Centre for the Study of Evolution, School of Biological Sciences, University of Sussex, United Kingdom
Correspondence: E-mail: m.r.q.woolfit{at}sussex.ac.uk.
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Abstract |
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Key Words: symbiosis yeast-like symbionts phylogeny AT bias relative rate likelihood ratio test
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Introduction |
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Two early tests of this prediction, using sequence data from organisms assumed to differ in population size, found that species with smaller Ne had faster rates of sequence evolution (Wu and Li 1985; DeSalle and Templeton 1988). However, each analysis was based on a single comparison, which limits the conclusions which can be drawn from them, a problem shared by several more recent studies (Easteal and Collet 1994; Kliman et al. 2000; Weinreich 2001). Spradling, Hafner, and Demastes (2001) compared data from 21 species of rodents and found an inverse relationship between Ne and rate of evolution, but their analysis did not use phylogenetically independent comparisons (see below). Johnson and Seger's (2001) study compared rates of evolution in mainland and island species of birds and found an increase in substitution rate in island species, which are assumed to have restricted population sizes. However, many of their comparisons overlapped on the phylogeny, and so they provide relatively few statistically independent data points to test the hypothesis.
Endosymbiotic microorganisms provide a further test of the effect of Ne on substitution rates (Moran 1996). These mutualistic bacteria and fungi live sequestered within the cells of eukaryotic hosts, and are maternally transmitted by the infection of eggs or embryos (Buchner 1965). This mode of transmission results in endosymbiotic lineages remaining sequestered within a host lineage, with strictly vertical transmission, dividing the population into isolated subpopulations which do not exchange genetic material. Relatively few endosymbionts are thought to be transmitted in each inoculationfor various Buchnera species, the number is estimated to be between 40 and several thousand individuals (Buchner 1965; Rispe and Moran 2000; Mira and Moran 2002), a small percentage of the population found in each adult host (Mira and Moran 2002). Although bottleneck size has not been quantified for other endosymbiont species, it seems likely that many of them undergo a similar degree of population reduction. These two characteristics of endosymbiont biology, population subdivision and recurrent severe reductions in population size (bottlenecks), both act to reduce the effective population size of these species (Rispe and Moran 2000).
Asexuality is also expected to reduce effective population size, because of the increased linkage of the genome (Felsenstein 1974; Pamilo, Nei, and Li 1987; Lynch and Blanchard 1998). It is not known whether bacterial endosymbionts are sexual, although it is probable that Buchnera at least is largely clonal, as it has lost several genes that facilitate recombination, including recA (Tamas et al. 2002). Vertically transmitted endosymbionts are in any case effectively clonal, as any recombination can only occur between very closely related individuals.
These factorspopulation subdivision, recurrent bottlenecks and effective asexualityare expected to lead to an increased rate of fixation of slightly deleterious mutations within endosymbiont lineages. Previous analyses of sequence data from the endosymbionts of aphids (Buchnera) and psyllids (Candidatus Carsonella) have shown that, as predicted, these bacteria exhibit higher rates of substitution than free-living bacteria (Moran 1996; Thao et al. 2000), as well as other predicted effects of increased drift, such as lack of adaptive codon bias and greatly increased genomic A + T content (Clark et al. 1992; Brynnel et al. 1998; Wernegreen and Moran 1999). Genomic base composition reflects a balance between mutational biases and selection for translational efficiency. Species with large effective population sizes are expected to exhibit strong bias toward optimal codon usage. In smaller populations, however, in which translational selection is weaker, patterns of codon usage largely reflect drift and mutational biases, as has been shown in Buchnera and Candidatus Carsonella (e.g. Spaulding and von Dohlen 1998; Wernegreen and Moran 1999).
The cause of a mutational bias toward A + T in Buchnera and Candidatus Carsonella is not known, although it has been suggested that host dependence itself might result in an A + T bias (Rocha and Danchin 2002). In an analysis based on 50 bacterial whole genomes (which did not take into account phylogenetic nonindependence) it was found that average genomic base composition differed significantly between host-dependent (62% A + T) and free-living or facultatively pathogenic bacteria (51% A + T). Their explanation for this, that host-dependent bacteria must compete with their host for scarce metabolic resources, and thus experience a reduced availability of metabolically more expensive cytosine and guanine, offers a possible mechanism for the A + T mutation bias observed in these two endosymbiont species.
Reports of faster substitution rates, and in some cases other indicators of increased genetic drift, in endosymbionts of aphids, psyllids, whiteflies, tsetse flies, and mealybugs (Moran 1996; Spaulding and von Dohlen 2001) suggest that reduced Ne may affect the rate and pattern of molecular evolution in these species. However, all but one of the mutualistic endosymbionts previously examined are -Proteobacteria, and several are very closely related (Clark et al. 1992; Munson, Baumann, and Moran 1992; Spaulding and von Dohlen 1998). Therefore it is not clear whether the increased substitution rate detected is due to reduced effective population size caused by the endosymbiotic lifestyle, or to some other influence on rate in this lineage of
-Proteobacteria. To tease apart possible explanations, it is necessary to analyze data from many more groups of endosymbionts, and to take into account information on phylogenetic relationships.
Species cannot be treated as independently derived data points because they form part of a phylogenetic hierarchy. Therefore similarity between species can vary according to their level of relatedness, rather than randomly, as would be expected if species traits were independently acquired (Harvey and Pagel 1991). Cross-species correlations of traits which do not account for this nonindependence are subject to inflated type I and type II errors and may be misleading (Martins and Garland 1991; Harvey and Rambaut 1998). The method of phylogenetically independent contrasts (Felsenstein 1985) corrects for species nonindependence by using a phylogeny to identify a set of independent comparisons between pairs of species, nodes, or clades which differ in the trait of interest (e.g., endosymbiosis). Any difference in other traits between the two species, nodes, or clades (e.g., rate of molecular evolution) must have arisen since their last common ancestor, and independently of any other pair. Each comparison then contributes one independent data point to a statistical analysis. The importance of using phylogenetically independent comparisons was recognized in Moran's (1996) original analysis of rate acceleration in endosymbionts; however, data were available for only a few comparisons.
It is important to note that an assumption of independent origin of endosymbiosis is not sufficient to consider comparisons phylogenetically independent. Endosymbiosis may have arisen four or more times in the Enterobacteriaceae, but because there is only one known sister group for all four lineages (see fig. 1, -Proteobacteria 5), we cannot exclude the possibility that an event which affected the rate of molecular evolution (mutation of a gene coding for an error-correcting enzyme, for example) could have occurred on the branch ancestral to this group, causing a change in evolutionary rate in all endosymbiotic lineages within it. In this case it would be misleading to count each of these four lineages as an independent data point in terms of rate change, even though they probably do represent four independent transitions to endosymbiosis (Buchner 1965; Lambert and Moran 1998).
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Methods |
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Phylogenetic Analysis
Sequences were selected from six major groups of bacteria (the -, ß-,
-, and
-Proteobacteria, Flavobacteria, and Spirochaetes) and one group of fungi (Ascomycetes). Each of these seven data sets was aligned and analyzed separately. Sequences were aligned by eye using Se-Al (Rambaut 2002). Each alignment consisted of approximately 1,600 bp. Regions of sequence which could not be aligned unambiguously (typically <120 bp in total) were excluded from the analysis. For each of the seven data sets, phylogenies were constructed in PAUP* (Swofford 2002) using two methods, maximum likelihood (ML) and LogDet. Maximum likelihood phylogenies were constructed with an HKY +
model of nucleotide substitution (Hasegawa, Kishino, and Yano 1985; Yang 1994) with the transition-transversion ratio (ti/tv) and the gamma shape parameter (
) estimated from the data. The HKY model of substitution allows base frequencies of the four nucleotides to vary, but it assumes that these frequencies are approximately constant across the sequences being compared. Because some endosymbiont sequences have very biased base compositions (Clark, Moran, and Baumann 1999), and this can affect topology reconstruction (Mooers and Holmes 2000), a second model, LogDet, was also used. The LogDet transformation is robust to variations in base composition across the tree (Lockhart et al. 1994).
The topologies of the maximum likelihood and LogDet trees were compared and phylogenetically independent comparisons, each consisting of a monophyletic group of endosymbionts and a closely related group of nonendosymbionts, were chosen only if they were supported by both trees (see fig. 1 and table 1 for these comparisons). Although some comparisons differ in the number of species in the endosymbiotic and nonendosymbiotic groups, this is not expected to bias our estimation of rates. Maximum likelihood methods, unlike parsimony (Sanderson 1990), explicitly model multiple substitutions per site and are therefore expected to be robust to "node density effect" (Bromham et al. 2002). Both methods of rate estimation require outgroups; we chose the most closely related definite outgroup for each comparison.
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Likelihood Ratio Tests
For each comparison, the hypothesis of a difference in substitution rate between the endosymbiotic and nonendosymbiotic clades was tested using a likelihood ratio test (Felsenstein 1981; Huelsenbeck and Rannala 1997). Using Rhino (Rhino: phylogenetic models for estimating rates and dates, version 1.02, available at http://evolve.zoo.ox.ac.uk/software/Rhino/main.html), substitution rates were estimated under two models, both using an HKY + model of substitution (see fig. 2). In the first model, an average substitution rate for all taxa in the comparison was estimated, whereas in the second model, one substitution rate was estimated for the endosymbiont clade and another for the other species in the comparison. To test whether the two rates were significantly different, twice the difference of the log likelihoods of the two models was compared to a chi-squared distribution with one degree of freedom. The substitution rates estimated under the second model were then compared to determine whether the endosymbiont rate was higher than that of the nonendosymbionts and outgroup.
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A + T Content of Endosymbiont 16S Sequences
To test whether endosymbiont 16S genes tend to have higher A + T contents than those of their free-living relatives, the sequence data and comparisons used in the Rhino analysis (see table 1) were used. For each independent comparison, the average A + T content of the 16S gene was calculated for the endosymbiont clade and the nonendosymbiont clade, and the values were compared to determine which was higher. A signed-ranks test was performed across all comparisons (table 3).
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Results |
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Substitution Rate
Both Rhino and relative rate tests showed that endosymbionts tend to have significantly faster rates of substitution than their free-living relatives (Rhino: 10/13 comparisons, P = 0.014; relative rate: 9/13 comparisons, P = 0.05; table 2). Likelihood ratio tests indicated that, of the 10 positive comparisons in the Rhino analysis, nine had significantly higher rates of evolution for the endosymbiotic clade than the rest of the species in the comparison. None of the three negative comparisons were significant. Results were consistent between Rhino and relative rate tests for all but one comparison (-Proteobacteria 2). For this comparison, a nonsignificant increase in substitution rate in the endosymbiont clade was detected in the Rhino analysis, while in the relative rate test the nonendosymbiotic species had a very marginally higher rate.
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A + T Content
Endosymbiont 16S genes were not significantly more A + T rich than those of nonendosymbionts (signed-ranks test: 9/13 comparisons, P = 0.076; table 3). However, our analysis of whole bacterial genomes using phylogenetically independent comparisons showed that host-dependent bacteria (both symbiotic and parasitic) tend to have higher genomic A + T content than non-host-dependent bacteria (signed-ranks test: 6/7 comparisons, P = 0.0139; table 4). The difference in base composition between host-dependent and non-host-dependent bacteria is smallest for the one negative comparison (Corynebacterium glutamicum versus Mycobacterium leprae).
We tested whether the different results for the 16S and whole genome data sets could be due to constraint on A + T content in the 16S gene, by comparing the change in A + T in the 16S and the whole genome between pairs of bacterial species. For all 15 comparisons, the difference in A + T content between the pairs of species was greater across the whole genome than in the 16S gene (sign test P = 0.00003; table 5), indicating that the base composition of the 16S gene differs in a biased manner from that of the whole genome.
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Discussion |
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Our results are also consistent with Ohta's (1972) prediction that decreased Ne will lead to an increase in substitution rate. There are, however, several other possible explanations for this increase in rate. A change in selective regime, either increasing the strength of positive selection or relaxing selective constraint, could increase the rate of fixation of mutations. Positive selection leading to increased fixation of nonsynonymous mutations in genes involved in host interaction, such as those coding for outer membrane proteins, has been demonstrated for some parasites (Haydon et al. 2001; Urwin et al. 2002). However, this is less likely to occur in beneficial symbioses, in which conflict between host and symbiont is expected to be reduced (Jiggins, Hurst, and Yang 2002), and in fact Buchnera has lost several genes required for biosynthesis of cell-surface components involved in cell defense, suggesting that it does not need to defend itself against the host immune system (Shigenobu et al. 2000).
It is more plausible that endosymbionts might experience a relaxation of purifying selection across loci, as their environment is in some ways more stable than that of free-living microorganisms. It is not possible to distinguish between the effects of decreased selective constraints and reduction in Ne based on the 16S data presented here. However, these two processes are expected to affect intraspecific variation in different wayspolymorphism will increase under relaxed selection, but decrease with a reduction in Ne. A study designed to investigate this question by comparing intraspecific variation in three genes found that polymorphism was significantly lower in Buchnera than in enteric bacteria (Funk, Wernegreen, and Moran 2001), suggesting that, at least for this endosymbiont, rate acceleration is more likely to be the result of changes in Ne than reduced constraint.
Another possible cause of rate acceleration is an increase in the mutation rate in endosymbionts, as suggested by Itoh, Martin, and Nei (2002). Such a change in mutation rate has been hypothesized for several lineages of mutualistic fungi, due to an increase in desiccation and UV-exposure associated with moving from a subterranean niche while free-living to growing on the substrate once in symbiosis with an alga or liverwort (Lutzoni and Pagel 1997). It is not clear whether the many lineages of endosymbionts considered here have experienced a similar common ecological change which could trigger an increase in the rate of mutation, as various species of endosymbionts are housed intracellularly and intercellularly in different kinds of organs and many parts of the host body, in a taxonomically diverse range of host species.
The effects of increased mutation rate can be distinguished from those of reduced Ne by examining the types of mutations which are being fixed in the population. An increase in overall mutation rate, without any concomitant change in selective regime, will lead to an increase in the fixation of both synonymous and nonsynonymous mutations. A decrease in Ne, however, will result in synonymous mutations continuing to fix at the same rate, whereas a larger proportion of nonsynonymous mutations will be under the selection threshold and will drift to fixation. The ratio of synonymous to nonsynonymous substitutions is therefore expected to remain constant under the first hypothesis, and increase under the second. Estimating separate synonymous and nonsynonymous substitution rates is not possible for 16S sequences, but several studies of protein-coding genes in Buchnera indicate that an increase in this ratio is found across a wide range of genes in this endosymbiont (Moran 1996; Brynnel et al. 1998; Clark, Moran, and Baumann 1999; Wernegreen, Richardson, and Moran 2001; Moya et al. 2002). Once protein coding genes are sequenced from a wider range of endosymbiont taxa, it will be possible to perform similar analyses to determine how general these results are. However, considering the data currently available, it seems likely that the increased substitution rates in endosymbiont 16S genes observed here are due to a reduction in effective population size.
A + T Content of Endosymbionts and Other Host-Dependent Bacteria
The A + T-rich genomes of Buchnera and Wigglesworthia (the endosymbiont of tsetse flies) have been adduced as further evidence of genetic drift resulting from reduced Ne in endosymbiont species. The genomic A + T content of these species ranges from 74% to 78% (Akman et al. 2002; van Ham et al. 2003), and this A + T enrichment is thought to be the result of the increased dominance of mutational bias over selection for translational efficiency and preservation of gene function (Clark, Moran, and Baumann 1999; Shigenobu et al. 2001; Palacios and Wernegreen 2002). This pattern of A + T-biased base composition has been considered to be a common property of vertically transmitted endosymbiont lineages (Moran and Baumann 2000). However, a significant increase in A + T content in endosymbiont 16S sequences is not observed in our analysis.
A probable explanation for the absence of significant base compositional change is that selection for function and rRNA secondary structure stability is overcoming mutational biases in these sequences (Muto and Osawa 1987; Sueoka 1988). Although endosymbiont 16S genes have accumulated a number of slightly deleterious mutations which have destabilized their rRNA secondary structure (Lambert and Moran 1998), this degeneration may be held in check to some extent by selection against increased A + T counteracting the mutational bias toward A + T. This interpretation is consistent with the results of our analysis comparing A+ T content in 16S genes and whole genomes, for which we found that the base composition of 16S genes varies significantly less than that of the rest of the genome for a wide range of bacteria (see table 5). This suggests that the rate acceleration observed for endosymbionts in this analysis would be more pronounced at other loci, as has been observed for Buchnera (Clark, Moran, and Baumann 1999).
Further sequence data will be required to determine whether the pattern of A + T-biased base composition observed in Buchnera and Wigglesworthia genomes, and in several genes in Candidatus Carsonella, is a general feature of endosymbiont genomes. There are a number of plausible causes of such a bias (reviewed in Moran 2002), including biased nucleotide pools favoring A and T, as suggested by Rocha and Danchin (2002). This hypothesis requires further examination, as host-dependent bacteria vary both in their nucleotide biosynthesis ability and in their location within the host, and so would not be expected to display a consistent base compositional response to host-dependence (Moran 2002). Our comparative analysis of bacterial whole genomes found, however, that host-dependent bacteria have significantly increased A + T content, which is consistent with this hypothesis. Although only seven independent comparisons were analyzed, the results suggest that the observed association between host-dependence and base composition is not an artifact of phylogenetic structure. More genomic data would be needed to confirm this pattern, and to examine the mechanism involved.
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Acknowledgements |
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Footnotes |
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