Recombination events between endosymbiont strains:
Recombination in endosymbiont genomes is pervasive and such events
significantly add to the diversification of these bacteria (Jiggins, von
Der Schulenburg, Hurst, & Majerus, 2001). To check for incidence of
recombination, we first analyzed the overall rates of recombination in
the Wolbachia sequences with both ClonalFrame and RDP4. Both
analyses showed a rate of nucleotide substitutions due to
recombination/point mutation (r/m ) of around 2.4 (95% confidence
interval between 1.4- 3.7) which represents intermediate rates of
recombination (Vos & Didelot, 2009). This also indicates that
recombination introduces twice more nucleotide substitutions as compared
to point mutation in the Wolbachia dataset. Unsurprisingly, the Φ
test in SplitsTree also showed significant evidence of recombination
(p <0.001) for the same Wolbachia sequences
(Figure S3). However, for Cardinium and Arsenophonus , RDP4
did not indicate any evidence of recombination. This was probably due to
the use of a single gene (16S rRNA gene) for these two
bacteria.
To enumerate the recombination events within the Wolbachiasequences, we first looked at phylogenetic trees to check if single gene
phylogenies of all the 5 MLST genes (Figure S4) differ significantly
with the concatenated MLST trees (Figure 4). The next level of analysis
was to use sliding window algorithms in RDP4 to locate recombination
breakpoints wherever possible. All of these recombination events were
then evaluated and confirmed manually. These analyses yielded several
possible recombination events elaborated below.
Recombination between supergroups : Several cases of acquisition
of a gene or gene segment from different supergroup were detected.
Phylogenetic and network analysis of concatenated MLST dataset (Figure
4) showed Wolbachia ST-N2, infecting morph0343 (Hymenoptera-
Encyrtidae), to cluster with B supergroup. But individual gene trees
revealed that the coxA fragment of ST-N2 clusters with A
supergroup (Figure 4) and has the allelic profile of 7. This
phylogenetic disparity suggests that coxA gene of ST-N2 was
acquired via recombination from a supergroup A Wolbachia .
Curiously enough, coxA allele 7 is also found in two otherWolbachia infected hosts, ST-565 of morph0294 (Hymenoptera-
Platygastridae) and ST-544 of morph0076 (Araneae- Orthobula ),
both with supergroup A infections (Table 1). Although it is impossible
to know which Wolbachia strains originally underwent
recombination and gave rise to the recombinant allele 7 of coxA ,
yet the presence of the same allele within the community suggests that
the recombination event could have involved members within this
ecological community.
Similarly, another case of recombination was observed where a B
supergroup Wolbachia ST-560, of morph0214 (Hemiptera-Muellerianella ), had the coxA gene fragment (allele
profile 2) from the A supergroup (Figure 4). This recombinantcoxA allele 2 also share sequence similarity with ST-550 and
ST-571, where coxA alleles are different by only two base pairs
(coxA allele profile 305) indicating that perhaps this is also
another case of recombination happening within the community.
Another case of recombination between supergroups was found with another
MLST gene, gatB , but between supergroups A and F. TheWolbachia ST-552 (supergroup F), infecting morph0148 (Araneae-Zelotes ), had a recombinant gatB , where the last 190 bp
fragment came from the A supergroup.
As the concatenated MLST tree
(Figure 4) shows, ST-552 clusters with F supergroup, but the individualgatB gene tree shows it to be from the A supergroup. This 190 bp
fragment differ by only one base pair with ST-544 infecting morph0076
(Araneae- Orthobula ). This is also indicative of a possible
recombination between these two Wolbachia STs belonging to two
different supergroups.
Recombination within supergroups : The pervasive recombination
necessitated the development of the MLST scheme for Wolbachia(Baldo et al., 2006) as single gene phylogenies were unable to properly
represent the evolutionary history of a particular strain. In this
scheme, alleles of any of the five different genes are given the same
nomenclature if they share sequence identity. As table 1 shows, many of
the morphospecies also share the same alleles. In fact, instead of the
maximum possible number of unique alleles (180) that could have been
present across the 5 MLST loci of the 36 infected morphospecies, there
is only 136. This is indicative of acquisition of same alleles by
recombination and are therefore, examples of within-supergroup
recombination events whereby MLST fragments are exchanged across
endosymbionts.
Next, we tried to identify intergenic (i.e ., within a particular
MLST gene) recombination happening within a supergroup. Since, this
detection is dependent on the algorithms present in RDP4 these estimates
are inherently conservative. Most of these algorithms scans for above
than expected sequence divergence in the given dataset. Therefore,
recombination events happening between closely related strains and/or
between regions with low variation will not be recorded as significant
events by these algorithms.
There can be two types of intergenic recombination events. First,
different MLST fragments (e.g ., between coxA andgatB of two different strains) can combine to form a chimeric
gene and secondly, recombination can happen within the same MLST genes
(e.g ., within coxA of two different strains). Our analysis
did not find any examples of the former. This is unsurprising as all the
MLST fragments are housekeeping genes and such chimeric variants will be
under strong negative selection. However, eight instances of
recombination within same MLST gene were found (Table 2), all within
supergroup A.