915 3. . S. . . . . . . S. . . . graminum. . 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. . . . . . . . . . . S. . . . S. . . . . .435 2. . . . graminum. . . . . . . . . . . . S. graminum. . . .0547 6. graminum.417 3. . . .484 3. . Schlechtendalia chinensis . . . . . . . . . . .9 2. . Melaphis rhois . S. graminum. . . . . . . . . .401 4. . . noxia .734 2. . . . . .227 3. . . graminum. . . . . . . . S. . . . graminum. . . . . . . chinensis . . . . . . . . . . noxia . . . .8356 3. . . Diuraphis noxia . . . . . . . . . . . . . . . . . . . . . . . . graminum. . . . S. . S. . . S. . . . . . . . . . . . . . S. . chinensis . . . . . . . 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. . . . . . . . . .81 3. . . . . . . . . . Critical values for these two-class x2 tests are 2. . . . . . . . . . . .734 5. . . . . graminum. . . . chinensis . . . . . . .531 2. .784 3. . . . . .9269 6. . . . . . graminum. . Thelaxes suberi . . . . .368 8. . . . . S. . . . . pisum . . . . graminum. . . .or A. . . . . S. . .708 2. . . . . . . . . . . . . . T. . . . . . . . . . . . . . . . . graminum. . . . . . . . . .0635 3. . . . . Acyrthosiphon pisum . . . . S. . . . .452 3. . . . . . . . . . .624 3. . . . . . . . .814 4. . . . Downloaded from http://mbe. graminum. . . S. . . . . . . .125 3. . . . Sitobion avenae . . . . . . . S. . S. . . . . . . chinensis . .044 4. . . . chinensis . . . . . .2069 2. . . graminum. . . . . . . . . .756 2. . graminum. . . . . . . . . . . . . . . . S. . . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . . . . . . .5667 4. S. . . padi . S. . . . . . . . .5566 5. . graminum. . graminum. . . . . . . . . . . . . . . . . . .754 2. graminum. . . .613 3. . . .265 3. . .7299 2.2 3 2. . . . . . . . . . . . . . . . . .532 3. . . . S.1) and 3. . . . . . . . . . . . . . . . . . . . . . . A. . . .039 2. . . .174 9. . . . . . . graminum. . . . graminum. graminum. . . . . . . . . graminum. . . . . . . .317 6. . . chinensis . . . . . . . . . . .45 3.777 3. graminum. . . . . . . . . . . . . . . . .8263 7. . . . S. . . . . . .2248 3. S. . . . .817 3. . . . graminum. . . . . . . . . . . . .5219 5. . .7696 7. S. . . . . . . . . . S. . . .5715 9. . . . D. . . . . pisum . . . suberi . . . T. . . . . . graminum. . .158 2. . . . . . . . . suberi . . . . . . . . . . . D. . S. . .841 (P . . . . . . . . . . .995 4 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. .8242 5. . Pemphigus betae . . .Ending Codons Across Several Buchnera Loci for Each of Eight Fourfold Degenerate Families Locus Schizaphis graminum . . . . . . . . . .0218 3. . . . . . graminum. . . . . . . . . . A. . . . . . . . . . . . .858 3. . . . . . . 2015 Taxona Amino Acid No. . . . . . . A. . . . . . . . A. graminum. . . . . . . . . . . .7963 3. . . . . S. . . . . graminum. . . A. . . . . . . . . . . . . . . . S. . . . . . Rhopalosiphum maidis . . . . . . . . . . . . S. . . .Evidence for Drift in Endosymbionts 89 Table 2 Significant Nonrandom Use of U. . . . .803 2. . . . . . . . . . avenae .76 3.487 3. . . . S. .2636 5. . . . . .184 3. . . pisum . . . . . . . . . pisum . .2 3. . . . . S. . . . suberi . . chinensis . . . . . S. . . . avenae . . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . S.424 2. . . . .9462 3. . . . . pisum . . . . . . . . . . . . . . graminum. . . . . . R. . . . . . 0. . . . . . . . .05). . . . . . . . . . . . S. . S. S. . . graminum. . . . . . . S. . . . . . . . . pisum . rhois . . . . . . .oxfordjournals. . . . .293 4. . . . . . .875 2. . . . . . . . graminum. . .27 4. . M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .706 (P . . . . .org/ by guest on August 5. . . . . . . S. . . T. . . . . . . of Amino Acid Residues in Protein . graminum. . . . . . . . . . . . . . 0. S. . . . . . . . . . . S. .

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

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

. This assumption may be violated by differing levels of codon bias in Buchnera and E.015 0. high ratios of nonsynonymous to synonymous divergence indicate that replacement substitutions accumulate at faster rates in a particular lineage (Brynnel et al. 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. Given the higher divergence among Uroleucon isolates.org/ by guest on August 5.. R. distinct trpB clades were considered separately... . . .. .. . .. a b Downloaded from http://mbe..025 0.051 8 1 7 2 5 1 5 2 6 0.. . nodes do not affect the results obtained. This approximation of nonsynonymous divergence divided by synonymous divergence is considerably higher for the Uroleucon isolates. trpE .. .. .. G. Each clade.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. . . . .. For the purposes of mapping nucleotide changes at each codon position. . . .. .12 0.. . .) 0.083 0. groES .8 43.09 5. . .6 24. however..77 0. . . b The x2 value expresses the deviation of the observed from the expected number of loci with preference for A-ending codons.154 0. .047 0.42 2.. S . . . Therefore.162 to 0.. gave comparable values for the ratio of the tree length at first and second positions. divided by the length at third positions adjusted for the number of sites.019 0. .. . coli versus Buchnera Under the assumption that rates of synonymous substitutions reflect mutation rates. ... .077 0. . .9 NO.. fig. . . .. trpA . 32 8 119 64 15 14 46 36 39 0. OF BUCHNERA LOCIa TOTAL LOCI x2 VALUEb 7. . .. . .01 0. .031 0.3 45.. . that the resolved trees presented are not intended to represent exact relationships among E. However.077 0. trpD . .. 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. tuf . coli or Buchnera isolates.8 31. ..031 0..07 0. . fig. . . ..123 0. P . The ECOR and Buchnera trees each include nodes with relatively weak support (see bootstrap values. .. . . Estimates of Synonymous Substitution in E. . It should be noted. 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. .... .0 37.. . L . . T . .. trpG . .. . . .. . c groEL had the greatest overrepresentation in the pool of significant tests compared to its frequency in the original sample. of x2 Tests Fraction of Total x2 Tests Performeda No.. . .0328 across the E. .. 4). The total number of significant x2 tests was 65. .041 0..015 0... .oxfordjournals. .. . 1998).. .060 0.13 8..092 A total of 772 x2 tests were performed. .018 0. 4). 37 52 33 37 32 72 41 55 53 47 37 48 23 31 54 51 90 99 70 85 55 103 95 106 39.92 Wernegreen and Moran Table 3 Number of Buchnera Loci Showing a Slight Preference for U. Because of low levels of divergence within each group. .. . V. . . .. . coli may depress rates of synonymous substitution and thus elevate Ka/Ks estimates.. coli strains (0.. . .64 P VALUE (. however. Significant x2 Tests Fraction of Significant x2 Testsb groELc .0 46.. .. . relative to the E. . ...01 0.....010 0. trpB .. these relatively weak ..285 across subsets of the Buchnera trpB phylogeny and 0. leu genes .32 6. .. .. 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. .6 42. .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.. nucleotide changes were summed across genealogies of trpB instead of calculated pairwise (fig. . .108 0. . coli. . coli strains and Buchnera associated with the aphid genus Uroleucon. . . 4). coli trpB phylogeny.

taxon 1 is Buchnera of Schizaphis graminum. .16 0. . . in Buchnera. ilvI . 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. gapA . Conclusions Because of their small population sizes and limited opportunities for recombination. observed duplications of leucine genes and their occurrence on plasmids may represent an adaptation for overexpression (Bracho et al.21 0. . . . While its role Downloaded from http://mbe. .vs. which do not synthesize tryptophan and which depend on Buchnera to provide this essential nutrient (Baumann et al.22 0. .22 0. . . gidA. dnaJ. Lissen. . Baumann et al. . . .42 0. .36** 1.01*** 4.05.34 0. The protein is known in E. since groEL is apparently single copy in at least one Buchnera species in which it is overexpressed. .07 0. .17 0.23 0.17 0. . .12 0.20 0. .0 2. putida H. . .6 1. .53 0. .84*** 6. . .11 0. . Taxon 3 is a more distantly related reference taxon. . . dnaN .36 0.21 0.7 2. Baumann. .24*** 1.21 0. . . influenzae Haemophilus ducreyi Vibrio cholerae Pseudomonas putida P.65*** 5. vertically inherited endosymbionts provide a good model system to test the effects of increased drift on sequence evolution in bacteria.38 0.37 0. influenzae P.and second. leuD.9 2. This increased copy number is considered an adaptation that benefits the aphid hosts. Rouhbakhsh et al.58 0.14 zb K01/K02 7. 1998).13** 3.45 0.8 2.4 1. . the symbiont of Acyrthosiphon pisum (Ohtaka and Ishikawa 1993). .09 0. .56 0. and taxon 2 is always Escherichia coli. and Baumann 1994. . . influenzae H. .01. the first and limiting enzyme in the tryptophan biosynthetic pathway.3 1. relative-rate tests and comparisons of Ka/Ks ratios support previous conclusions that Buchnera lineages experience rapid sequence evolution at nonsynonymous sites.26*** 3. tpiA .14 0.07 0. . Goloubinoff et al. both mutualistic (Aksoy 1995.36 0. influenzae H. .4 3. rpoB . which are limiting in the plant phloem on which aphids feed. In this study. groEL comprises about 10% of the total protein produced (Ishikawa 1984.9 2.55 0. Hara et al. .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. . . . .06 0. In addition.4 4. . comparisons of changes at first. . A set of loci that might be suspected of experiencing unusual selection in Buchnera are those encoding enzymes for biosynthesis of essential amino acids. . Douglas 1998). . Baumann et al. . .24 0. coli.05 0. . . . are amplified in tandem repeats on multicopy plasmids (Lai. influenzae H. . .12 0.oxfordjournals. leuC. .22 0. .07 0. 2015 ilvC . . . . 0. . . b z scores were calculated as described by Muse and Weir (1992).25 0.31 0. secB. .45 0.29 0.0001. 1998a). 1997. rpsA. .25 0. .29 0.23* 3. 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.16 0. Gatenby.30 0. . . . .06 0. . and Lorimer 1989). and translocation of other polypeptides (Bochdareva.36 0. .44 0.41 0. c ‘‘(pt)’’ indicates that only a partial sequence is available in GenBank.05 0. . .20 2. . 1990).7 5. However. . groEL is known to be constitutively overexpressed in several intracellular bacteria. assembly. 0. ilvD . .14 0.34** 3.37** 5. In addition. . 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. . .9 1. .25 0. compared to their free-living relative.29 0.10 0.32 0.25 0. 1998b). dnaG(pt)c . and Clark 1996) and pathogenic (Garduno et al. atpD . . influenzae Pseudomonas aeruginosa Azotobacter vinelandii A.66*** 1. . . .33 5. . .20 0. and Girshovich 1988. . .3 6. influenzae H.7 2. relative to synonymous sites.18 0.21 4. . .org/ by guest on August 5. which are Buchnera of Acyrthosiphon pisum. Function . . influenzae H.40 0. In addition. 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. aroA . . .59 0. influenzae 0. 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. . except for dnaJ and dnaK. 1996. The mechanism for this overexpression is uncertain but likely involves changes in gene regulation rather than a gene duplication. . .6 3.18 0.3 2. .71 1. Goloubinoff. . . . .38*** 4. ** P . .21 0. E.5 2.18*** 3.42 0.09 0. .37 0.06 0. genes for anthranilate synthase (trpEG). . putida H. .30 0.0 3.6 a In each test. .58 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. . . 1989.27 0. . dnaK .33 0. In several Buchnera lineages. influenzae H. coli) also suggest higher rates of fixation at replacement sites. .31 0. 1995. cysE.21 0. . 1997.77 0.12 0. . In Buchnera.43 0. vinelandii Aeromonas salmonicida H. .02 0. . . . 0. .28 0. . .04 0.20 0.83*** 2.21*** 6. *** P . van Ham et al.6 4. Probabilities for one-tailed t-test (H :K 0 01 # K02) are * P . Baumann. . . Buchnera provisions host insects with these nutrients. . 1995.10 0. coli to be a heat shock chaperonin involved in the folding. .24 0. Baumann. 1997. leuB . . . Likewise. . .14 0.00*** 6. third-codon positions across very shallow taxonomic levels (Buchnera isolates of Uroleucon and members of the ECOR collection of E.48*** 7. leuA . . .

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

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

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