A Conservative Test of Genetic Drift in the Endosymbiotic Bacterium Buchnera: Slightly Deleterious Mutations in the Chaperonin groEL

Corresponding author: Jennifer J. Wernegreen, Marine Biological Laboratory, 7 MBL St., Woods Hole, MA 02543., jwernegreen{at}mbl.edu (E-mail)

Communicating editor: Z. YANG

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The obligate endosymbiotic bacterium Buchnera aphidicola shows elevated rates of sequence evolution compared to free-living relatives, particularly at nonsynonymous sites. Because Buchnera experiences population bottlenecks during transmission to the offspring of its aphid host, it is hypothesized that genetic drift and the accumulation of slightly deleterious mutations can explain this rate increase. Recent studies of intraspecific variation in Buchnera reveal patterns consistent with this hypothesis. In this study, we examine inter- and intraspecific nucleotide variation in groEL, a highly conserved chaperonin gene that is constitutively overexpressed in Buchnera. Maximum-likelihood estimates of nonsynonymous substitution rates across Buchnera species are strikingly low at groEL compared to other loci. Despite this evidence for strong purifying selection on groEL, our intraspecific analysis of this gene documents reduced synonymous polymorphism, elevated nonsynonymous polymorphism, and an excess of rare alleles relative to the neutral expectation, as found in recent studies of other Buchnera loci. Comparisons with Escherichia coli generally show patterns predicted by their differences in N_e. The sum of these observations is not expected under relaxed or balancing selection, selective sweeps, or increased mutation rate. Rather, they further support the hypothesis that drift is an important force driving accelerated protein evolution in this obligate mutualist.

SEVERAL features characterize genome evolution in Buchnera aphidicola, the obligate bacterial endosymbiont of aphids. First, Buchnera shows extreme reduction of genome size compared to Escherichia coli, the most closely related free-living species in the -proteobacteria. Buchnera genomes range in size from 450 kb (GIL et al. 2002 ) to 641 kb (SHIGENOBU et al. 2000 ), while those of natural E. coli isolates vary from 4.5 to 5.5 Mb (BERGTHORSSON and OCHMAN 1995 ). The genomes of Buchnera are also extremely AT biased, at 26% GC (SHIGENOBU et al. 2000 ). In addition, Buchnera experiences elevated rates of sequence evolution across the genome, especially at nonsynonymous sites (MORAN 1996 ; ROUHBAKHSH et al. 1997 ; CLARK et al. 1999 ; WERNEGREEN et al. 2001 ). Similar patterns of genome reduction and increased evolutionary rates have been documented in other obligate endosymbionts of insects (e.g., AKSOY 2000 ; CLARK et al. 2001 ; WERNEGREEN et al. 2002 ), and accelerated 16S rDNA evolution also characterizes the maternally transmitted symbionts of mollusks (PEEK et al. 1998 ). Various studies suggest that reduced effective population sizes (N_e) and increased genetic drift may underlie these observed changes in the mode and tempo of molecular evolution (FUNK et al. 2001 ; MIRA and MORAN 2002 ).

Specific aspects of their endosymbiosis with aphids may contribute to reduced N_e in Buchnera. The exclusive occurrence of these bacteria within aphid cells and a lack of any free-living stage reflect their reciprocally obligate relationship, in which Buchnera provides essential amino acids to, and receives nutrients from, the host (SHIGENOBU et al. 2000 ). Maternal transmission of Buchnera ensures its inheritance by host offspring, but inflicts a population bottleneck since only a few bacterial cells infect each developing egg or embryo (BUCHNER 1965 ; MIRA and MORAN 2002 ). Congruence among Buchnera and host phylogenies indicates the high fidelity and evolutionary stability of this transmission mode throughout the 150–200 million years of this mutualism (MUNSON et al. 1991 ). Furthermore, the wind-borne colony founding and rapid clonal population growth of aphids (HALES et al. 1997 ) results in bottlenecks that reduce the N_e of host and endosymbiont alike and produce distinct polymorphism patterns at aphid mitochondrial genes (FUNK et al. 2001 ; ABBOT and MORAN 2002 ). An apparent lack of horizontal transfer among Buchnera strains (BUCHNER 1965 ; FUNK et al. 2000 ; WERNEGREEN and MORAN 2001 ; TAMAS et al. 2002 ) may accentuate the effects of genetic drift caused by bacterial and aphid population bottlenecks.

An elevated rate of fixation of slightly deleterious mutations under bottleneck-induced drift may generally explain the increased rates of nonsynonymous divergence observed in endosymbionts, including Buchnera. However, alternative processes must also be considered. For example, the mutualistic endosymbiotic lifestyle may relax selective constraints at specific genes that are redundant in the host cell or may relax selection across the genome as a result of decreased maximum replication rates or diminished severity of the intracellular environment compared to that experienced by related free-living bacteria. Effects of relaxed selection can resemble those of decreased N_e because both will reduce the parameter N_es and thus increase substitution rates, as predicted by the nearly neutral theory of molecular evolution (OHTA 1973 , OHTA 1992 ). Alternatively, elevated mutation pressure due to the loss of DNA repair genes in small endosymbiont genomes may drive rate acceleration (ANDERSSON and ANDERSSON 1999 ; SHIGENOBU et al. 2000 ; AKMAN et al. 2002 ; TAMAS et al. 2002 ). Positive selection may also elevate evolutionary rates, but such selection typically acts at specific loci and is not expected to produce the genome-wide rate acceleration seen in Buchnera (WERNEGREEN and MORAN 1999 ).

Fully distinguishing the effects of drift, relaxed selection, and increased mutation pressure on sequence variation is difficult, since these forces often have similar effects and may act simultaneously. For example, recent studies of interspecific divergence (WERNEGREEN and MORAN 1999 ) and intragenomic variation (PALACIOS and WERNEGREEN 2002 ) indicate that mutation bias and drift largely shape codon usage in Buchnera, in contrast to the adaptive codon bias seen in E. coli. Interspecific comparisons show elevated ratios of nonsynonymous to synonymous substitutions (d_N/d_S) across Buchnera genes of varied functional categories (CLARK et al. 1999 ; WERNEGREEN and MORAN 1999 ). Increased mutation pressure can be ruled out as a sole explanation for these observations because it should affect d_N and d_S similarly and thus not influence their ratio. However, these patterns are predicted by both relaxed selection and genetic drift, so their contributions cannot be distinguished by interspecific approaches.

Population genetic analyses can more fully distinguish the contributions of drift, selection, and mutational pressure because each of these forces has distinct predicted effects on variation within species. Reduced N_e is expected to reduce levels of neutral polymorphism due to a reduction in the time to fixation or loss under genetic drift, but should increase levels of slightly deleterious polymorphism (for which |s| 1/N_e; OHTA 1992 ) because a greater number of mutations will fall into this category. These weakly deleterious mutations would otherwise be quickly removed by selection in large populations but are free to persist and fluctuate under drift in small populations. Likewise, under reduced N_e, ratios of nonsynonymous to synonymous changes are expected to be higher within than between species, because even those slightly deleterious nonsynonymous mutations that fluctuate for a time within species under drift are most often eliminated by selection prior to fixation (MCDONALD and KREITMAN 1991 ). Unlike relaxed selection or increased mutation rates, the hypothesis of bottleneck-induced drift also predicts an excess of young (and therefore rare) alleles, since few mutations will predate the bottleneck or have had sufficient time to rise to high frequency within populations (TAJIMA 1989 ).

Applying the population genetic approach, intraspecific studies of Buchnera from two aphid species (Uroleucon ambrosiae and Pemphigus obesinymphae) demonstrated predicted effects of bottlenecks and genetic drift on patterns and levels of polymorphism (FUNK et al. 2001 ; ABBOT and MORAN 2002 ). These studies found extremely low levels of synonymous polymorphism and a significant excess of young, rare alleles compared to that expected under a neutral equilibrium model. They also detected an excess of nonsynonymous polymorphisms at a minority of assayed Buchnera genes (one of four loci).

The current study extends these prior investigations through comparative and intraspecific analyses of nucleotide variation in the chaperonin gene groEL in Buchnera and E. coli. In E. coli, groEL assists in protein folding (FAYET et al. 1989 ) and prevents misfolding under conditions of environmental stress (BOCHDAREVA et al. 1988 ). groEL is constitutively overexpressed in Buchnera (BAUMANN et al. 1996 ) and accounts for 10% of all proteins produced (ISHIKAWA 1984 ; HARA et al. 1990 ). In Buchnera, groEL may buffer against the accumulation of slightly deleterious amino acid substitutions that would otherwise cause conformational problems across the proteome (MORAN 1996 ). This compensatory process has been demonstrated experimentally in E. coli, using simulated vertical transmission events, mutation accumulation, and induced groEL overexpression (FARES et al. 2002B ). groEL of Buchnera has also acquired phosphotransferase activity as a novel histidine kinase (MORIOKA et al. 1993 , MORIOKA et al. 1994 ; MATSUMOTO et al. 1999 ). As predicted from its critical functions, groEL in Buchnera experiences stronger purifying selection than other Buchnera genes do (PALACIOS and WERNEGREEN 2002 ). Despite this, groEL nonetheless experiences accelerated protein evolution and evolves 2.4 times faster in Buchnera than in E. coli (MORAN 1996 ).

This study compares patterns of polymorphism and divergence at groEL with those reported for several additional Buchnera genes sampled in the previous complementary study (FUNK et al. 2001 ). We also examine site-specific synonymous and nonsynonymous substitution rates in groEL across the phylogeny of Buchnera associated with different Uroleucon species to evaluate purifying selection at groEL compared to other Buchnera loci. This interspecific analysis also allows us to test for positive selection, which was recently invoked in a study of groEL from divergent Buchnera lineages (FARES et al. 2002A ). The known functional importance of Buchnera groEL makes this chaperonin a strong candidate for a conservative test of the drift hypothesis. That is, detecting the signature of genetic drift at groEL would provide especially strong evidence that reduced N_e and drift play a general and dominant role in endosymbiont protein evolution.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Samples:
Although criteria for defining bacterial species are controversial, any workable species concept must consider the ecological range of a particular bacterial lineage (COHAN 2002 ). Buchnera of all aphids are technically considered the same species (B. aphidicola), but symbionts of different aphid species do not transfer and may be considered distinct populations ecologically and genetically. That is, the fixation of a mutation in Buchnera may occur throughout a particular aphid host species, but not beyond this ecological boundary. Therefore, for the purpose of this study, an "intraspecific" sample of Buchnera refers to endosymbionts of the same aphid host species, and "interspecific" refers to endosymbionts of different aphid host species.

The intraspecific data set of Buchnera includes groEL sequences of the 21 geographically widespread North American isolates described in FUNK et al. 2001 . Each of these isolates is derived from a single individual of the aphid species U. ambrosiae (Table 1). Patterns of polymorphism at groEL were compared to those of dnaN, leuBC, and trpEG sequences analyzed previously (FUNK et al. 2001 ). Buchnera from various Uroleucon host species were included in the interspecific analyses of newly collected groEL sequences and previously published dnaN, leuBC, and trpEG sequences (WERNEGREEN et al. 2001 ; Table 1). Genomic DNA of these diverse Uroleucon species was kindly provided by N. A. Moran. Collection information and original DNA extraction methods for the Buchnera strains can be found in FUNK et al. 2001 and MORAN et al. 1999 .

The E. coli data set included nine isolates from the ECOR E. coli reference strain collection (OCHMAN and SELANDER 1984 ). The E. coli strains used in this study were deliberately selected to span distinct genetic groups within the ECOR collection, similar to other E. coli population genetic studies (HALL and SHARP 1992 ; NELSON and SELANDER 1992 ; BOYD et al. 1994 ; GUTTMAN and DYKHUIZEN 1994 ). Strains were chosen to represent major divisions (A–E) as indicated by multilocus enzyme electrophoresis (MLEE) analysis of the ECOR collection (HERZER et al. 1990 ). Isolates were kindly provided by H. Ochman (University of Arizona). Our sample included ECOR isolates 4 and 17 (group A), 29 (group B1), 51 and 60 (group B2), 46 and 50 (group D), and 31 and 37 (group E). This nonrandom sample is not directly comparable to the sample of Buchnera-U. ambrosiae isolates, which were collected without any prior knowledge of their genetic differentiation. However, the E. coli sample does provide a useful reference point to compare overall levels of variation between species of free-living and endosymbiotic bacteria. Patterns of nucleotide variation in groEL were compared to those in other genes (celC, gapA, gutB, mdh, pabB, and putP) analyzed previously in E. coli, using similar sampling across the ECOR collection. Sequences and alignments for these genes were retrieved from http://lifesci.rutgers.edu/~heylab/ProgramsandData/sites_data_sets.htm; all sequences are also available from GenBank, using accession numbers supplied in the original publications.

Molecular techniques:
Gene amplification and sequencing of Buchnera loci other than groEL were described previously (FUNK et al. 2001 ; WERNEGREEN et al. 2001 ). In this study, groEL sequences of E. coli and Buchnera were obtained through polymerase chain reaction (PCR) amplification, TA cloning of certain products, and automated sequencing as described below.

E. coli groEL: Cultures of Luria broth were inoculated with single colonies of freshly streaked ECOR isolates and incubated for 18 hr at 37° and 250 rpm. Genomic DNA was extracted using the DNeasy tissue kit (QIAGEN, Chatsworth, CA). We used PCR to amplify a 2.1-kb region of the groE operon with E. coli-specific primers designed for this study: ECgrES-42F (5'-AAACCACGTAAGCTCCGGCG-3') and EcgrEL+35R (5'-ACCCCCAGACATTTCTGCC-3'). PCR reactions were performed at 25 µl and contained one-tenth volume of diluted DNA, PCR buffer [Fisher or Promega (Madison, WI)], 2.5 mM MgCl₂ (Promega), 1.0 mM dNTPs (Invitrogen, San Diego), 0.4 pmol/µl each primer, and 0.04 units of Taq polymerase (Fisher or Promega) and were brought to volume using sterile _ddH₂O. All PCR reactions were performed in a PTC-200 gradient thermocycler (MJ Research, Watertown, MA) using initial denaturation of 94° for 2 hr, 35 cycles of 95° for 20 sec, 61° for 50 sec, 72° for 1 min, followed by a final extension at 72° for 7 min. E. coli PCR products were confirmed on agarose gels and cloned using the TOPO TA cloning kit and Top 10 One Shot chemically competent cells (Invitrogen) according to manufacturer's instructions. Clones were purified using Qiaquick PCR purification kit (QIAGEN) and were quantified by gel electrophoresis and spectrophotometry.

Buchnera-Uroleucon groEL: A region of groES and groEL of Buchnera was amplified from aphid DNA samples prepared in previous studies (MORAN et al. 1999 ; FUNK et al. 2001 ). Buchnera-specific PCR primers were designed to span a 2-kb region of the groE operon: uroGroES1F (5'-GAAAATTCGTCCGTTGCATG-3') and uroG1640R (5'-ATCATTCCGCCCATACC-3'). PCR reactions were performed as above, but with a reaction volume of 50 µl and an annealing temperature of 55°. PCR products were confirmed on agarose gels prior to purification using the Qiaquick kit (QIAGEN).

TA clones and PCR products of groEL genes were sequenced using appropriate primers on an ABI 3700 automated sequencer using Big Dye v3.0 (Applied Biosystems, Foster City, CA). Internal sequencing primers in both forward and reverse orientations were designed on the basis of the external reads. Sequences were assembled and edited using PHRED, PHRAP, and CONSED. All DNA assemblies were checked by eye and any ambiguous base calls were changed to N. Edited groEL sequences totaled 1644 bp for E. coli and 1569 bp for Buchnera. Bacterial isolates sampled and GenBank accession numbers are given in Table 1.

Data analysis:
Sequences were aligned using both MacClade 4.04 (MADDISON and MADDISON 2000 ) and Se-Al v2.0a11 (RAMBAUT 2002 ) and edited by eye. Alignments for all data sets were unambiguous. Estimates of nucleotide variation were calculated using DNASP (ROZAS and ROZAS 1999 ). These included , the average pairwise nucleotide diversity, and _w, the number of segregating sites for haploid genomes. Both and _w are estimates of the neutral parameter ( = 2N_eµ for haploid, maternally inherited genomes, where N_e is the female effective population size). In addition, we calculated the absolute number of synonymous and nonsynonymous polymorphisms and used these to estimate K, the average pairwise divergence between two species. We applied multiple tests of neutrality of sequence evolution, including Tajima's D (TAJIMA 1989 ), Fu and Li's D* (FU and LI 1993 ), Fu and Li's F* (FU and LI 1993 ), and Fu's Fs (FU 1997 ). Each of these statistics tests the prediction that two estimators of (e.g., and _w) should be equivalent in an equilibrium population that is evolving neutrally (KREITMAN 2000 ).

We applied the McDonald-Kreitman test (MK test; MCDONALD and KREITMAN 1991 ) and calculated the neutrality index (NI; Rand AND KANN 1996 ) to compare the ratios of synonymous to nonsynonymous mutations within Buchnera-U. ambrosiae and between this species and Buchnera-U. rudbeckiae. The null hypothesis of neutrality predicts that the two ratios will be equal. Buchnera-U. rudbeckiae was used for comparison because this aphid host is closely related to U. ambrosiae (FUNK et al. 2001 ; WERNEGREEN and MORAN 2001 ) and was used as the outgroup in the previous study (FUNK et al. 2001 ). The same tests were performed for E. coli groEL, using Salmonella typhimurium (GenBank accession no. U01039) as an outgroup (BRENNER 1984 ; DAUGA 2002 ).

Ratios of nonsynonymous (d_N) to synonymous (d_S) substitution rates provide an index for the strength and nature of selection at a given locus. We used the program codeml from the PAML package (YANG 2000 ) to estimate site-specific d_S and d_N for the Uroleucon interspecific data set of groEL and the leuBC, trpEG, and dnaN data sets examined previously (WERNEGREEN et al. 2001 ; Table 1). Parameters were optimized across the phylogeny of Buchnera groEL (data not shown), which is consistent with published phylogenies of Buchnera-Uroleucon (CLARK et al. 1999 ; WERNEGREEN and MORAN 2001 ) and the host (MORAN et al. 1999 ). Parameter estimates were calculated using two nested likelihood models of sequence evolution. Model 0 assumes a single d_N/d_S () across all sites in a gene, while model 3 allows to vary among codon sites, with three site classes available. (Neither model allows variation in among branches in the phylogeny.) The significance of differences in the likelihoods of the two models was evaluated with the likelihood ratio test (HUELSENBECK and BULL 1996 ). When interpreting d_N/d_S, values >1 are generally considered evidence for positive selection, while values <1 suggest purifying selection (NIELSEN 2001 ). The power of site-specific estimates is particularly sensitive to the taxon sample size, as values can be overestimated for small samples such as the 10 species used in this study (SUZUKI and NEI 2002 ). This does not seriously compromise its use here, however, since we are primarily interested in the presence and relative strength of selection among Buchnera genes (all of which would be similarly affected by such overestimates), rather than in quantifying it in absolute terms.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Buchnera
Intraspecific analysis of Buchnera-U. ambrosiae: The sample of 21 Buchnera-U. ambrosiae groEL sequences represented only five distinct haplotypes and 12 segregating sites, 10 of which were singletons (Table 2 and Table 3). Buchnera groEL showed low nucleotide variation relative to other genes in Buchnera and to E. coli groEL. For example, nucleotide diversity per site (_tot) was 10-fold lower (0.10 for Buchnera) compared to that for E. coli (0.96; Table 2). Tests of neutrality in Buchnera groEL indicated an excess of rare alleles, with significantly negative values for Tajima's D for both silent and replacement sites and for Fu and Li's D* and F* (Table 4). The NI (Table 4) and MK test (Table 5) revealed a higher nonsynonymous to synonymous ratio for polymorphism than for divergence, and the MK test showed a significant deviation from the neutral expectation (G = 5.1, P = 0.024).

Interspecific analysis of d_N/d_S: The relatively low estimate of d_N/d_S (or ) at Buchnera groEL compared to those at other Buchnera genes implies low rates of nonsynonymous substitution due to strong purifying selection. The estimate in model 0 (a single value for all sites) was 10–25 times lower for groEL than for other loci (Table 6). The higher d_N/d_S observed at trpEG and leuABC corroborated previous results showing accelerated nonsynonymous substitutions at these amino acid biosynthetic genes in Buchnera-Uroleucon (WERNEGREEN et al. 2001 ). Likelihood estimates of site-specific substitution rates (model 3) fit the data better than model 0 does for every gene (Table 6), indicating significant variation in site classes. A proportion of sites in dnaN, leuBC, and trpEG showed > 1. In contrast, the highest estimated at groEL was still quite low (maximum = 0.1355) and represented a small fraction (5.7%) of the total sites. This very low at groEL indicates strong purifying selection against amino acid changes and provides no evidence of positive selection (i.e., > 1).

View this table: [in this window] [in a new window]   Table 6. Maximum-likelihood estimation of synonymous and nonsynonymous substitution rates ( = dN/dS) of Buchnera genes

E. coli
Each of the nine E. coli isolates represented a unique haplotype at groEL because, as in other population genetic studies of E. coli (see above), we selected isolates that span the known genetic diversity of the ECOR strain collection. Fifty percent of segregating sites were singletons and, as mentioned above, E. coli showed much higher levels of nucleotide diversity than did Buchnera at groEL (Table 2). Compared to other genes in E. coli, however, groEL showed low nucleotide diversity and extreme codon bias (Table 2). Tests of neutrality based on mutation spectra were nonsignificant in E. coli (Table 4), except for Tajima's D estimate for replacement mutations. Nevertheless, the relatively high NI (3.6; Table 4) and a significant MK test result (G = 4.4, P = 0.036; Table 5) indicate elevated ratios of nonsynonymous to synonymous polymorphism relative to divergence.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Molecular evolutionary rates in Buchnera are elevated at both synonymous and nonsynonymous sites, but the rate acceleration is greater at nonsynonymous sites (MORAN 1996 ; WERNEGREEN and MORAN 1999 ). In addition, endosymbionts such as Buchnera experience substitutions in the 16S rDNA gene that destabilize the secondary structure of the 16S rRNA molecule and further suggest the accumulation of deleterious changes by genetic drift (LAMBERT and MORAN 1998 ). Previous intraspecific analyses (WERNEGREEN and MORAN 1999 ; FUNK et al. 2001 ; ABBOT and MORAN 2002 ) are completely consistent with the hypothesis that genetic drift underlies this observed rate increase. The present study extends these investigations and evaluates whether drift offers an explanation that is sufficiently general and powerful to account for variation at an overexpressed chaperonin, groEL.

Evolution of groEL—comparisons within Buchnera:
Consistent with its functional importance in the symbiosis, we observed low d_N/d_S at Buchnera groEL compared to other Buchnera genes. Likelihood estimates of substitution rates between Buchnera species reveal only a small fraction (5.7%) of sites with ratios as high as 0.1355, in contrast to > 1 for 2.6 and 7.7% of sites in the biosynthetic genes leuBC and trpEG, respectively (Table 6). A miniscule fraction of sites at dnaN (0.9%) showed > 1. The action of positive selection at leuBC and trpEG is unclear, given the relatively small taxon sample available (SUZUKI and NEI 2002 ). However, this comparison highlights the variable selective pressures experienced by different Buchnera loci and the exposure of groEL to comparatively strong purifying selection.

Our population genetic analysis of groEL adds to the growing evidence that strong effects of genetic drift in small endosymbiont populations explain unusual patterns of genetic variation in Buchnera. Our pertinent findings from Buchnera-U. ambrosiae include low levels of synonymous polymorphism, the apparent accumulation of slightly deleterious mutations suggested by MK tests, and an excess of young, rare alleles and singletons that is reflected in significant values of Tajima's D and Fu and Li's D* and F*. All these observations are consistent with the expected effects of drift under the repeated bottlenecking caused by bacterial transmission and aphid demographics. Such bottlenecks result in (1) a loss of allelic diversity; (2) a high proportion of extant alleles that have had insufficient time to rise to appreciable frequencies (TAJIMA 1989 ); and (3) a genome-wide decrease in the efficacy of selection, so that an increasing proportion of mutations fall into the nearly neutral category (OHTA 1992 ) and are observed as nonsynonymous polymorphisms.

Such slightly deleterious amino acid changes would be quickly removed in large populations where selection is more effective, but may fluctuate under genetic drift in small populations, thus contributing to elevated polymorphism (OHTA 1992 ). However, even these slightly deleterious nonsynonymous mutations are likely to be eliminated by selection prior to fixation (MCDONALD and KREITMAN 1991 ; BROOKFIELD and SHARP 1994 ), such that ratios of nonsynonymous to synonymous changes within species should exceed those between species. The common observation of this pattern in mitochondrial genes, for example, has recently been interpreted as indicating the unexpectedly high frequency of slightly deleterious alleles in the mitochondrial genome (Rand et al. 1994 , Rand et al. 2000 ; Rand AND KANN 1996 ). The large NI value of Buchnera groEL relative to other, less conserved, Buchnera genes also supports previous findings of greater ratios of nonsynonymous to synonymous polymorphism than divergence in more conserved genes (Rand AND KANN 1996 ; HASEGAWA et al. 1998 ).

Many explanatory alternatives to drift exist, but none are completely compatible with the sum of our findings. These alternatives are summarized here for the sake of completeness. First, although excess nonsynonymous polymorphism might be explained by relaxed selection, this mechanism should yield similar increases in nonsynonymous divergence, which is not observed. This discrepancy might be a consequence of a recent relaxation of selection that is restricted to the focal study species (here, Buchnera-U. ambrosiae) and has not affected the outgroup lineage (here, U. rudbeckiae; NACHMAN et al. 1996 ). However, this hypothesis is inconsistent with the general rate of acceleration observed across Buchnera lineages associated with diverse aphid host taxa (CLARK et al. 1999 ).

Second, although a recent selective sweep can also explain low synonymous polymorphism and left-skewed allele distributions (TAJIMA 1989 ), it cannot explain the excess of nonsynonymous intraspecific polymorphisms observed in the MK tests (Table 5).

Third, balancing selection (POLLEY and CONWAY 2001 ) may explain excess nonsynonymous polymorphism, but also predicts an excess of alleles at intermediate frequency rather than the excess of rare Buchnera alleles observed here and previously (FUNK et al. 2001 ; MIRA and MORAN 2002 ).

Fourth, it has been proposed that the elevated substitution rate in Buchnera might entirely reflect increased mutation rates across the genome (ITOH et al. 2002 ). However, increased mutation pressure alone cannot explain the elevated d_N/d_S documented extensively for Buchnera (MORAN 1996 ; BRYNNEL et al. 1998 ; CLARK et al. 1999 ; WERNEGREEN and MORAN 1999 ). Increased mutation rates should affect both nonsynonymous and synonymous sites equally and thus leave their ratio unchanged. Furthermore, elevated mutation rate cannot explain our observations of low synonymous polymorphism levels, skewed allele distributions, and significant MK test results.

Evolution of groEL—Buchnera vs. E. coli:
Previous studies have compared patterns of sequence evolution in Buchnera and E. coli, due to their close phylogenetic relationship and extreme differences in life histories and population sizes (CLARK et al. 1999 ; WERNEGREEN and MORAN 1999 ). The effective population size of E. coli has been estimated at 2 x 10⁸ (HARTL et al. 1994 ) and 2.5 x 10⁹ (OCHMAN and WILSON 1987 ), while N_e of Buchnera is estimated to be 10⁷ for both Buchnera-U. ambrosiae (FUNK et al. 2001 ) and Buchnera-P. obesinymphae (ABBOT and MORAN 2002 ). Unlike Buchnera, E. coli experiences limited recombination among strains and is globally distributed across diverse hosts. As discussed above, we sampled E. coli to deliberately span distinct genetic (MLEE) groups within the ECOR collection, as done in other E. coli population genetic studies (HALL and SHARP 1992 ; NELSON and SELANDER 1992 ; BOYD et al. 1994 ; GUTTMAN and DYKHUIZEN 1994 ).

This sample allows us to compare overall levels of genetic variation between Buchnera and E. coli at groEL and to compare this chaperonin with other loci previously sampled from each species. At groEL, E. coli shows 5-fold higher levels of nonsynonymous polymorphism than Buchnera does (_non = 0.16 and 0.03, respectively) and 10-fold higher levels of synonymous polymorphism (_syn = 3.35 and 0.32), consistent with the predicted negative relationship between N_e and nucleotide polymorphism (Rand AND KANN 1996 ). Further, _non/_syn is higher in Buchnera (0.10) groEL than in E. coli (0.05), consistent with decreased synonymous polymorphism and/or increased (slightly deleterious) nonsynonymous polymorphism in this bottlenecked endosymbiont. For both species, groEL is relatively conserved compared to other genes (Table 2). The low nonsynonymous divergence between E. coli and S. typhimurium at groEL (K_A = 0.007) compared to other loci sampled (mean K_A = 0.039 for 67 pairwise comparisons) indicates exceptionally strong purifying selection at this chaperonin (SHARP 1991 ). In E. coli, groEL shows extreme codon bias (0.77 codon adaptation index; SHARP and LI 1987A ), consistent with its high expression level and demonstrated functional importance, and the large N_e of E. coli.

Contrary to expected patterns of sequence variation in large populations, E. coli groEL, like that of Buchnera, showed an excess of nonsynonymous polymorphism, as indicated by the significant MK test (Table 5). Like Buchnera, E. coli also exhibited a significant excess of rare alleles at replacement sites relative to the neutral expectation. However, the clonal and subdivided population structure of E. coli (MILKMAN 1973 ; WHITTAM et al. 1983 ) and our own nonrandom selection of genetically divergent and ecologically diverse strains for analysis may partially explain these patterns. For example, this sampling scheme may have predisposed us to find nonsynonymous mutations that had been fixed in local populations by either drift or divergent selection. Indeed, all of the nine nonsynonymous mutations in our sample of E. coli groEL are singletons unique to six isolates representing major ECOR divisions. In addition, selection on codon usage at high expression genes in E. coli may have influenced synonymous variation and thus affected the ratios of nonsynonymous to synonymous changes (SHARP and LI 1987B ). Thus, any tentative explanations for the unexpected MK and Tajima's D results will require further analysis of additional genes and of closely related isolates within the ECOR groups. However, potential inflation of nonsynonymous polymorphism in E. coli would actually bias against the conclusions we draw from our comparison of _non/_syn in Buchnera and E. coli. That is, if our sampling strategy overestimated nonsynonymous polymorphism in E. coli, then _non/_syn would be elevated in E. coli. Despite this potential bias, _non/_syn is nonetheless greater in Buchnera than in E. coli, consistent with the effects of a decreased N_e and repeated bottlenecks.

In sum, our study documents patterns of nucleotide variation that are highly consistent with an important role for genetic drift in the nearly neutral molecular evolution of a highly constrained Buchnera locus. Our population genetic approach allows us to further demonstrate these patterns to be inconsistent with explanations based on alternative evolutionary mechanisms. These results further support the hypothesis that population bottlenecks play a generally important role in the molecular evolution of bacterial endosymbionts.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY372289, AY372290, AY372291, AY372292, AY372293, AY372294, AY372295, AY372296, AY372297, AY372298, AY372299, AY372300, AY372301, AY372302, AY372303, AY372304, AY372305, AY372306, AY372307, AY372308, AY372309, AY372310, AY372311, AY372312, AY372313, AY372314, AY372315, AY372316, AY372317, AY372318 and AY372485, AY372486, AY372487, AY372488, AY372489, AY372490, AY372491, AY372492, AY372493.

	ACKNOWLEDGMENTS

We are grateful to Seth Bordenstein, Adam Lazarus, and two anonymous reviewers for comments on the manuscript and Roger Milkman for helpful discussion. We thank Jonas Sandström and Nancy Moran for collecting the Uroleucon samples used in the interspecific analysis and Paul Baumann for DNA extractions of several of these isolates. This work was made possible by support to J.J.W. from the National Institutes of Health (R01 GM62626-01), the National Science Foundation (DEB 0089455), the National Aeronautics and Space Administration Astrobiology Institute (NCC2-1054), and the Josephine Bay Paul and C. Michael Paul Foundation.

Manuscript received April 22, 2003; Accepted for publication October 8, 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ABBOT, P. and N. A. MORAN, 2002  Extremely low levels of genetic polymorphism in endosymbionts (Buchnera) of aphids (Pemphigus). Mol. Ecol. 11:2649-2660.[Medline]

AKMAN, L., A. YAMASHITA, H. WATANABE, K. OSHIMA, and T. SHIBA et al., 2002  Genome sequence of the endocellular obligate symbiont of tsetse flies, Wigglesworthia glossinidia.. Nat. Genet. 32:402-407.[Medline]

AKSOY, S., 2000  Tsetse—a haven for microorganisms. Parasitol. Today 16:114-118.[Medline]

ANDERSSON, J. O. and S. G. ANDERSSON, 1999  Insights into the evolutionary process of genome degradation. Curr. Opin. Genet. Dev. 9:664-671.[Medline]

BAUMANN, L., P. BAUMANN, and M. A. CLARK, 1996  Levels of Buchnera aphidicola chaperonin groEL during growth of the aphid Schizaphis graminum.. Curr. Microbiol. 32:279-285.

BERGTHORSSON, U. and H. OCHMAN, 1995  Heterogeneity of genome sizes among natural isolates of Escherichia coli.. J. Bacteriol. 177:5784-5789.[Abstract/Free Full Text]

BOCHDAREVA, E. S., N. M. LISSEN, and A. S. GIRSHOVICH, 1988  Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature 336:254-257.[Medline]

BOYD, E. F., K. NELSON, F. S. WANG, T. S. WHITTAM, and R. K. SELANDER, 1994  Molecular genetic basis for allelic polymorphism in malate dehydrogenase (mdh) in natural populations of Escherichia coli and Salmonella enterica.. Proc. Natl. Acad. Sci. USA 91:1280-1284.[Abstract/Free Full Text]

BRENNER, D., 1984 Enterobacteriaceae. Williams & Wilkins, Baltimore.

BROOKFIELD, J. F. Y. and P. M. SHARP, 1994  Neutralism and selection face up to DNA data. Trends Genet. 10:109-111.[Medline]

BRYNNEL, E. U., C. G. KURLAND, N. A. MORAN, and S. G. ANDERSSON, 1998  Evolutionary rates for tuf genes in endosymbionts of aphids. Mol. Biol. Evol. 15:574-582.[Abstract]

BUCHNER, P., 1965 Endosymbiosis of Animals With Plant Microorganisms. Interscience Publishers, New York.

CLARK, M. A., N. A. MORAN, and P. BAUMANN, 1999  Sequence evolution in bacterial endosymbionts having extreme base compositions. Mol. Biol. Evol. 16:1586-1598.[Abstract]

CLARK, M. A., L. BAUMANN, M. L. THAO, N. A. MORAN, and P. BAUMANN, 2001  Degenerative minimalism in the genome of a psyllid endosymbiont. J. Bacteriol. 183:1853-1861.[Abstract/Free Full Text]

COHAN, F. M., 2002  What are bacterial species? Annu. Rev. Microbiol. 56:457-487.[Medline]

DAUGA, C., 2002  Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies. Int. J. Syst. Evol. Microbiol. 52:531-547.[Abstract]

FARES, M. A., E. BARRIO, B. SABATER-MUNOZ, and A. MOYA, 2002a  The evolution of the heat-shock protein groEL from Buchnera, the primary endosymbiont of aphids, is governed by positive selection. Mol. Biol. Evol. 19:1162-1170.[Abstract/Free Full Text]

FARES, M. A., M. X. RUIZ-GONZALEZ, A. MOYA, S. F. ELENA, and E. BARRIO, 2002b  Endosymbiotic bacteria: groEL buffers against deleterious mutations. Nature 417:398.[Medline]

FAYET, O., T. ZIEGELHOFFER, and C. GEORGOPOULOS, 1989  The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J. Bacteriol. 171:1379-1385.[Abstract/Free Full Text]

FU, Y. X., 1997  Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics 147:915-925.[Abstract]

FU, Y. X. and W. H. LI, 1993  Statistical tests of neutrality of mutations. Genetics 133:693-709.[Abstract]

FUNK, D. J., L. HELBLING, J. J. WERNEGREEN, and N. A. MORAN, 2000  Intraspecific phylogenetic congruence among multiple symbiont genomes. Proc. R. Soc. Lond. B Biol. Sci. 267:2517-2521.[Medline]

FUNK, D. J., J. J. WERNEGREEN, and N. A. MORAN, 2001  Intraspecific variation in symbiont genomes: bottlenecks and the aphid-Buchnera association. Genetics 157:477-489.[Abstract/Free Full Text]

GIL, R., B. SABATER-MUNOZ, A. LATORRE, F. J. SILVA, and A. MOYA, 2002  Extreme genome reduction in Buchnera spp.: toward the minimal genome needed for symbiotic life. Proc. Natl. Acad. Sci. USA 99:4454-4458.[Abstract/Free Full Text]

GUTTMAN, D. S. and D. E. DYKHUIZEN, 1994  Detecting selective sweeps in naturally occurring Escherichia coli.. Genetics 138:993-1003.[Abstract]

HALES, D. F., J. TOMIUK, K. WOHRMANN, and P. SUNNUCKS, 1997  Evolutionary and genetic aspects of aphid biology: a review. Eur. J. Entomol. 94:1-55.

HALL, B. G. and P. M. SHARP, 1992  Molecular population genetics of Escherichia coli: DNA sequence diversity at the celC, crr, and gutB loci of natural isolates. Mol. Biol. Evol. 9:654-665.[Abstract]

HARA, E., T. FUKATSU, K. KAKEDA, M. KENGAKU, and C. OHTAKA et al., 1990  The predominant protein in an aphid endosymbiont is homologous to an E. coli heat shock protein. Symbiosis 8:271-283.

HARTL, D. L., E. N. MORIYAMA, and S. A. SAWYER, 1994  Selection intensity for codon bias. Genetics 138:227-234.[Abstract]

HASEGAWA, M., Y. CAO, and Z. YANG, 1998  Preponderance of slightly deleterious polymorphism in mitochondrial DNA: nonsynonymous/synonymous rate ratio is much higher within species than between species. Mol. Biol. Evol. 15:1499-1505.[Free Full Text]

HERZER, P. J., S. INOUYE, M. INOUYE, and T. S. WHITTAM, 1990  Phylogenetic distribution of branched RNA-linked multicopy single-stranded DNA among natural isolates of Escherichia coli. J. Bacteriol. 172:6175-6181.[Abstract/Free Full Text]

HUELSENBECK, J. and J. BULL, 1996  A likelihood ratio test to detect conflicting phylogenetic signal. Syst. Biol. 45:92-98.

ISHIKAWA, H., 1984  Characterization of the protein species synthesized in vivo and in vitro by an aphid endosymbiont. Insect Biochem. Mol. Biol. 14:417-425.

ITOH, T., W. MARTIN, and M. NEI, 2002  Acceleration of genomic evolution caused by enhanced mutation rate in endocellular symbionts. Proc. Natl. Acad. Sci. USA 99:12944-12948.[Abstract/Free Full Text]

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

LAMBERT, J. D. and N. A. MORAN, 1998  Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria. Proc. Natl. Acad. Sci. USA 95:4458-4462.[Abstract/Free Full Text]

MADDISON, D., and W. MADDISON, 2000 MacClade: Analysis of Phylogeny and Character Evolution. Sinauer Associates, Sunderland, MA.

MATSUMOTO, K., M. MORIOKA, and H. ISHIKAWA, 1999  Phosphocarrier proteins in an intracellular symbiotic bacterium of aphids. J. Biochem. 126:578-583.[Abstract/Free Full Text]

MCDONALD, J. H. and M. KREITMAN, 1991  Adaptive protein evolution at the Adh locus in Drosophila.. Nature 351:652-654.[Medline]

MILKMAN, R., 1973  Electrophoretic variation in Escherichia coli from natural sources. Science 182:1024-1026.[Abstract/Free Full Text]

MIRA, A. and N. A. MORAN, 2002  Estimating population size and transmission bottlenecks in maternally transmitted endosymbiotic bacteria. Microbiol. Ecol. 44:137-143.[Medline]

MORAN, N. A., 1996  Accelerated evolution and Muller's rachet in endosymbiotic bacteria. Proc. Natl. Acad. Sci. USA 93:2873-2878.[Abstract/Free Full Text]

MORAN, N., M. KAPLAN, M. GELSEY, T. MURPHY, and E. SCHOLES, 1999  Phylogenetics and evolution of the aphid genus Uroleucon based on mitochondrial and nuclear DNA sequences. Syst. Entomol. 24:85-93.

MORIOKA, M., H. MURAOKA, and H. ISHIKAWA, 1993  Chaperonin produced by an intracellular symbiont is an energy-coupling protein with phosphotransferase activity. J. Biochem. 114:246-250.[Abstract/Free Full Text]

MORIOKA, M., H. MURAOKA, K. YAMAMOTO, and H. ISHIKAWA, 1994  An endosymbiont chaperonin is a novel type of histidine protein kinase. J. Biochem. 116:1075-1081.[Abstract/Free Full Text]

MUNSON, M. A., P. BAUMANN, M. A. CLARK, L. BAUMANN, and N. A. MORAN et al., 1991  Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families. J. Bacteriol. 173:6321-6324.[Abstract/Free Full Text]

NACHMAN, M. W., W. M. BROWN, M. STONEKING, and C. F. AQUADRO, 1996  Nonneutral mitochondrial DNA variation in humans and chimpanzees. Genetics 142:953-963.[Abstract]

NELSON, K. and R. K. SELANDER, 1992  Evolutionary genetics of proline permease gene (putP) and the control region of the proline utilization operon in populations of Salmonella and Escherichia coli.. J. Bacteriol. 174:6886-6895.[Abstract/Free Full Text]

NIELSEN, R., 2001  Statistical tests of selective neutrality in the age of genomics. Heredity 86:641-647.[Medline]

OCHMAN, H. and R. K. SELANDER, 1984  Standard reference strains of Escherichia coli from natural populations. J. Bacteriol. 157:690-693.[Abstract/Free Full Text]

OCHMAN, H., and A. C. WILSON, 1987 Evolutionary history of enteric bacteria, pp. 1649–1654 in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, edited by H. E. UMBARGER. American Society for Microbiology, Washington, DC.

OHTA, T., 1973  Slightly deleterious mutant substitutions in evolution. Nature 246:96-98.[Medline]

OHTA, T., 1992  The nearly neutral theory of molecular evolution. Annu. Rev. Ecol. Syst. 23:263-286.

PALACIOS, C. and J. J. WERNEGREEN, 2002  A strong effect of AT mutational bias on amino acid usage in Buchnera is mitigated at high expression genes. Mol. Biol. Evol. 19:1575-1584.[Abstract/Free Full Text]

PEEK, A. S., R. C. VRIJENHOEK, and B. S. GAUT, 1998  Accelerated evolutionary rate in sulfur-oxidizing endosymbiotic bacteria associated with the mode of symbiont transmission. Mol. Biol. Evol. 15:1514-1523.[Free Full Text]

POLLEY, S. D. and D. J. CONWAY, 2001  Strong diversifying selection on domains of the Plasmodium falciparum apical membrane antigen 1 gene. Genetics 158:1505-1512.[Abstract/Free Full Text]

RAMBAUT, A., 2002 Se-Al Sequence Alignment Editor. Oxford University Press, London/New York/Oxford.

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

RAND, D. M., M. DORFSMAN, and L. M. KANN, 1994  Neutral and non-neutral evolution of Drosophila mitochondrial DNA. Genetics 138:741-756.[Abstract]

RAND, D. M., D. M. WEINREICH, and B. O. CEZAIRLIYAN, 2000  Neutrality tests of conservative-radical amino acid changes in nuclear- and mitochondrially-encoded proteins. Gene 261:115-125.[Medline]

ROUHBAKHSH, D., M. A. CLARK, L. BAUMANN, N. A. MORAN, and P. BAUMANN, 1997  Evolution of the tryptophan biosynthetic pathway in Buchnera (aphid endosymbionts): studies of plasmid-associated trpEG within the genus Uroleucon.. Mol. Phylogenet. Evol. 8:167-176.[Medline]

ROZAS, J. and R. ROZAS, 1999  DNASP, version 3: an integrated program for molecular population genetics and molecular evolutionary analyses. Bioinformatics 15:174-175.[Abstract/Free Full Text]

SHARP, P. M., 1991  Determinants of DNA sequence divergence between Escherichia coli and Salmonella typhimurium: codon usage, map position, and concerted evolution. J. Mol. Evol. 33:23-33.[Medline]

SHARP, P. M. and W. H. LI, 1987a  The Codon Adaptation Index—a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 15:1281-1295.[Abstract/Free Full Text]

SHARP, P. M. and W. H. LI, 1987b  The rate of synonymous substitution in enterobacterial genes is inversely related to codon usage bias. Mol. Biol. Evol. 4:222-230.[Abstract]

SHIGENOBU, S., H. WATANABE, M. HATTORI, Y. SAKAKI, and H. ISHIKAWA, 2000  Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407:81-86.

SOKAL, R. R., and F. J. ROHLF, 1991 Biometry, Ed. 3. W. H. Freeman, New York.

SUZUKI, Y. and M. NEI, 2002  Simulation study of the reliability and robustness of the statistical methods for detecting positive selection at single amino acid sites. Mol. Biol. Evol. 19:1865-1869.[Abstract/Free Full Text]

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

TAMAS, I., L. KLASSON, B. CANBACK, A. K. NASLUND, and A. S. ERIKSSON et al., 2002  50 million years of genomic stasis in endosymbiotic bacteria. Science 296:2376-2379.[Abstract/Free Full Text]

WERNEGREEN, J. J. and N. A. MORAN, 1999  Evidence for genetic drift in endosymbionts (Buchnera): analyses of protein-coding genes. Mol. Biol. Evol. 16:83-97.[Abstract]

WERNEGREEN, J. J. and N. A. MORAN, 2001  Vertical transmission of biosynthetic plasmids in aphid endosymbionts (Buchnera). J. Bacteriol. 183:785-790.[Abstract/Free Full Text]

WERNEGREEN, J. J., A. O. RICHARDSON, and N. A. MORAN, 2001  Parallel acceleration of evolutionary rates in symbiont genes underlying host nutrition. Mol. Phylogenet. Evol. 19:479-485.[Medline]

WERNEGREEN, J. J., A. B. LAZARUS, and P. H. DEGNAN, 2002  Small genome of Candidatus Blochmannia, the bacterial endosymbiont of Camponotus, implies irreversible specialization to an intracellular lifestyle. Microbiology 148:2551-2556.[Abstract/Free Full Text]

WHITTAM, T. S., H. OCHMAN, and R. K. SELANDER, 1983  Multilocus genetic structure in natural populations of Escherichia coli. Proc. Natl. Acad. Sci. USA 80:1751-1755.[Abstract/Free Full Text]

YANG, Z., 2000 PAML: Phylogenetic Analysis by Maximum Likelihood. University College, London.

This article has been cited by other articles: