The Rate of Recombination in Wolbachia Bacteria

Francis M. Jiggins

Department of Genetics, University of Cambridge, UK

Knowledge of the rate at which recombination occurs is critical for understanding the evolution and population genetics of bacteria, because it both generates novel combinations of beneficial alleles and also prevents the accumulation of deleterious mutations. Furthermore, it may influence our interpretation of evolutionary patterns, for example, during comparative analyses of phylogenies.

I have examined the presence and rate of recombination in a group of predominantly vertically (maternally) transmitted bacterial symbionts, arthropod Wolbachia. It is expected that vertical transmission through one sex will reduce the frequency with which different bacterial strains will come into contact and may therefore reduce the rate of recombination. I assessed this effect by comparing arthropod Wolbachia data with smaller data sets from related obligate horizontally and vertically transmitted bacteria.

All the three taxa studied belong to the Rickettsiaceae, a family of alpha-proteobacterial symbionts that live within the cell cytoplasm of their hosts. Our main focus was on the Wolbachia infections of arthropods, which mostly either distort the host sex ratio toward females (the transmitting sex) or induce cytoplasmic incompatibility (O'Neill et al. 1992Citation ; Rousset et al. 1992Citation ; Stouthamer et al. 1993Citation ; Hurst et al. 1999Citation ). I also investigated Cowdria ruminantium, a tick-borne parasite of ruminants, and Wolbachia from nematode worms, which are mutualists (Bandi et al. 1999Citation ).

The rate of horizontal transmission, and hence the hypothesized opportunity for recombination, is highest in C. ruminantium, the vertebrate parasite. Horizontal transmission is thought to be entirely absent in mutualistic Wolbachia infections because the phylogenies of the hosts and their symbionts are identical (Bandi, Anderson, and Blaxter 1998Citation ). An intermediate rate horizontal transmission is thought to occur in arthropod Wolbachia. Transmission is predominantly maternal because within a given host species there is typically strong linkage disequilibrium between the host mitochondria and Wolbachia (Montchamp-Moreau, Ferveur, and Jacques 1991Citation ; Turelli, Hoffmann, and McKechnie 1992Citation ; Grandjean et al. 1993Citation ; Ballard 2000Citation ; Schulenburg et al. 2002Citation ; but see Huigens et al. 2000Citation for an exception). But the bacterial and host phylogenies are largely incongruent, and distantly related Wolbachia strains may infect the same host, indicating that rare horizontal transmission does occur (Werren, Windsor, and Guo 1995Citation ; Schilthuizen and Stouthamer 1997Citation ; Zhou, Rousset, and O'Neill 1998Citation ).

Therefore, the hypothesis that the rate of recombination is determined by the frequency with which multiple strains coinfect a single host generates the prediction that the rate of recombination will be zero in nematode Wolbachia, intermediate in arthropod Wolbachia, and highest in Cowdria.

Recombination has been detected in two studies of arthropod Wolbachia. In the first, the phylogenies of different genes were shown to be incongruent, indicating recombination between the genes (Jiggins et al. 2001bCitation ). The second report described a gene sequence that was the product of recombination between bacteria that infect a parasitoid and its host (Werren and Bartos 2001Citation ). Therefore, current data provides evidence for just two recombination events. I have extended this analysis by describing both the taxonomic distribution of recombination and estimating the rate at which it occurs.

I analyzed aligned DNA sequences from two genes. The first was wsp from Wolbachia and its homologue map1 in Cowdria, both of which encode surface proteins. The second was the cell cycle gene ftsZ, for which data is only available from Wolbachia. Three separate alignments of wsp from the nematode worm Wolbachia (10 sequences), the A-group arthropod Wolbachia (17 sequences) and B-group arthropod Wolbachia (35 sequences) were used. A fourth alignment was made of the wsp homologue map1 from C. ruminantium (14 sequences). These alignments have EMBL accession numbers ranging from ALIGN_000198 to ALIGN_000200 (note that the final alignment includes both A- and B-group Wolbachia which I analyzed separately). The arthropod wsp alignment contains two regions that were omitted because they contain insertions and deletions that make the alignment of homologous sites uncertain. These regions were included in the data set as missing data, making the sequences the same length as that from strain wRi (accession number AF020070). Three separate alignments were made of the ftsZ gene from nematode Wolbachia and from A- and B-group arthropod Wolbachia. These alignments omitted the 3'-end of the gene because it contained insertions and deletions that made alignment uncertain.

Recombination can be detected by a decline in linkage disequilibrium with distance. This is because as the distance between two sites decreases, there will have been fewer recombination events to break down linkage disequilibrium. The linkage disequilibrium between pairs of sites was estimated using two different measures, r2 and |D'|, and the correlation with distance was calculated using Pearson's coefficient (Awadalla, Eyre-Walker, and Maynard-Smith 1999Citation ; Jorde and Bamshad 2000Citation ). The significance of the negative correlation was calculated by a Mantel test. The position of the sites was randomized and the statistic recalculated a minimum of 1,000 times. The significance was taken as the proportion of times the correlation coefficient was the same or more negative than the observed value. The analysis only included sites with two segregating alleles and was performed using the program LDhat (McVean 2001aCitation ).

There was a significant decline in linkage disequilibrium with distance in both arthropod Wolbachia and C. ruminantium but not in nematode worm Wolbachia (table 1 ). In general, the r2 measure of linkage disequilibrium detected recombination more often than the |D'| measure, which is consistent with previous analyses which have shown r2 to be more powerful (McVean 2001bCitation ). The analysis was then repeated including only those sites at which both alleles occurred at frequencies of over 10%. The exclusion of rare alleles is expected to make the test more powerful because common alleles tend to be older and are therefore more likely to show evidence of recombination (Awadalla, Eyre-Walker, and Maynard-Smith 1999Citation ). As expected, this increased the significance of the tests for recombination in all cases for arthropod Wolbachia and Cowdria sequences (table 2 ). However, the exclusion of rare alleles had no consistent effects on the analysis of the nematode sequences in which recombination was still not detected (table 2 ). Finally, the analysis was repeated using only those sites identified as being informative about recombination by a coalescent method (McVean 2001bCitation ). The exclusion of uninformative sites did not appear to consistently alter the power of the tests to detect recombination (data not shown).


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Table 1 Recombination in the Rickettsiaceae. Analysis includes all the sites with two alleles segregating

 

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Table 2 Recombination in the Rickettsiaceae. Analysis includes sites with two alleles segregating and frequencies >0.1

 
A rough guide to the relative rates at which recombination occurs can be gained by comparing the correlation coefficient of linkage disequilibrium and distance. This correlation was greater in arthropod Wolbachia than in Cowdria, which suggests that 2Ner may be higher in Wolbachia.

The second approach used to test both for recombination and estimate the rate of recombination was an approximate likelihood method based on coalescent theory (McVean, Awadalla, and Fearnhead 2002Citation ). This method estimates the value of the population rate of recombination, 2Ner, where Ne is the effective population size and r the rate of crossing over within an individual for each generation. It is this statistic that determines the effect of recombination on patterns of linkage disequilibrium. The presence or absence of recombination can be tested for using a permutation test. This method also detected recombination in arthropod Wolbachia and C. ruminantium sequences but not in nematode Wolbachia (tables 1 and 2).

The population rate of recombination, 2Ner, was then estimated by this method (tables 1 and 2). The estimate in nematode worms ranged from zero to five. The rate was higher in Cowdria (18) and in arthropod Wolbachia (20–28). This again suggests that the population rate of recombination in arthropod Wolbachia may be similar to that in its horizontally transmitting relative. The estimated value of 2Ner was consistent across both the different genes and between the A- and B- group arthropod Wolbachia (tables 1 and 2). In all four arthropod Wolbachia data sets (two genes, two groups) the estimated value of 2Ner fell between 18 and 28. The estimates were insensitive to the exclusion of rare alleles (table 2 ).

Our a priori hypothesis was that the rate of recombination would be lower in taxa with lower rates of horizontal transmission. This is supported in the case of nematode Wolbachia, which do not recombine. But the estimate of 2Ner in arthropod Wolbachia (20–28) is similar to that of the horizontally transmitting relative Cowdria (18). These are typical of the rates of recombination estimated by this method for human pathogens. For example 2Ner = 41 in the bacterium Helicobacter pylori and ranges from 0.84 to over 100 in various pathogenic viruses (McVean, Awadalla, and Fearnhead 2002Citation ).

The final approach used was to estimate the minimum number of recombination events Rm in the data by parsimony (Hudson and Kaplan 1985Citation ). This method uses an infinite sites model, which assumes an absence of homoplasy in the data. But some regions of these genes are highly variable and most probably include numerous homoplasies, meaning that these results must be treated with great caution. The number of recombination events that can be detected depends in part on the number of sequences, so for this analysis several alignments of 14 wsp-map1 sequences were used. This approach gave a slightly higher estimate of Rm in the Cowdria map1 gene (Rm = 51) compared with three alignments of the same number of arthropod Wolbachia wsp sequences (Rm = 26–35).

One possible source of error (except for in the parsimony analysis) comes from the smaller number of nematode Wolbachia sequences analyzed. Therefore I generated 10 replicate data sets of arthropod sequences containing the same number of sequences as the nematode alignments. Recombination was only detected in nine of the 10 replicates using the likelihood and linkage disequilibrium methods.

The maximum likelihood estimate of the rate of recombination (2Ner) in arthropod Wolbachia was consistent across both Wolbachia groups and both genes used. This suggests that this estimate is reasonably insensitive to any biases in the sampling of sequences or factors specific to either gene such as positive selection on the surface protein gene (Jiggins, Hurst, and Yang 2002Citation ). But Wolbachia bacteria are likely to have complex population demographics that are not accounted for in the coalescent model. This means that the values of 2Ner should be treated with caution. But given the agreement across data sets and methods of analysis, I can conclude that the population rate of recombination in arthropod Wolbachia is similar to that in Cowdria.

Early arthropod Wolbachia research often made the unstated assumption that there was no recombination. Recently, however, there were two reports of recombination, and in each case a single recombination event could explain the data (Jiggins et al. 2001b;Citation Werren and Bartos 2001Citation ). Our analysis has extended this to show that recombination occurs across both the A and B groups, across gene regions, and at a rate similar to horizontally transmitted pathogens. This surprisingly high rate of recombination may be because r is larger than expected and because different strains frequently come into contact and recombine. In particular, horizontal transmission between host species may be far more common than we realize, perhaps because most transmission events do not establish in the novel host. Alternatively, the effective population size may be very large. In this respect, it is notable that in excess of 20% of all insects may be infected with this bacterium (Werren, Windsor, and Guo 1995Citation ; West et al. 1998Citation ; Jiggins et al. 2001aCitation ).

An alternative explanation of these results is that the rate of recombination in arthropod Wolbachia is low, but natural selection favors the spread of recombinants. This could occur because of selection for evolutionary novelty in parasites (Cowdria and arthropod Wolbachia) but not mutualists (nematode Wolbachia). Our data do not provide any support for this hypothesis because the recombination rate in the conserved cell cycle gene ftsZ is similar to that in the surface protein gene wsp. The wsp gene is known to be under positive selection in parasitic but not mutualistic Wolbachia (Jiggins, Hurst, and Yang 2002Citation ).

The implications of such high rates of recombination in the evolution of Wolbachia are considerable. Recombination means that novel beneficial mutations will be able to spread across different genetic backgrounds. This may enable different bacterial strains to change the phenotypic effects they have on their host rapidly (Hurst, Jiggins, and Pomiankowski 2002Citation ). Similarly, strains that induce cytoplasmic incompatibility may rapidly combine and change their crossing type. Previous analyses have assumed that such changes required two separate mutations to occur in the same bacterium before they are favored by selection (Charlat, Calmet, and Mercot 2001Citation ). The high rate of recombination also has important consequences for biologists wanting to study these bacteria. For example, phylogenies reconstructed from gene sequence data must be treated with caution. This makes it difficult to date events in the evolution of these bacteria or reconstruct past patterns of evolution.

Acknowledgements

This research was funded by an Emmanuel College (Cambridge) Research Fellowship and a NERC small grant. Gil McVean gave helpful advice on the analysis.

Footnotes

Adam Eyre-Walker, Reviewing Editor

Keywords: Recombination ftsZ gene Rickettsiaceae Wolbachia Cowdria wsp gene Back

Address for correspondence and reprints: Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK. E-mail: fmj1001{at}mole.bio.cam.ac.uk Back

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Accepted for publication May 21, 2002.